WO2007120166A2 - Miniaturized in vitro protein expression array - Google Patents

Miniaturized in vitro protein expression array Download PDF

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WO2007120166A2
WO2007120166A2 PCT/US2006/026752 US2006026752W WO2007120166A2 WO 2007120166 A2 WO2007120166 A2 WO 2007120166A2 US 2006026752 W US2006026752 W US 2006026752W WO 2007120166 A2 WO2007120166 A2 WO 2007120166A2
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protein
expression
nucleic acid
array
detection
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PCT/US2006/026752
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French (fr)
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WO2007120166A3 (en
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Zhonghui Hugh Fan
Shuguang Jin
Qian Mei
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University Of Florida Research Foundation, Inc.
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Publication of WO2007120166A2 publication Critical patent/WO2007120166A2/en
Publication of WO2007120166A3 publication Critical patent/WO2007120166A3/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/08Ergonomic or safety aspects of handling devices
    • B01L2200/082Handling hazardous material
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14Process control and prevention of errors
    • B01L2200/142Preventing evaporation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • B01L2300/0654Lenses; Optical fibres
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • B01L2300/0663Whole sensors
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0829Multi-well plates; Microtitration plates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867Multiple inlets and one sample wells, e.g. mixing, dilution
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0472Diffusion

Definitions

  • Detection and identification of toxic agents are important for medical diagnostics, food/water safety testing, and biological warfare defense. Methods to detect them include immunoassays, sensors, mass spectrometry, and genetic analysis. Nucleic acid-based genetic analysis involves DNA amplification that offers high sensitivity and unambiguous identification. However, nucleic acid-based genetic analysis is not applicable to agents that contain no nucleic acids.
  • ricin which is listed as a Category B bioterrorism agent according to the Centers for Disease Control and Prevention.
  • Immunoassay is advantageous over other methods due to its simplicity and rapid analysis, but it requires an antibody that is specific to the agent of interest. Therefore, it can not be used for detecting unknown or new agents because of the fact that antibody is simply not available.
  • the ability to detect agents that have not been identified previously becomes more important. There is thus a need in the art to develop assays for the detection of toxins and other biological agents.
  • a sensor array is designed for detecting inhibition of biological synthesis of pre- characterized proteins which indicates presence of a toxic analyte.
  • the sensor array comprises an array of units; each unit is for expression of one protein and thus functions as one sensor.
  • the sensor array expresses a group of pre-characterized proteins in different expression system, thus the response pattern (or signature) of an analyte due to different inhibitory effects is registered and used as a tool for detection and identification. New agents are identified by comparing the response pattern with signatures of known agents in a pre- collected database.
  • a sensor for detecting toxins comprises a substrate; at least one well formed in or on said substrate; at least a first in vitro transcription and translation (IVT) unit disposed in said well, said first IVTs comprising a DNA template including a coding sequence which is transcribed into messenger RNA using an RNA polymerase; a eukaryotic or prokaryotic lysate providing ribosomes for protein translation by said messenger RNA, wherein said first IVT expresses a first protein, and a detector for detecting a signal related to a concentration of said first protein, wherein a level of said signal is reduced when a target toxin which inhibits expression of said first protein by said IVT is present as compared to when said target toxin is not present.
  • IVTT in vitro transcription and translation
  • a sensor array for detecting multiple toxins comprises a substrate; a plurality of wells formed in or on said substrate; a first and at least a second in vitro transcription and translation (IVT) unit disposed in a first and at least a second of said plurality of said wells, respectively, said first and second IVTs each comprising a DNA template including a coding sequence which is transcribed into messenger RNA using an RNA polymerase; a eukaryotic or prokaryotic lysate providing ribosomes for protein translation by said messenger RNA, wherein said first IVT expresses a first protein and said second IVT expresses a second protein different from said first protein, and a detector for detecting a signal related to a concentration of said proteins, wherein a level of said signal is reduced when a target toxin capable of inhibiting protein translation for said IVTs is present as compared to when said target toxin is not present.
  • IVTT in vitro transcription and translation
  • a sensor array in which each sensor comprises at least a feeding chamber; a reaction chamber; a membrane for feeding nutrients and removing by products.
  • a channel connects a feeding chamber and a reaction chamber and a pumping mechanism is used for supplying nutrients.
  • a pumping mechanism is used for removing byproducts and/or a pumping mechanism is used for supplying nutrients and removing byproducts.
  • a pumping mechanism comprises at least one of: osmosis, capillary force, pneumatic pump, syringe pump, electrokinetic pump, piezoelectric pump, acoustic, or other pumps.
  • a method of identifying inhibitors of protein expression comprises expressing a nucleic acid sequence encoded by an expression vector in a reaction mixture wherein the nucleic acid is transcribed and translated in presence or absence of a candidate drug; measuring protein expression in the presence or absence of a candidate drag; and, identifying inhibitors of protein expression.
  • the reaction mixture comprises polymerases, nucleotides, amino acids.
  • a surrogate cell array comprises a microcompartment wherein each microcompartment comprises a reaction chamber, feeding chambers, and a channel or a cavity to connect each microcompartment.
  • the reaction chamber is a miniaturized reaction chamber having a diameter of about 0.1 mm to 30 mm.
  • the feeding chamber comprises amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer.
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • the reaction chamber comprises a cell-free expression system, such as for example, in vitro transcription and translation (TVT).
  • TVT in vitro transcription and translation
  • a channel or a cavity connected to the chambers comprises a dialysis membrane with the molecular weight cutoff of about 1 kDa to about 20
  • the dialysis membrane has a molecular weight cut off of about 10 KDa.
  • the channel or cavity diameter is about 5 ⁇ m up to 10 mm.
  • the feeding chamber is dimensionally proportioned to about 2 to 100 times larger than the dimensions of the reaction chamber.
  • a miniaturized array comprises a reaction chamber dimensionally proportioned to contain a volume of fluid 10 times less than a feeding chamber.
  • the reaction chamber is dimensionally proportioned to contain about
  • the chambers comprising the surrogate cell array are made from at least one material selected from the group consisting of polypropylene, polycarbonate, polystyrene, vinyl, acrylic, plastics, metal and glass.
  • the surrogate cell array comprises a plurality of microcompartments and channels.
  • the new detection systems can include pattern recognition software.
  • the software compares the target molecule binding pattern corresponding to the unknown sample with binding patterns corresponding to known compounds. From these comparisons, the software can determine the composition of the sample, or deduce information about the source of the sample.
  • the systems can be used to detect the existence of characteristic compounds, or "molecular fingerprints," associated with certain chemicals or conditions. For example, the systems can be used for human drug testing by detecting the presence of drugs. The systems can also be used for pollution monitoring by detecting compounds characteristic of the discharge of certain toxic pollutants.
  • Figure IA is a schematic representation showing three dimensional view of a miniaturized, 2x3 solution array for ricin detection. The units were laid out according to the standards of 96-well plates (i.e., 9 mm pitch).
  • Figure IB is a schematic representation of a cross-sectional view of one unit of the array in Figure IA.
  • Figure 2 is a schematic representation showing a surrogate cell array for in vitro protein expression.
  • the layout is presented in the format of 96-well microplates.
  • An exploded view of one cell is shown at the bottom, consisting of a reaction chamber, two feeding chambers, a channel connecting the chambers, and a dialysis membrane. The design is not to scale.
  • FIG. 3 A shows Green Fluorescent Proteins (GFP) expression confirmed by Western blotting.
  • Lane 1 pre-stained protein markers; 2: negative control (containing all reagents except for the DNA template); 3: recombinant GFP (rGFP) purchased; 4: GFP expressed. Expressed GFP contains a stretch of six histidines at its C-terminal, causing its molecular weight slightly larger than rGFP.
  • Figure 3B shows CAT expression confirmed by Western blotting.
  • Lane 1 protein markers; 2: negative control; 3: CAT expressed.
  • Figure 3 C shows luciferase expression confirmed by luminescence detection.
  • Lane 1 negative control; 2: luciferase expressed.
  • FIG. 4A shows the inhibitory effects of tetracycline (TC) on the expression yield of GFP in E. coli expression system.
  • Lane 1 of Western blotting analysis pre-stained protein markers; 2: expression with 3000 ng/ ⁇ L of TC; 3: 300 ng/ ⁇ L; 4: 30 ng/ ⁇ L; 5: 3 ng/ ⁇ L; 6: 0.3 ng/ ⁇ L 7: positive control (no TC); 8: negative control; 9: rGFP purchased.
  • Figures 4B- 4G shows the effects of TC ( Figures 4B, 4D and 4F or cycloheximide (CH) ( Figures 4C, 4E, 4G) on the expression yield of GFP ( Figures 4B, 4C) in E. coli expression system, of CAT ( Figures 4D, 4E) in E. coli expression system, and of luciferase in rabbit reticulocyte expression system. All x axes are the concentration (ng/ ⁇ L) of toxin in log scale. Y axes are the amount of expressed protein either normalized to the positive control ( Figures 4B-4E) or in log scale of luminescence signal ( Figures 4F, 4G).
  • FIG. 5 A and 5B shows the response pattern of the 3x4 IVT sensor array for two toxin simulants, tetracycline (TC, Figure 5A) and cycloheximide (CH, Figure 5B).
  • the experiments were carried out in two of the 2x3 well devices.
  • the signals for the positive control were from the first row of 3 wells in the device, in which GFP, CAT, and luciferase were expressed in their respective expression systems. These wells were free of toxins.
  • the signals for the negative control were from the second row of 3 wells in the device, in which the expression vector was not added.
  • the signals for the samples were from the remaining two rows of 3 wells in the device, in which either 17 ng of CH or 25 ng of TC was added into the protein expression system.
  • Figure 6 A is a graph showing normalized production yield of luciferase as a function of the expression time in the device or in a microcentrifuge tube. A linear DNA vector was used.
  • Figure 6B is a graph showing GFP expression yield as a function of time. Expression took place in microcentrifuge tube (closed circles) or in a device with dialysis membrane (open circles). The error bars were obtained from 3 repeats.
  • Figure 7 is a schematic representation illustrating signal amplification as a result of the inhibitory effects of ricin on the production of every copy of protein. Thousands of copies of a protein can be expressed from each copy of RNA.
  • Figure 8 is a graph showing the calibration curve for ricin detection. Protein expression yield, indicated by luminescence, decreased with the concentration of ricin A chain (solid circles). However, the expression yield remained the same when the ricin A chain was heat denatured and its toxicity was deactivated (open circles). Experiments were carried out in the device and the expression time was 4 hours. The error bars are the standard deviation obtained from three repeat experiments. Lines are the best fit of linear regression of the experimental results.
  • Figure 9 A is a graph showing luciferase production as a function of the expression time in a miniaturized device or in a microcentrifuge tube. The expression yield is indicated by the luminescence. A circular DNA vector was used.
  • Figure 9B is a graph showing protein expression yield, indicated by luminescence, decreased with the concentration of ricin A chain when the expression time was fixed at 5 minutes (solid circles). However, the expression yield remained the same when the ricin' s toxicity was deactivated by heat denature (open circles).
  • Figure 10 is a graph showing the comparison among ricin A chain, B chain, whole ricin, and ricin treated with 2-mercaptoethanol.
  • FIG. 11 is a schematic illustration of a surrogate cell array for protein expression. The area shadowed with diagonal lines indicates a subset of 24 wells. The row A is for the positive controls, B for the negative controls, and C-H for the samples. The inset in the expanded view shows transcription and translation in each surrogate cell.
  • Detection and identification of toxins are important for medical diagnostics, food/water safety testing, and biological warfare defense.
  • Methods to detect toxic agents include immunoassay, sensors, mass spectrometry, and genetic analysis.
  • Nucleic acid-based genetic analysis involves DNA amplification that offers high sensitivity and unambiguous identification. However, it is not applicable to agents that contain no nucleic acids.
  • ricin which is listed as a Category B bioterrorism agent according to the Centers for Disease Control and Prevention.
  • Immunoassay is advantageous over other methods due to its simplicity and rapid analysis, but it requires an antibody that is specific to the agent of interest. Therefore, it can not be used for detecting unknown or new agents because of the fact that antibody is simply not available. With the increasing ability to modify and engineer potential warfare agents, the ability to detect agents that have not been identified previously becomes more important.
  • the array is based on the mechanism of toxin actions.
  • One of mechanisms toxins use to cause toxic effects is to inhibit protein synthesis in cells.
  • ricin acts on the 28S ribosomal subunit and prevents the binding of elongation factor-2, a critical protein in the process of protein translation. This interaction inactivates ribosomes, inhibits protein synthesis, and leads to cell death.
  • elongation factor-2 a critical protein in the process of protein translation. This interaction inactivates ribosomes, inhibits protein synthesis, and leads to cell death.
  • a number of potent biological toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin, diptheria toxin and exotoxin A. Since each type of toxin has unique mechanism of toxicity, it is possible to exploit the mechanism of toxin actions for toxin detection.
  • exon and intron are art-understood terms referring to various portions of genomic gene sequences.
  • Exons are those portions of a genomic gene sequence that encode protein.
  • Introns are sequences of nucleotides found between exons in genomic gene sequences.
  • the terms “rare” or “low copy numbers” refer to nucleic acid molecules that are less than about 300 copies per cell.
  • moderate or “medium copy numbers” refer to nucleic acid molecules that are about 300-1,000 copies per cell.
  • unsaturant or “high copy numbers” refer to nucleic acid molecules that are about
  • Amplification relates to the production of additional copies of a nucleic acid sequence.
  • nucleic acid molecule or “polynucleotide” will be used interchangeably throughout the specification, unless otherwise specified.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides
  • RNA molecules deoxyribonucleosides
  • DNA molecules deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA--DNA, DNA-RNA and
  • RNA--RNA helices are possible.
  • RNA molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • fragment or segment as applied to a nucleic acid sequence, gene or polypeptide, will ordinarily be at least about 5 contiguous nucleic acid bases (for nucleic acid sequence or gene) or amino acids (for polypeptides), typically at least about 10 contiguous nucleic acid bases or amino acids, more typically at least about 20 contiguous nucleic acid bases or amino acids, usually at least about 30 contiguous nucleic acid bases or amino acids, preferably at least about 40 contiguous nucleic acid bases or amino acids, more preferably at least about 50 contiguous nucleic acid bases or amino acids, and even more preferably at least about 60 to 80 or more contiguous nucleic acid bases or amino acids in length.
  • “Overlapping fragments” as used herein, refer to contiguous nucleic acid or peptide fragments which begin at the amino terminal end of a nucleic acid or protein and end at the carboxy terminal end of the nucleic acid or protein. Each nucleic acid or peptide fragment has at least about one contiguous nucleic acid or amino acid position in common with the next nucleic acid or peptide fragment, more preferably at least about three contiguous nucleic acid bases or amino acid positions in common, most preferably at least about ten contiguous nucleic acid bases amino acid positions in common.
  • oligonucleotide or “primers” are used interchangeably throughout the specification and include linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorthiorate, methylphosphonate, and the like.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • phosphorthiorate phosphorthiorate
  • methylphosphonate and the like.
  • Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, Ho ⁇ gsteen or reverse Ho ⁇ gsteen types of base pairing, or the like.
  • the oligonucleotide may be composed of a single region or may be composed of several regions. For example, hinge regions comprising different lengths and base composition.
  • the oligonucleotide may be "chimeric", that is, composed of different regions.
  • "chimeric" compounds are oligonucleotides, which comprise two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties.
  • the desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties.
  • the chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.
  • the oligonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers.
  • the spacers are intended to constitute a covalent "bridge” between the regions and have in preferred cases a length not exceeding about 100 carbon atoms.
  • the spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
  • the term "monomers” typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units.
  • Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.
  • nucleobase covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered “non- naturally occurring” have subsequently been found in nature. Thus, “nucleobase” includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof.
  • nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5- methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S.
  • nucleobase is intended to cover every and all of these examples as well as analogues and tautomers thereof.
  • Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.
  • nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
  • nucleosides in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429-4443, Toulme, J.
  • Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
  • the term "stability" in reference to duplex or triplex formation generally designates how tightly an antisense oligonucleotide binds to its intended target sequence; more particularly, “stability” designates the free energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability.
  • oligonucleotides are selected that have melting temperatures of at least 45 0 C when measured in 100 mM NaCl, 0.1 mM EDTA and 10 niM phosphate buffer aqueous solution, pH 7.0 at a strand concentration of both the oligonucleotide and the target nucleic acid of 1.5 ⁇ M.
  • duplex or triplex formation will be substantially favored over the state in which the antigen and its target are dissociated.
  • a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes.
  • modified oligonucleotides, e.g. comprising LNA units, of the invention form perfectly matched duplexes and/or triplexes with their target nucleic acids.
  • the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Tm Thermal Melting Point
  • stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C. for short probes (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • probe or “capture probe” are defined as a nucleic acid, capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e. A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids (PNA) in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • PNA peptide nucleic acids
  • target nucleic acid refers to a nucleic acid (often derived from a biological sample), to which the probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified.
  • the target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target.
  • target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from context.
  • stringent conditions refers to conditions under which a probe will hybridize to its target subsequence, but with only insubstantial hybridization to other sequences or to other sequences such that the difference may be identified. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • downstream when used in reference to a direction along a nucleotide sequence means in the direction from the 5' to the 3' end.
  • upstream means in the direction from the 3' to the 5' end.
  • the term "gene” means the gene and all currently known variants thereof and any further variants which may be elucidated.
  • variant of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic”, “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type target genes.
  • Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base.
  • SNPs single nucleotide polymorphisms
  • the presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.
  • mRNA means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.
  • complementary means that two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence.
  • the complementary sequence of the oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence.
  • alleles or variants thereof can be identified.
  • a BLAST program also can be employed to assess such sequence identity.
  • complementary sequence as it refers to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule by the base-pairing rules. More particularly, the term or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% of the nucleotides of the other strand, usually at least about 98%, and more preferably from about 99 % to about 100%.
  • Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software, for example the BLAST program.
  • the "percentage of sequence identity” or “sequence identity” is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the polynucleotide sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical subunit (e.g. nucleic acid base or amino acid residue) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Percentage sequence identity when calculated using the programs GAP or BESTFIT is calculated using default gap weights.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and WunschJ MoI. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sd.
  • a "heterologous” component refers to a component that is introduced into or produced within a different entity from that in which it is naturally located.
  • a polynucleotide derived from one organism and introduced by genetic engineering techniques into a different organism is a heterologous polynucleotide which, if expressed, can encode a heterologous polypeptide.
  • a promoter or enhancer that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter or enhancer.
  • a "promoter,” as used herein, refers to a polynucleotide sequence that controls transcription of a gene or coding sequence to which it is operably linked.
  • a large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources, are well known in the art and are available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • an "enhancer,” as used herein, refers to a polynucleotide sequence that enhances transcription of a gene or coding sequence to which it is operably linked.
  • enhancers from a variety of different sources are well known in the art and available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • a number of polynucleotides comprising promoter sequences (such as the commonly-used CMV promoter) also comprise enhancer sequences.
  • "Operably linked” refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter controls transcription of the coding sequence. Although an operably linked promoter is generally located upstream of the coding sequence, it is not necessarily contiguous with it.
  • An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. Operably linked enhancers can be located upstream, within or downstream of coding sequences.
  • a polyadenylation sequence is operably linked to a coding sequence if it is located at the downstream end of the coding sequence such that transcription proceeds through the coding sequence into the polyadenylation sequence.
  • a "detectable marker gene” is a gene that allows cells carrying the gene to be specifically detected (e.g., distinguished from cells which do not carry the marker gene).
  • a large variety of such marker genes are known in the art. Preferred examples thereof include detectable marker genes which encode proteins appearing on cellular surfaces, thereby facilitating simplified and rapid detection and/or cellular sorting.
  • the lacZ gene encoding beta-galactosidase can be used as a detectable marker, allowing cells transduced with a vector carrying the lacL gene to be detected by staining.
  • a "selectable marker gene” is a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selective agent.
  • an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be positively selected for in the presence of the corresponding antibiotic.
  • Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated.
  • a variety of such marker genes have been described, including bifunctional (i.e. positive/negative) markers (see, e.g., WO
  • patient or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • Substrate or “probe substrate” refers to a solid phase onto which an adsorbent can be provided (e.g., by attachment, deposition, etc.).
  • Eluant or "washing solution” refers to an agent that can be used to mediate adsorption of a marker to an adsorbent. Eluants and washing solutions are also referred to as
  • Selectivity threshold modifiers Eluants and washing solutions can be used to wash and remove unbound materials from the substrate surface.
  • Detect refers to identifying the presence, absence or amount of the object to be detected.
  • Detectable moiety refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, 35 S, fluorescent dyes, 6xHis, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety in a sample.
  • Energy absorbing molecule refers to a molecule that absorbs energy from an ionization source in a mass spectrometer thereby aiding desorption of analyte from a surface. Depending on the size and nature of the analyte, the energy absorbing molecule can be optionally used. Energy absorbing molecules used in MALDI are frequently referred to as
  • CHCA dihydroxybenzoic acid
  • sample is used herein in its broadest sense.
  • a sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
  • a sample also comprises any chemical compound; any environmental agent such as toxins, pollutants and the like, water samples, air samples, and soil samples.
  • substantially purified refers to nucleic acid molecules or proteins that are removed from their natural environment and are isolated or separated, and are at least about
  • Substrate refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
  • reaction mixture refers to an amplification mixture, a DNA dependent DNA polymerase, an RNA polymerase, the triphosphate nucleotides and the triphosphate deoxynucleotides, a DNA dependent RNA polymerase, a cellular translation extract, the mixtures necessary for amplification, transcription and translation and optionally one or several substances permitting revelation of the activity of the reporter molecule.
  • the system can be used as a platform technology to develop therapeutic drug, identify targets for future use on biosensors and evaluate cellular responses to toxic/pathogenic insult at the molecular level.
  • the systematic and quantitative study of the interplay among a large number of the constituents in the range outside the typical concentration in a biological cell can be used to quickly identify the target molecules of any toxins that inhibit protein expression.
  • the target information will lead to the development of therapeutics and ultimately clinical diagnostic assays for use in hospital emergency rooms. Currently, no such capability exists.
  • the approach in this invention provides a unique approach to detect unknown or genetically modified agents.
  • the multiplexed surrogate cell array offers a means for high-throughput protein expression that is needed by the emerging proteomics research. As more and more new genes are being identified, there is a considerable need to determine the function and properties of the proteins encoded by these genes. To match high-throughput gene discovery, methods to produce a large number of proteins in parallel are needed.
  • the invention will also contributes to basic sciences and researches by understanding nanoliter-scale transcription and translation, expanding the applications of miniaturization, and bringing forward biosensor array technology.
  • the surrogate cell array system comprises an array of microcompartments; each microcompartment functions as a surrogate cell, consisting of a reaction chamber, two feeding chambers, and a channel to connect them as shown in Figures 1 and 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs.
  • the feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis.
  • the reaction chamber contains cell-free expression system with other reagents as in the feeding chambers.
  • the channel connected to the chambers provides a means to supply nutrients and remove byproducts. The selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at between about 1 KDa up to 100 KDa, which at the same time allows entry of nutrients into the feeding chamber.
  • Device Design The selective removal of small molecule byproduct
  • the device comprises an array of units; each unit consists of a reaction chamber, two feeding chambers, and a channel to connect them as shown in Figure 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs.
  • the feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis.
  • the reaction chamber contains cell-free expression system with other reagents as in the feeding chambers.
  • the channel connected to the chambers provides a means to supply nutrients and remove byproducts.
  • the selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at ⁇ 10 KDa, which at the same time allows all small molecules in the feeding chamber passing by.
  • the incorporation of membrane is important because of two facts: (1) the flow of a feeding solution leads to higher expression yield compared to static conditions, because protein synthesis will not terminate earlier due to fast depletion of energy sources (ATP and GTP); (2) removal of small molecule byproducts is also very important for the high yield expression of proteins in a cell- free medium, inhibition of protein synthesis resulting from the small molecular byproducts such as hydrolysis products of triphosphates is eliminated.
  • the device possesses appropriate geometric configuration to achieve desired properties for in vitro protein expression.
  • the dimension of the microchannel is in the range of 1 micron to 1 centimeter, depending on the flow rate required for supplying nutrients and removing byproduct.
  • the feeding chambers must be sufficiently large compared to the reaction chamber so that they function as reservoirs. In the current design, the volume of the reaction chamber is 10 nanoliter-100 microliter whereas that of the feed chambers is about 100 nanoliter-1 mililiter.
  • One of the mechanisms for supplying nutrients from the feeding chambers to the reaction chamber and for removing byproducts from the reaction chamber to the feeding chambers is osmosis, a net flow resulting from the difference in chemical potential of solutes between two solutions separated by the membrane.
  • the degree of the potential difference is determined by their difference in solute activities, which are correlated with solute concentrations.
  • the osmotic pressure, ⁇ , resulting from a byproduct in the reaction chamber can be approximately calculated by Van't Hoff s Law assuming it is in the limit of infinite dilution (Levine, 1988 Physical Chemistry. New York: McGraw-Hill Book Co.).
  • Cb is the concentration of the byproduct
  • R is gas constant
  • T is the temperature.
  • Another mechanism for manipulating fluids is the capillary force due to surface tension, especially when the dimension is very small.
  • volume flow rate in the channel, Q can be calculated using the equation below based on a fully developed flow in the channel.
  • D is the channel diameter
  • p viscosity
  • L is the distance between chambers.
  • Other pumping mechanisms may also be used for supplying nutrients and removing byproducts, including pneumatic pumps, syringe pumps, electrokinetic pumps, piezoelectric pumps, acoustic and other pumps.
  • in vitro protein expression was selected for detecting toxins that inhibit protein synthesis.
  • One of mechanisms toxins use to cause toxic effects is to inhibit protein synthesis in cells.
  • ricin acts on the 28S ribosomal subunit and prevents the binding of elongation factor-2, a critical protein in the process of protein translation. This interaction inactivates ribosomes, inhibits protein synthesis, and leads to cell death.
  • elongation factor-2 a critical protein in the process of protein translation.
  • This interaction inactivates ribosomes, inhibits protein synthesis, and leads to cell death.
  • a number of potent biological toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin, diphtheria toxin and exotoxin A. Since each type of toxin has unique mechanism of toxicity, it is possible to exploit the mechanism of toxin actions for toxin detection.
  • IVT in vitro transcription and translation
  • the device comprises an array of IVT units; each unit is for expression of one protein and thus functions as one sensor.
  • the array expresses a group of pre-characterized proteins in different expression system; the proteins and expression systems are judiciously selected so that protein synthesis in each unit is inhibited or affected differently by different type of toxins.
  • the response pattern (or signature) of a toxin due to different inhibitory effects is registered and used as a tool for detection and identification.
  • New agents are identified by comparing the response pattern with signatures of known agents in a pre-acquired database.
  • Three proteins were first synthesized in two types of expression systems. The first protein is Green Fluorescent Protein (GFP), a widely-used fluorescent molecule with known DNA sequence and crystal structure. Protein expression was carried out by combining DNA template, E. coli lysate, and a reaction mix consisting of T7-RNA polymerase, nucleotides, amino acids, and other reagents. GFP product was confirmed by fluorescence spectrometry and Western blotting. The result of Western blotting is shown in Figure 3 A.
  • GFP Green Fluorescent Protein
  • the second protein is chloramphenicol acetyl-transferase (CAT), an enzyme responsible for bacterial resistance to an antibiotic drug, chloramphenicol.
  • CAT was expressed in the same E. coli expression system; success of the protein expression was confirmed using Western blotting as shown in Figure 3B.
  • the third protein is luciferase, an enzyme from firefly tails that catalyzes the production of light in the reaction between luciferin and adenosine triphosphate (ATP). Synthesis of luciferase was carried out using rabbit reticulocyte expression system. Detection of the expression product was achieved by monitoring the intensity of luminescence after mixing with luciferin, ATP, and other reagents, as shown in Figure 3C.
  • TC tetracycline
  • CH cycloheximide
  • Figure 4 shows the expression yield of GFP, CAT, and luciferase when a series of concentrations of TC or CH were added in two protein expression systems.
  • GFP synthesis was completely inhibited when 3000 ng/ ⁇ L of TC was used. Partial inhibition was observed when a series of lower concentrations (300 ng/ ⁇ L to 0.3 ng/ ⁇ L) of TC was added. This result suggests that a qualitative and quantitative relationship exists between the expression yield and toxin amount.
  • GFP has been used for visualization, tracking, and quantification of a variety of proteins in cells after they are fused together.
  • An increase of fluorescence signal in an IVT unit indicates the production of GFP or GFP-fused proteins.
  • Quantitative information may be obtained by comparing the fluorescence signals of sample units and of reference units, which include both positive and negative controls in the array device. Any variation or adverse effects will be cancelled out between control and sample units. The magnitude of the signal can be correlated to the amount of proteins produced in the device.
  • DNA templates containing a coding sequence (reporter gene) for expressing an additional stretch of six histidines (6xHis) at the C-terminal of the protein of interest can also be used.
  • 6xHis six histidines
  • a variety of biological assays are available for detecting the amount of 6xFfis tag fused with a protein.
  • luminescence detection can also be used by fusing luciferase with proteins of interest, as demonstrated in the examples which follow.
  • Other examples include commercially available vectors with his tags, such as: E.
  • coli pET vectors-Novagen
  • Baculovirus Pharmingen
  • Yeast Invitrogen expression vectors containing His/fusion protein tags
  • the reporter gene is a gene which can be transcribed and translated in vitro in the presence of sequences which regulate its expression.
  • the protein that the reporter gene codes for can be detected by any technique known to a person skilled in the art.
  • the reporter gene can be the gene of the protein GFP (Green Fluorescent Protein), as described in the examples which follow, or that of the beta-lactamase (TEM-I).
  • GFP Green Fluorescent Protein
  • TEM-I beta-lactamase
  • the GFP Green Fluorescent Protein
  • it the fluorescent emission which is measured.
  • beta- lactamase it is the activity of this enzyme which is measured by incubating a fraction of the translation reaction in a buffer containing nitrocephine.
  • Nitrocephine is a chromogenic beta- lactamine which has the property of changing color from yellow to red when it is hydrolyzed by a beta-lactamase.
  • Any other reporter gene can be contemplated in the process of the invention, such as beta-galactosidase, beta-glucuronidase, luciferase, peroxidase or a microperoxidase, etc.
  • the reporter gene advantageously encodes for an enzyme.
  • the specificity of the labeling of the target substance can be carried out by any direct or indirect method known to a person skilled in the art.
  • the target substance is directly combined with the gene and with the elements necessary for the expression of said reporter gene in vitro. It relates, for example, to a recombinant nucleic acid molecule where the target substance is a nucleic sequence included in said recombinant nucleic acid molecule equally including the reporter gene and the sequences necessary for its in vitro expression.
  • the target substance is combined with a reporter gene and with the sequences necessary for its expression in vitro, by the intermediary of a specific ligand of the of the target substance.
  • This ligand is combined with the reporter gene and with the elements necessary for its expression in vitro. It is therefore the contacting of this ligand with the target substance which permits the carrying out of the specific labeling of the target substance.
  • It relates for example to an antibody labeled by the reporter gene and the sequences necessary for its expression in vitro which is capable of specifically recognizing a target substance composed of an antigen.
  • a target/ligand couple substance is understood as for example: an antigen/antibody, a nucleic sequence/a nucleic sequence, a probe, a receptor/a receptor ligand, etc.
  • the labeling of the specific ligand of the target substance can be as previously a direct or indirect labeling.
  • the combination of the reporter gene and a target sequence corresponding to a protein allows several embodiments.
  • the bonding of a nucleic acid molecule composed of a reporter gene and of the sequences necessary for its expression in vitro, on a protein can be carried out by techniques known to a person skilled in the art making use of bonding compounds such as: streptavidine/biotin (Kipriyanov et al., (1995). Hum Antibodies Hybridomas 6 (3), 93-101); a peptide corresponding to polylysine (Avrameas et al., (1998). PNAS 95 (10), 5601-6; Curiel et al., (1992).
  • the device is constructed using solid substrates.
  • Solid- state substrates for use can include any solid material.
  • Preferred materials include but not limited to: plastic materials including polystyrene, polymethylmethacrylate (PMMA), polythylene terephthalate polycarbonate, polydimethylsiloxane (PDMS), poly(cyclic olefin), and a variety of copolymers.
  • Solid substrates can have any useful form including, micro well plates, thin films or membranes, beads, bottles, dishes, fibers, optical fibers, woven fibers, chips, compact disks, shaped polymers, particles and microparticles.
  • an array comprises surrogate cells for in vitro protein expression.
  • Each cell comprises elements for biological protein synthesis, nutrient supply, removal of inhibitory byproducts, and flow controls.
  • In vitro protein expression is produced by implementing the coupled transcription/translation reactions in the surrogate cell.
  • a method of determining the interplay among the constituents required for protein expression is provided. Quantitative studies are performed by generating a panel of protein expression conditions by supplementing purified key components. The in vitro process allows for varying the components and their concentration ratios of the reconstituted components including initiation factors, elongation factors, release factors, and many enzymes. The effects of the amount of these components and their concentration ratios on the protein expression yield are determined. [0109] In another preferred embodiment, a method of identifying molecular responses of the surrogate cell array to toxins is provided.
  • orchratoxin A a mycotoxin produced by fungi that displays toxic effect on human and animals — for demonstrating the capability of the surrogate cell array, since its exact mechanism is not known.
  • OTA orchratoxin A
  • Toxic substances can be produced by an animal, plant or microbe.
  • a mechanism toxins use to cause toxic effects is through inhibition of protein synthesis in cells.
  • ricin a bioterrorism agent — blocks protein synthesis by inactivating ribosomes.
  • a number of other potent toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin produced by Shigella dysenteriae, Shiga-like toxin by enteropathogenic E. coli, diphtheria toxin from Corynebacterium diphtheriae and exotoxin A of Pseudomonas aeruginosa.
  • staphylococcal enterotoxin B inflicts toxic effect through activation of inflammation (superantigen)
  • botulinum inhibits the release of neurotransmitter
  • cholera toxin activates adenylcyclase activity
  • perfringin toxin causes pore-formation leading to the leakage of the cell content.
  • the surrogate cell array is modified to study different toxicity mechanisms, which can be used for toxin detection and identification based on the array response pattern as well as for searching for therapeutics.
  • OTA orchratoxin A
  • OTA a mycotoxin produced by fungi.
  • OTA is a mycotoxin produced by Penicillium and Aspergillus.
  • Penicillium and Aspergillus Several major mechanisms have been shown as involved in the toxicity of OTA, including inhibition of protein synthesis. However, the exact mechanism is not known.
  • the method uses the surrogate cell arrays to obtain quantitative information about the inhibitory effects of OTA on protein synthesis and identify its target molecules.
  • Artificial Cells Biological cells can be altered by modern techniques such as cloning and mutation. However, genetic engineering comes with limitations in cost and efficiency, as well as possible toxicity of the engineered product to the cell. As a result, artificial cells or cell surrogates have been developed to overcome the limitations. Artificial cells offer an alternative method for studying the functions of a variety of molecules with biological significance, for understanding the regulation of cellular activities, and for searching for the synthetic and metabolic pathways. In addition, artificial polymer cells have been employed for encapsulating medicines for drug delivery and organ transplant. Similarly, liposome is one of popular methods for delivering therapeutics at targeted locations. Artificial cells or cell surrogates will have significant impacts on pharmacology, medical diagnostics, and the related biological fields.
  • plastics such as for example, used in typical tissue culture, are used for fabricating the cell array device because of the following reasons.
  • plastics such as for example, used in typical tissue culture, are used for fabricating the cell array device because of the following reasons.
  • plastics such as for example, used in typical tissue culture, are used for fabricating the cell array device because of the following reasons.
  • a wide range of plastics are available to be selected for a biological assay of interest.
  • the compatibility between plastics and chemical/biological reagents is evidenced by the fact that many labwares such as microcentrifuge tubes and microplates are made of plastics.
  • a variety of plastic materials including polystyrene, polymethylmethacrylate (PMMA), polycarbonate, polydimethylsiloxane (PDMS), and poly(cyclic olefin) have been investigated for microfabrication and microscale assays.
  • Plastic parts made by techniques such as injection molding or embossing can be quite inexpensive: the manufacturing cost of an injection- molded compact disc (CD). Therefore, plastic devices can be made so cheaply as to be disposable after a single use. This could have tremendous impact in applications where cross-contamination of sequential samples is of concern.
  • Devices are fabricated following the method we described previously (Mei, Q.; Fredrickson, C. K.; Jin, S.; Fan, Z. H. Anal Chem 2005, 77, 5494-5500) though they are modified and optimized .
  • One modification is incorporation of dialysis membrane.
  • dialysis membranes that are commercially available, a polycarbonate membrane with an appropriate pore sizes or the like, is used. Thermal lamination of polycarbonate with other plastic layers is routinely practiced in industry; thermally laminating the membrane with plastic substrates forms the array device.
  • the apparatus detects biological agents such as toxins, nerve gases, bacterial agents and the like.
  • biological agents such as toxins, nerve gases, bacterial agents and the like.
  • An example of detection of ricin is shown in the examples which follow.
  • the ricin detection method is based on ricin' s inhibitory effects on protein synthesis.
  • Biological synthesis (expression) of a protein includes the steps of gene transcription and protein translation; and these reactions can be coupled into one-step operation and carried out in a cell-free medium.
  • Ricin is known to inhibit protein synthesis by interaction with 28S ribosome RNA; the inhibitory effect is exploited as the sensing mechanism in this invention. For each copy of RNA, thousands of copies of proteins can be produced.
  • An array of protein expression units is developed to accommodate positive/negative controls and multiple samples.
  • the array device contains a solution without any reagent captured on a solid surface, offering flexibility without comprising the activities of biomolecules.
  • the miniaturized solution array possesses a mechanism to supply nutrients continuously and remove byproducts, leading to higher protein expression yields and thus larger detection signals (lower detection limit) when ricin is present.
  • the production of green fluorescent protein and luciferase in the solution array is described in the examples which follow.
  • a calibration curve has been obtained between the luciferase expression yield and the ricin concentration, showing a detection limit of 1 pg/ ⁇ L of ricin.
  • the array device is also demonstrated for measuring the toxicity level of ricin after physical or chemical treatment.
  • Methods to detect ricin at a low concentration include enzyme-linked immunosorbent assay (ELISA) and immunoassay using radioactive labeling. Although offering high sensitivity, ELISA involves several labor-intensive and time-consuming steps. For radioimmunoassay, the handling and disposal of radioisotopes are environmental challenges.
  • IVT in vitro transcription and translation
  • IVT Due to the absence of cellular control mechanisms, IVT overcomes the limitations experienced by cell-based recombinant protein production, including poor expression yield, low solubility, cytotoxicity, or susceptibility to proteolysis. IVT has been demonstrated for various applications, including protein chips and drug screening.
  • IVT has also been implemented in micro fluidic devices. For instance, Nojima et al. synthesized an mRNA by flowing two reactants from two inlets and mixing them through a Y-shaped structure into one outlet (Nojima, T. et al., Bioprocess Engineering 2000, 22, 13- 17). The product was collected from the outlet and then analyzed off-the-device. Although the work showed the feasibility to implement cell-free transcription in a microfluidic device, the key drawback of this device is the use of excessive accessories including external pumps and valves, and the lack of integration, making it difficult to be implemented in a high- throughput format. IVT has also been demonstrated in the microplate format. Angenendt et al.
  • the device of the invention allows for the screening of inhibition of protein expression from any encoded nucleic acid sequence.
  • the nucleic acids are preferably expressed in an expression vector.
  • the vector comprising the desired nucleic acid sequence preferably has at least one such nucleic acid sequence.
  • the vector may comprise more than one such nucleic acid sequence, or combinations of allelic variants.
  • Genetic constructs suitable for use include regulatory elements necessary for gene expression of a nucleic acid molecule.
  • the elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal.
  • enhancers may be required for gene expression of the sequence of choice, variants or fragments thereof.
  • Initiation codons and stop codons are generally considered to be part of a nucleotide sequence that encodes the immunogenic target protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence.
  • a wide variety of expression vectors may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, nonchromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El 5 pCRl, pBR322, pMal-G2, pET, pGEX (Smith et al., 1988, Gene 67: 31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNA' s, e.g., the numerous derivatives of ⁇ phage, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • phage DNA' s e.g., the numerous derivatives of ⁇ phage
  • both non-fusion transfer vectors such as but not limited to pVL941 (BamHl cloning site; Summers), pVL1393 (BamHl, Smal, Xbal, EcoRl, Notl, Xmalll, BgIII, and Pstl cloning site; Invitrogen), ⁇ VL1392 (BgIII, Pstl, Notl, Xmalll, EcoRI, Xbal, Smal, and BamHl cloning site; Summers and Invitrogen), and pBlueBacIII (BamHl, BgIII, Pstl, Ncol, and HindIII cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc700 (BamHl and Kpnl cloning site, in which the BamHl recognition site begins with the
  • Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g., any expression vector with a DHFR expression vector, or a DHFR/methotrexate co- amplification vector, such as pED (Pstl, Sail, Sbal, Smal, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991).
  • DHFR dihydrofolate reductase
  • a glutamine synthetase/methionine sulfoximine co-amplification vector such as pEE14 (HindIII, Xbal, Smal, Sbal, EcoRI, and BcII cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech).
  • a vector that directs episomal expression under control of Epstein Barr Virus can be used, such as pREP4 (BamHl, Sfil, Xhol, Notl, Nhel, HindIII, Nhel, PvuII, and Kpnl cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHl, Sfil, Xhol, Notl, Nhel, HindIII, Nhel, PvuII, and Kpnl cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, HindIII, Notl, Xhol, Sfil, BamHl cloning site, inducible m ' ethallothionein Ha gene promoter, hygromycin selectable marker: Invitrogen
  • Selectable mammalian expression vectors for use in the invention include pRc/CMV (Hindlll, BstXI, Notl, SbaL and Apal cloning site, G418 selection; Invitrogen), pRc/RSV (Hindlll, Spel, BstXI, Notl, Xbal cloning site, G418 selection; Invitrogen), and others.
  • Vaccinia virus mammalian expression vectors for use according to the invention include but are not limited to pSCl 1 (Smal cloning site, TK- and ⁇ -gal selection), pMJ601 (Sail, Smal, AfII, Narl, BspMII, BamHI, Apal, Nhel, SacII, Kpnl, and Hindlll cloning site; TK- and ⁇ -gal selection), and pTKgptFIS (EcoRI, Pstl, Sail, Accl, Hindll, Sbal, BamHI, and Hpa cloning site, TK or XPRT selection).
  • pSCl 1 Mal cloning site, TK- and ⁇ -gal selection
  • pMJ601 Smal, AfII, Narl, BspMII, BamHI, Apal, Nhel, SacII, Kpnl, and Hindlll cloning site
  • Yeast expression systems can also be used according to the invention to express Eph polypeptides.
  • the non- fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXI, EcoRI, BstXI, BamHI, Sad, Kpnl, and Hindlll cloning sit; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXI, EcoRI, BamHI, Sad, Kpnl, and Hindlll cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the present invention.
  • Promoters and polyadenylation signals examples include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metallothionein.
  • SV40 Simian Virus 40
  • MMTV Mouse Mammary Tumor Virus
  • HIV Human Immunodeficiency Virus
  • LTR HIV Long Terminal Repeat
  • ALV a virus
  • CMV Cytomegalovirus
  • EBV Epstein Barr Virus
  • RSV Rous Sarcoma Virus
  • polyadenylation signals useful to practice the present invention include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
  • the SV40 polyadenylation signal is in pCEP4 plasmid (Invitrogen, San Diego Calif), referred to as the SV40 polyadenylation signal.
  • additional elements include enhancers.
  • the enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • the invention is useful for identifying candidate drugs and to screen the effects of the drugs in protein expression for therapy.
  • the nucleic acid sequences used for detecting protein expression in the presence or absence of a candidate drug can be any nucleic acid sequence that may be involved in a disease, for example, over expression of tumor genes. These genes can include any allelic variants, mutants and the like. Sequences of different nucleic acids are accessible from various public databases such as GenBank. [0131] As more genes or variants thereof, are identified, oligonucleotide sequences are generated, or fragments thereof, may be employed as probes in the purification, isolation and detection of genes with similar sequences. Assays may be used for determining the sequence of an unknown nucleic acid; single nucleotide polymorphism (SNP) analysis; analysis of gene expression patterns from a particular species, tissue, cell type, etc.; gene identification; etc.
  • SNP single nucleotide polymorphism
  • Nonhomologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV- TK) gene and selecting against its nonhomologous insertion with the herpes drugs such as gancyclovir (GANC) or FIAU (l-(2-deoxy 2-fluoro-B-D-arabmofluranosyl)-5-iodouracii).
  • HSV- TK Herpes Simplex virus thymidine kinase
  • GANC gancyclovir
  • FIAU l-(2-deoxy 2-fluoro-B-D-arabmofluranosyl
  • isolated gene sequences of interest may be labeled and used to screen a cDNA library constructed from mRNA obtained from cells or tissues (e.g., stem cells, brain tissues) derived from the organism (e.g., mouse) of interest.
  • the hybridization conditions used should be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived.
  • the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions, as described in detail the Examples which follow.
  • Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N. Y.; and Ausubel, et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.
  • Another preferred method for identification of potential nucleic acids for screening of candidate drugs includes combining PCR with SAGE.
  • Serial Analysis of Gene Expression (SAGE) is based on the identification of and characterization of partial, defined sequences of transcripts corresponding to gene segments. These defined transcript sequence "tags" are markers for genes which are expressed in a cell, a tissue, or an extract, for example.
  • SAGE is based on several principles.
  • a short nucleotide sequence tag (9 to 10 bp) contains sufficient information content to uniquely identify a transcript provided it is isolated from a defined position within the transcript. For example, a sequence as short as 9 bp can distinguish about 262,144 transcripts given a random nucleotide distribution at the tag site, whereas estimates suggest that the human genome encodes about 80,000 to 200,000 transcripts (Fields, et al., Nature Genetics, 7:345 1994).
  • the size of the tag can be shorter for lower eukaryotes or prokaryotes, for example, where the number of transcripts encoded by the genome is lower.
  • a tag as short as 6-7 bp may be sufficient for distinguishing transcripts in yeast.
  • random dimerization of tags allows a procedure for reducing bias (caused by amplification and/or cloning).
  • concatenation of these short sequence tags allows the efficient analysis of transcripts in a serial manner by sequencing multiple tags within a single vector or clone.
  • serial analysis of the sequence tags requires a means to establish the register and boundaries of each tag.
  • the concept of deriving a defined tag from a sequence in accordance with the present invention is useful in matching tags of samples to a sequence database.
  • a computer method is used to match a sample sequence with known sequences.
  • the tags used herein uniquely identify genes. This is due to their length, and their specific location (3') in a gene from which they are drawn.
  • the full length genes can be identified by matching the tag to a gene data base member, or by using the tag sequences as probes to physically isolate previously unidentified genes from cDNA libraries.
  • the methods by which genes are isolated from libraries using DNA probes are well known in the art. See, for example, Veculescu et al., Science 270: 484 (1995), and Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, N. Y.).
  • the position of the hybridizing or matching region in the transcript can be determined. If the tag sequence is not in the 3' end, immediately adjacent to the restriction enzyme used to generate the SAGE tags, then a spurious match may have been made. Confirmation of the identity of a SAGE tag can be made by comparing transcription levels of the tag to that of the identified gene in certain cell types.
  • Analysis of gene expression is not limited to the above method but can include any method known in the art. All of these principles may be applied independently, in combination, or in combination with other known methods of sequence identification.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et al, FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov.
  • ESTs can also be used to identify nucleic acid molecules which are over expressed in a tumor cell.
  • ESTs from a variety of databases can be identified.
  • preferred databases include, for example, Online Mendelian Inheritance in Man (OMIM), the Cancer Genome Anatomy Project (CGAP), GenBank, EMBL, PIR, SWISS-PROT, and the like.
  • OMIM which is a database of genetic mutations associated with disease, was developed, in part, for the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • OMEVI can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/Omim/.
  • CGAP which is an interdisciplinary program to establish the information and technological tools required to decipher the molecular anatomy of a cancer cell.
  • CGAP can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/ncicgap/. Some of these databases may contain complete or partial nucleotide sequences.
  • alternative transcript forms can also be selected from private genetic databases.
  • nucleic acid molecules can be selected from available publications or can be determined especially for use in connection with the present invention.
  • nucleotide sequence of the nucleic acid molecule is determined by assembling a plurality of overlapping ESTs.
  • the EST database (dbEST), which is known and available to those skilled in the art, comprises approximately one million different human mRNA sequences comprising from about 500 to 1000 nucleotides, and various numbers of ESTs from a number of different organisms. dbEST can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/dbEST/index.html.
  • ESTs have applications in the discovery of new genes, mapping of genomes, and identification of coding regions in genomic sequences. Another important feature of EST sequence information that is becoming rapidly available is tissue-specific gene expression data. This can be extremely useful in targeting selective gene(s) for therapeutic intervention. Since EST sequences are relatively short, they must be assembled in order to provide a complete sequence. Because every available clone is sequenced, it results in a number of overlapping regions being reported in the database. The end result is the elicitation of alternative transcript forms from, for example, normal cells and tumor cells.
  • the resultant virtual transcript may represent an already characterized nucleic acid or may be a novel nucleic acid with no known biological function.
  • the Institute for Genomic Research (TIGR) Human Genome Index (HGI) database which is known and available to those skilled in the art, contains a list of human transcripts.
  • TIGR can be accessed through the world wide web of the Internet, at, for example, tigr.org. Transcripts can be generated in this manner using TIGR- Assembler, an engine to build virtual transcripts and which is known and available to those skilled in the art.
  • TIGR- Assembler is a tool for assembling large sets of overlapping sequence data such as ESTs, BACs, or small genomes, and can be used to assemble eukaryotic or prokaryotic sequences.
  • TIGR-Assembler is described in, for example, Sutton, et ah, Genome Science & Tech., 1995, 1, 9-19, which is incorporated herein by reference in its entirety, and can be accessed through the file transfer program of the Internet, at, for example, tigr.org/pub/software/TIGR. assembler.
  • PHRAP is used for sequence assembly within Find Neighbors and Assemble EST Blast.
  • PHRAP can be accessed through the world wide web of the Internet, at, for example, chimera.biotech. washington.edu/uwgc/tools/phrap.htm. Identification of ESTs and generation of contiguous ESTs to form full length RNA molecules is described in detail in U.S. application Ser. No. 09/076,440, which is incorporated herein by reference in its entirety.
  • alternative transcript information could be also retrieved from other gene databases, such as for example, LOCUSLINK, Alternative Splicing Database (ASD), and ASAP database.
  • ASD Alternative Splicing Database
  • primers used in the reaction mixture can comprise modified nucleobases.
  • the term “succeeding monomer” relates to the neighboring monomer in the 5'- terminal direction and the “preceding monomer” relates to the neighboring monomer in the 3 '-terminal direction.
  • Monomers are referred to as being "complementary” if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, pseudoisocytosine with G, etc.
  • Watson-Crick base-pairing rules e.g. G with C, A with T or A with U
  • other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, pseudoisocytosine with G, etc.
  • Preferred oligonucleotides of the invention also may have at least one non- modified nucleic acid located either at or within a distance of no more than three bases from the mismatch position(s) of a complementary oligonucleotide, such as at a distance of two bases from the mismatch position, e.g. at a distance of one base from the mismatch position, e.g. at the mismatch position.
  • primers for use in the disclosed amplification method can be oligonucleotides having sequence complementary to the target sequence. This sequence is referred to as the complementary portion of the primer.
  • the complementary portion of a primer can be any length that supports specific and stable hybridization between the primer and the target sequence under the reaction conditions. Generally, for reactions at 37°C, this can be, for example about 5 to about 35 nucleotides long or about 16 to about 24 nucleotides long. If whole genome amplification is desired, the primers can be from about 5 to about 60 nucleotides long, and in particular, can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 nucleotides long.
  • target sequences that are to be amplified and used in the screening of candidate drugs are of unknown sequence.
  • primers may be random, or of degenerate sequence (that is, use of a collection of primers having a variety of sequences), primer hybridization need not be specific. In such cases the primers need only be effective in priming synthesis. For example, in whole genome amplification specificity of priming is not essential since the goal generally is to amplify all sequences equally.
  • Sets of random or degenerate primers can comprise primers of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 nucleotides long or more.
  • Primers six nucleotides long are referred to as hexamer primers.
  • preferred primers for whole genome amplification are random hexamer primers. That is, random hexamer primers where every possible six nucleotide sequence is represented in the set of primers.
  • sets of random primers of other, particular lengths, or of a mixture of lengths preferably comprise every possible sequence the length of the primer, or, in particular, the length of the complementary portion of the primer.
  • Use of random primers is described in U.S. Pat. Nos. 5,043,272 and 6,214,587 the contents of which are hereby incorporated by reference in their entirety.
  • the primers can have one or more modified nucleotides.
  • modified primers are referred to herein as modified primers.
  • Modified primers have several advantages. First, some forms of modified primers, such as RNA/2'-O-methyl RNA chimeric primers, have a higher melting temperature (Tm) than DNA primers. This increases the stability of primer hybridization and will increase strand invasion by the primers. This will lead to more efficient priming. Also, since the primers are made of RNA, they will be exonuclease resistant. Such primers, if tagged with minor groove binders at their 5' end, will also have better strand invasion of the template dsDNA.
  • RNA primers can also be very useful for amplification of nucleic acid molecules from biological samples such as cells or tissue. Since the biological samples contain endogenous RNA, this RNA can be degraded with RNase to generate a pool of random oligomers, which can then be used to prime the polymerase for amplification of the DNA. This eliminates any need to add primers to the reaction. Alternatively, DNase digestion of biological samples can generate a pool of DNA oligonucleotide primers for RNA dependent DNA amplification. [0152] Chimeric primers can also be used.
  • Chimeric primers are primers having at least two types of nucleotides, such as both deoxyribonucleotides and ribonucleotides, ribonucleotides and modified nucleotides, or two different types of modified nucleotides.
  • One form of chimeric primer is peptide nucleic acid/nucleic acid primers (PNA/NAP).
  • PNA/NAP peptide nucleic acid/nucleic acid primers
  • 5'-PNA-DNA-3' or 5'-PNA-RNA-3' primers may be used for more efficient strand invasion and polymerization invasion.
  • the DNA and RNA portions of such primers can have random or degenerate sequences.
  • Other forms of chimeric primers are, for example, 5'-(2'-O- Methyl)RNA-RNA-3' or 5'-(2'-O-Methyl)RNA-DNA-3'.
  • nucleotide analogs are known and can be used in oligonucleotides.
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and 2- aminoadenin-9-yl.
  • a modified base includes but is not limited to locked nucleic acids (LNA), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other aikyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substit
  • Universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases substitute for the normal bases but have no bias in base pairing. That is, universal bases can base pair with any other base. Primers composed, either in whole or in part, of nucleotides with universal bases are useful for reducing or eliminating amplification bias against repeated sequences in a target sample. This would be useful, for example, where a loss of sequence complexity in the amplified products is undesirable. Base modifications often can be combined with for example a sugar modification, such as 2'-O-methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as U.S. Pat. Nos.
  • Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxyribose as well as synthetic modifications.
  • Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to ClO, alkyl or C2 to ClO alkenyl and alkynyl.
  • 2' sugar modifications also include but are not limited to ⁇ 0[(CH 2 ) n 0]m CH 3 , --O(CH 2 ) n OCH 3 , -0(CHa) n NHi --O(CH 2 ) n CH 3 , " -0(CH 2 J n -ONH 2 , and -O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
  • modifications at the 2' position include but are not limited to: Cl to ClO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH 2 and S.
  • Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Nucleotide analogs can also be modified at the phosphate moiety.
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkages between two nucleotides can be through a 3 '-5' linkage or a 2'-5' linkage, and the linkage can comprise inverted polarity such as 3 -5' to 5 -3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, U.S. Pat. Nos.
  • nucleotide analogs need only comprise a single modification, but may also comprise multiple modifipations within one of the moieties or between different moieties.
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize and hybridize to complementary nucleic acids in a Watson-Crick or Ho ⁇ gsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA).
  • PNA aminoethylglycine
  • U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al, Science 254:1497-1500 (1991)).
  • Primers can comprise nucleotides and can be made up of different types of nucleotides or the same type of nucleotides.
  • one or more of the nucleotides in a primer can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 10% to about 50% of the nucleotides can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 50% or more of the nucleotides can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; or all of the nucleotides are ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2''-
  • the nucleotides can comprise bases (that is, the base portion of the nucleotide) and can (and normally will) comprise different types of bases.
  • bases can be universal bases, such as 3-nitropyrrole or 5- nitroindole; about 10% to about 50% of the bases can be universal bases; about 50% or more of the bases can be universal bases; or all of the bases can be universal bases.
  • primers with complementary sequences to target nucleic acids are preferred. Primers may also comprise additional sequence at the 5' end of the primer that is not complementary to the target sequence. This sequence is referred to as the non-complementary portion of the primer.
  • the non-complementary portion of the primer serves to facilitate strand displacement during DNA replication.
  • the non- complementary portion of the primer can also include a functional sequence such as a promoter for an RNA polymerase.
  • the non-complementary portion of a primer may be any length, but is generally about 1 to 100 nucleotides long, and preferably about 4 to 8 nucleotides long. The use of a non-complementary portion is not preferred when random or partially random primers are used for example, in whole genome amplification.
  • the non-complementary portion of a primer can include sequences to be used to further manipulate or analyze amplified sequences.
  • a detection tag which is a specific nucleotide sequence present in the non-complementary portion of a primer.
  • Detection tags have sequences complementary to detection probes. Detection tags can be detected using their cognate detection probes. Detection tags become incorporated at the ends of amplified strands. The result is amplified DNA having detection tag sequences that are complementary to the complementary portion of detection probes. If present, there may be one, two, three, or more than three detection tags on a primer. It is preferred that a primer have one, two, three or four detection tags. Most preferably, a primer will have one detection tag. Generally, it is preferred that a primer have 10 detection tags or less.
  • a primer may have the same sequence or they may have different sequences, with each different sequence complementary to a different detection probe. It is preferred that a primer comprise detection tags that have the same sequence such that they are all complementary to a single detection probe. For some multiplex detection methods, it is preferable that primers comprise up to six detection tags and that the detection tag portions have different sequences such that each of the detection tag portions is complementary to a different detection probe. A similar effect can be achieved by using a set of primers where each has a single different detection tag.
  • the detection tags can each be any length that supports specific and stable hybridization between v the detection tags and the detection probe. For this purpose, a length of about 10 to about 35 nucleotides is preferred, with a detection tag portion about 15 to about 20 nucleotides long being most preferred.
  • the oligomer can comprise a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand that facilitates the direct of indirect detection of the oligomer or the immobilization of the oligomer onto a solid support.
  • groups are typically attached to the oligonucleotide when it is intended as a probe for in situ hybridization, in Southern hybridization, Dot blot hybridization, reverse Dot blot hybridization, or in Northern hybridization.
  • the spacer may suitably comprise a chemically cleavable group.
  • thermochemically active groups covers compounds which are able to undergo chemical reactions upon irradiation with light.
  • functional groups hereof are quinones, especially 6-methyl-l,4- naphtoquinone, anthraquinone, naphtoquinone, and 1,4-dimethyl-anthraquinone, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds.
  • thermally reactive group is defined as a functional group which is able to undergo thermochemically-induced covalent bond formation with other groups.
  • thermochemically reactive groups are carboxylic acids, carboxylic acid esters such as activated esters, carboxylic acid halides such as acid fluorides, acid chlorides, acid bromide, and acid iodides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.
  • carboxylic acids carboxylic acid esters such as activated esters
  • carboxylic acid halides such as acid fluorides, acid chlorides, acid bromide, and acid iodides
  • chelating group means a molecule that contains more than one binding site and frequently binds to another molecule, atom or ion through more than one binding site at the same time.
  • functional parts of chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA), aminophosphonic acid, etc.
  • reporter group means a group which is detectable either by itself or as a part of an detection series.
  • functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g.
  • dansyl (5-dimethylamino)-l-naphthalenesulfonyl
  • DOXYL N-oxyl-4,4- dimethyloxazolidine
  • PROXYL N-oxyl-2,2,5,5-tetramethyl ⁇ yrrolidine
  • TEMPO N-oxyl- 2,2,6,6-tetramethylpiperidine
  • dinitrophenyl acridines, coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems, Inc.), erythrosine, coumaric acid, umbelliferone, Texas red, rhodamine, tetram ethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-l-diazole (NBD), pyrene, fluorescein, Europium
  • paramagnetic probes ⁇ e.g. Cu 2+ , Mg 2+ ) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy
  • enzymes such as peroxidases, alkaline phosphatases, ⁇ galactosidases, and glycose oxidases
  • antigens antibodies
  • haptens groups which are able to combine with an antibody, but which cannot initiate an immune response by itself, such as peptides and steroid hormones
  • carrier systems for cell membrane penetration such as: fatty acid residues, steroid moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folic acid peptides for specific receptors, groups for mediating endocytose, epidermal growth factor (EGF), bradykinin, and platelet derived growth factor (PDGF).
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • ligand is a molecule, such as an antibody, hormone, or drug, that binds to a receptor.
  • a ligand can comprise a molecule, ion, or atom that is bonded to the central metal atom of a coordination compound.
  • Ligands can comprise functional groups such as: aromatic groups (such as benzene, pyridine, naphthalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, Ci-C 2O alky
  • DNA intercalators photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the "active/functional" part of the groups in question.
  • DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are typically represented in the form M-K- where M is the "active/functional" part of the group in question and where K is a spacer through which the "active/functional" part is attached to the 5- or 6-membered ring.
  • the group B in the case where B is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, has the form M-K-, where M is the "active/functional" part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand, respectively, and where K is an optional spacer comprising 1-50 atoms, preferably 1-30 atoms, in particular 1-15 atoms, between the 5- or 6-membered ring and the "active/functional" part.
  • spacer means a thermochemically and photochemically non-active distance-making group and is used to join two or more different moieties of the types defined above. Spacers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length (e.g. see Hermanson et. al., "Immobilized Affinity Ligand Techniques", Academic Press, San Diego, California (1992), p. 137-ff). Generally, the length of the spacers are less than or about 400 A, in some applications preferably less than 100 A.
  • the spacer thus, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulfur atoms.
  • the spacer K may comprise one or more amide, ester, amino, ether, and/or thioether functionalities, and optionally aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly- ⁇ -alanine, polyglycine, polylysine, and peptides in general, oligosaccharides, oligo/polyphosphates.
  • the spacer may consist of combined units thereof.
  • the length of the spacer may vary, taking into consideration the desired or necessary positioning and spatial orientation of the "active/functional" part of the group in question in relation to the 5- or 6-membered ring.
  • the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulphide groups cleavable under reductive conditions, peptide fragments cleavable by peptidases, etc. [0173] To further aid in detection and quantitation of nucleic acids amplified using the disclosed method, detection labels can be directly incorporated into amplified nucleic acids or can be coupled to detection molecules.
  • a detection label is any molecule that can be associated with amplified nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly.
  • Many such labels for incorporation into nucleic acids or coupling to nucleic acid probes are known to those of skill in the art.
  • Examples of detection labels suitable for use in the disclosed method are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands.
  • fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPYTM, Cascade BlueTM, Oregon GreenTM, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dyeTM, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • FITC fluorescein isothiocyanate
  • NBD nitrobenz-2-oxa-l,3-diazol-4-yl
  • AMCA
  • Examples of other specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy Fl, Brilliant Sulpho flavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin
  • Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection.
  • fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5 l -dichloro-6-carboxyrhodamine (JOE), T- chloro-5'-fluoro-7',8'-fused phenyl- l ⁇ -dichloro- ⁇ -carboxyfiuorescein (NED), and 2'-chloro- 7'-phenyl-l,4-dichloro-6-carboxyfiuorescein (VIC).
  • 6-FAM 6- carboxyfluorescein
  • TET 2',4',1,4,-tetrachlorofluorescein
  • HEX 2',4',5',7',1,4- hexachloro
  • Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ.; Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland, Ohio. [0176] Labeled nucleotides are a preferred form of detection label since they can be directly incorporated into the amplification products during synthesis.
  • detection labels that can be incorporated into amplified nucleic acids include nucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke, Mutation Research 290:217-230 (1993)), amino allyldeoxyuridine (Henegariu et ah, Nature Biotechnology 18:345-348 (2000)), 5-methylcytosine (Sano et al, Biochim. Biophys. Acta 951:157-165 (1988)), bromouridine (Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al, Proc. Natl. Acad.
  • nucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke, Mutation Research 290:217-230 (1993)), amino allyldeoxyuridine (Henegariu et ah, Nature Biotechnology 18
  • Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)).
  • a preferred nucleotide analog detection label for DNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma- Aldrich Co).
  • nucleotide analogs for incorporation of detection label into DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular Biochemicals).
  • a preferred nucleotide analog for incorporation of detection label into RNA is biotin- 16-UTP (biotin- 16-uridine-5'- triphosphate, Roche Molecular Biochemicals). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling. Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labeled probes.
  • Biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[l,2,- dioxetane-3-2'-(5'-chloro)tricyclo[3.3.1.1.sup.3,7 ]decane]-4-yl)phenyl phosphate; Tropix, Inc.).
  • suitable substrates for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[l,2,- dioxetane-3-2'-(5'-chloro)tricyclo[3.3.1.1.sup.3,7 ]decane]-4-yl
  • Labels can also be enzymes, such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases, that can be detected, for example, with chemical signal amplification or by using a substrate to the enzyme which produces light (for example, a chemiluminescent 1,2-dioxetane substrate) or fluorescent signal.
  • enzymes such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases
  • a substrate to the enzyme which produces light for example, a chemiluminescent 1,2-dioxetane substrate
  • fluorescent signal for example, a chemiluminescent 1,2-dioxetane substrate
  • detection labels that combine two or more of these detection labels are also considered detection labels. Any of the known detection labels can be used with probes, tags, and method to label and detect nucleic acid amplified using the disclosed method. Methods for detecting and measuring signals generated by detection labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection label coupled to the antibody. As used herein, detection molecules are molecules which interact with amplified nucleic acid and to which one or more detection labels are coupled.
  • oligonucleotide libraries may be employed as probes in the purification, isolation and detection of for instance pathogenic organisms such as viral, bacteria and fungi etc. Oligonucleotides also may be used as generic tools for the purification, isolation, amplification and detection of nucleic acids from groups of related species such as for instance rRNA from gram-positive or gram negative bacteria, fungi, mammalian cells etc.
  • nucleic Acid Fingerprints can be used to produce replicated strands that serve as a nucleic acid fingerprint of a complex sample of nucleic acid. Such a nucleic acid fingerprint can be compared with other, similarly prepared nucleic acid fingerprints of other nucleic acid samples to allow convenient detection of differences between the samples. The nucleic acid fingerprints can be used both for detection of related nucleic acid samples and comparison of nucleic acid samples. For example, the presence or identity of specific genes can be detected by producing a nucleic acid fingerprint of the test organism and comparing the resulting nucleic acid fingerprint with reference nucleic acid fingerprints prepared from known organisms.
  • Changes and differences in gene expression patterns can also be detected by preparing nucleic acid fingerprints of mRNA from different cell samples and comparing the nucleic acid fingerprints.
  • the replicated strands can also be used to produce a set of probes or primers that is specific for the source of a nucleic acid sample.
  • the replicated strands can also be used as a library of nucleic acid sequences present in a sample.
  • Nucleic acid fingerprints can be made up of, or derived from, for example, whole genome amplification of a sample such that the entire relevant nucleic acid content of the sample is substantially represented, or from multiple strand displacement amplification of selected target sequences within a sample.
  • Nucleic acid fingerprints can be stored or archived for later use.
  • replicated strands produced in the disclosed method can be physically stored, either in solution, frozen, or attached or adhered to a solid-state substrate such as an array. Storage in an array is useful for providing an archived probe set derived from the nucleic acids in any sample of interest.
  • informational content of, or derived from, nucleic acid fingerprints can also be stored. Such information can be stored, for example, in or as computer readable media.
  • nucleic acid fingerprints examples include nucleic acid sequence information (complete or partial); differential nucleic acid sequence information such as sequences present in one sample but not another; hybridization patterns of replicated strands to, for example, nucleic acid arrays, sets, chips, or other replicated strands. Numerous other data that is or can be derived from nucleic acid fingerprints and replicated strands produced in the disclosed method can also be collected, used, saved, stored, and/or archived.
  • Nucleic acid fingerprints can also comprise or be made up of other information derived from the information generated in the disclosed method, and can be combined with information obtained or generated from any other source. The informational nature of nucleic acid fingerprints produced using the disclosed method lends itself to combination and/or analysis using known bioinformatics systems and methods. [0183] Nucleic acid fingerprints of nucleic acid samples can be compared to a similar nucleic acid fingerprint derived from any other sample to detect similarities and differences in the samples (which is indicative of similarities and differences in the nucleic acids in the samples).
  • a nucleic acid fingerprint of a first nucleic acid sample can be compared to a nucleic acid fingerprint of a sample from the same type of organism as the first nucleic acid sample, a sample from the same type of tissue as the first nucleic acid sample, a sample from the same organism as the first nucleic acid sample, a sample obtained from the same source but at time different from that of the first nucleic acid sample, a sample from an organism different from that of the first nucleic acid sample, a sample from a type of tissue different from that of the first nucleic acid sample, a sample from a strain of organism different from that of the first nucleic acid sample, a sample from a species of organism different from that of the first nucleic acid sample, or a sample from a type of organism different from that of the first nucleic acid sample.
  • the same type of tissue is tissue of the same type such as liver tissue, muscle tissue, or skin (which may be from the same or a different organism or type of organism).
  • the same organism refers to the same individual, animal, or cell.
  • two samples taken from a patient are from the same organism.
  • the same source is similar but broader, referring to samples from, for example, the. same organism, the same tissue from the same organism, the same DNA molecule, or the same DNA library. Samples from the same source that are to be compared can be collected at different times (thus allowing for potential changes over time to be detected). This is especially useful when the effect of a treatment or change in condition is to be assessed. Samples from the same source that have undergone different treatments can also be collected and compared using the disclosed method.
  • a different organism refers to a different individual organism, such as a different patient, a different individual animal, different mono-cellular or multi-cellular organisms .
  • Different organism includes a different organism of the same type or organisms of different types.
  • a different type of organism refers to organisms of different types such as a dog and cat, a human and a mouse, or bacteria such as E. coli and Salmonella.
  • a different type of tissue refers to tissues of different types such as liver and kidney, or skin and brain.
  • a different strain or species of organism refers to organisms differing in their species or strain designation as those terms are understood in the art.
  • Expression patterns can be evaluated by qualitative and/or quantitative measures.
  • Certain techniques for evaluating protein expression yield data that are predominantly qualitative in nature. That is, the methods detect differences in expression that classify expression into distinct modes without providing significant information regarding quantitative aspects of expression.
  • a technique can be described as a qualitative technique if it detects the presence or absence of expression of a protein, i.e., as measured by detectable labels.
  • some methods provide data that characterize expression in a quantitative manner. That is, the methods relate expression on a numerical scale, e.g., a scale of 0-5, a scale of 1-10, a scale of +-+++, from grade 1 to grade 5, a grade from a to z, or the like. It will be understood that the numerical, and symbolic examples provided are arbitrary, and that any graduated scale (or any symbolic representation of a graduated scale) can be employed in the context of the present invention to describe quantitative differences in nucleotide sequence expression. Typically, such methods yield information corresponding to a relative increase or decrease in expression.
  • any method that yields either quantitative or qualitative expression data is suitable for evaluating expression of protein in the presence or absence of a candidate drug.
  • the recovered data e.g., the expression profile is a combination of quantitative and qualitative data.
  • the expression profiles are typically recorded in a database.
  • the database is a relational database accessible by a computational device, although other formats, e.g., manually accessible indexed files of expression profiles as photographs, analogue or digital imaging readouts, spreadsheets, etc. can be used.
  • the expression patterns, expression profiles (collective expression patterns), and molecular signatures (correlated expression patterns) are stored digitally and accessed via a database.
  • the database is compiled and maintained at a central facility, with access being available locally and/or remotely.
  • the algorithms optionally additionally query additional samples against the existing database to further refine the association between a molecular signature and disease criterion.
  • the data set comprising the one (or more) molecular signatures is optionally queried against an expanding set of additional or other disease criteria.
  • the RTS 100 E. coli HY kit two expression vectors containing the genes encoding Green Fluorescent Protein (GFP) and chloramphenicol acetyl-transferase (CAT), and anti-6xHis were obtained form Roche Diagnostics GmbH (Mannheim, Germany).
  • GFP Green Fluorescent Protein
  • CAT chloramphenicol acetyl-transferase
  • anti-6xHis were obtained form Roche Diagnostics GmbH (Mannheim, Germany).
  • TNT Quick Coupled Transcription/Translation system T7 luciferase DNA vector, luciferase assay reagent, and nuclease- free water were from Promega Corporation (Madison, WI).
  • Acrylamide-bisacrylamide (electrophoretic grade, 5% C), tetramethylethylenediamine, sodium dodecyl sulfate (SDS), ammonium persulfate, tris(hydroxymethyl)aminomethane (Tris), glycine, sodium chloride, glycerol, bromophenol blue, ⁇ -mercaptoethanol, Tween-20, tetracycline, and cycloheximide were purchased from Fisher Scientific (Atlanta, GA). Polyvinylidene difluoride (PVDF) membranes (0.2 ⁇ m), and filter papers were from Bio-Rad Laboratories (Hercule, CA).
  • PVDF Polyvinylidene difluoride
  • biotinylated secondary antibody and streptavidin-alkaline phosphatase were from Amersham Biosciences (Piscataway, NJ) while recombinant Green Fluorescent Protein (rGFP) and rabbit anti-GFP polyclonal antibody were from BD Biosciences (Palo Alto, CA).
  • the phosphatase staining solution (Bromo-chloro-indoryl phosphate/Nitro Blue Tetrazolium, BCIP/NBT) was obtained from KPL (Gaithersburg, MD). Protein Expression.
  • RTS 100 reaction solution was composed of 12 ⁇ L E. coli lysate, 10 ⁇ L reaction mix (proprietary composition, supplied in the kit by the manufacturer), 12 ⁇ L amino acids without methionine, 1 ⁇ L methionine, 5 ⁇ L reconstitution buffer (proprietary composition, supplied in the kit by the manufacturer), and 10 ⁇ L nuclease-free water containing 1 ⁇ g GFP or CAT vector.
  • the reaction solution was then incubated in a microcentrifuge tube at 3O 0 C for 4 hours.
  • the reaction solution was stored at 4 0 C for additional 24 hours for the maturation of GFP.
  • luciferase synthesis was prepared by combining 40 ⁇ L TNT Quick master mix (proprietary composition, supplied in the kit by the manufacturer), 1 ⁇ L methionine, and 9 ⁇ L nuclease-free water containing 1 ⁇ g luciferase vector. Incubation was performed in a microcentrifuge tube at 30 °C for 1.5 hours.
  • toxin inhibition assay in a microcentrifuge tube, a stock solution of tetracycline and that of cycloheximide were prepared at 15 ⁇ g/ ⁇ L and 10 ⁇ g/ ⁇ L, respectively. A series of amounts of tetracycline or cycloheximide were added into protein expression mixture. The concentrations of toxins used are listed in the figures or text. To save reagents and match with miniaturized devices, 8 ⁇ L of prokaryotic or eukaryotic expression solution was used, making the total volume of each inhibition assay at 10 ⁇ L. For each set of experiments, a positive control (without inhibitor) and a negative control (without the expression vector) have also been included.
  • FIG. 1A and IB A miniaturized device with an array of 2x3 wells was designed and fabricated for demonstrating toxin detection.
  • the device was made from acrylic (Lucite International, Cordova, TN) and the wells were created by a milling machine (Flashcut CNC, Menlo Park, CA). The distances between wells (center to center) are 9 mm, matching the standards for 96-well microplates defined by the Society for Biomolecular Screening and accepted by the American National Standards Institute; this arrangement insures compatibility with a variety of commercial fluid dispensing systems and plate readers.
  • each well The diameter and depth of each well are 2.7 mm and 2.3 mm, respectively, providing the total well volume of -13 ⁇ L. This is about 25 times smaller than the wells in conventional 96-well microplates. The decrease in the well size will significantly reduce reagent consumption for high-throughput assays. The size of the well is also in agreement with our goal to integrated microfiuidic components. After fabrication, the device was sterilized by exposing to UV light for 30 minutes that ensured the consistency of the protein expression.
  • the gel was removed from the glass plates and then equilibrated in the transfer buffer, which is comprised of 48 mM Tris, 39 mM glycine, and 20% V/V methanol.
  • PVDF membrane was pre-soaked with methanol, followed by soaking in the transfer buffer for 1 hour.
  • the Mini Trans-Blot system (BioRad) was set up with pre-wetted fiber pad, filter paper, gel, and PVDF membrane according to the instruction from the manufacturer. The cassette and ice-cooling unit were placed in the tank that was filled with the transfer buffer.
  • the PVDF membrane were removed from the trans-blot apparatus and blocked with 5% w/v non-fat dried milk in Tris-buffered saline (TBS) solution (with 0.05% Tween-20) for 1 hour at room temperature. After being washed three times (5 minutes each time) with TBS solution, the membrane was incubated for 1 hour at room temperature with 1 ⁇ g/mL anti-GFP polyclonal antibody for GFP product or 0.3 ⁇ g/mL anti-6xHis monoclonal antibody for CAT product, respectively. At the end of conjugation, the membrane was washed three times and then incubated with 1.5 ⁇ g/mL biotinylated secondary antibody at room temperature for 1 hour.
  • TBS Tris-buffered saline
  • the membrane Upon completion of the incubation, the membrane was rinsed again with TBS solution for three times, followed by incubation at ambient temperature for 30 minutes with a solution of streptavidin-alkaline phosphatase (1:2000 dilution from the stock solution). After being washed, the membrane was immersed in chromogenic substrate (BCIP/NBT) for 3 minutes, followed by rinsing with water (to stop reaction). Images of protein bands were acquired with a color laser scanner (Canon); proteins bands were quantified using Image J from the National Institute of Health (Image J is developed on Mac OS X using its built in editor and Java compiler, plus the BBEdit editor and the Ant build tool. The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA. http://rsb.info.nih.gov/ij).
  • luciferase expressed by IVT was achieved by SIRIUS luminometer from Berthold (Pforzheim, Germany). The luminometer was programmed to have a two- second delay, followed by a five-second measurement of luciferase activity. The expression product of 2 ⁇ L was added to a luminometer tube containing 40 ⁇ L of luciferase assay reagent and mixed evenly. The tube was then placed in the luminometer; and the data were acquired.
  • the device comprises an array of units; each unit consists of a reaction chamber, two feeding chambers, and a channel to connect them as shown hi Figure 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs.
  • the feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis.
  • the reaction chamber contains cell-free expression system with other reagents as in the feeding chambers.
  • the channel connected to the chambers provides a means to supply nutrients and remove byproducts.
  • the selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at -10 KDa, which at the same time allows all small molecules in the feeding chamber passing by.
  • the incorporation of membrane is important because of two facts: (1) the flow of a feeding solution leads to higher expression yield compared to static conditions, because protein synthesis will not terminate earlier due to fast depletion of energy sources (ATP and GTP); (2) removal of small molecule byproducts is also very important for the high yield expression of proteins in a cell-free medium. Inhibition of protein synthesis due to the small molecular byproducts such as hydrolysis products of triphosphates is eliminated.
  • the device possesses appropriate geometric configuration to achieve desired properties for in vitro protein expression.
  • the dimension of the microchannel is in the range of 1 micron to 1 centimeter, depending on the flow rate required for supplying nutrients and removing byproduct.
  • the feeding chambers must be sufficiently large compared to the reaction chamber so that they function as reservoirs.
  • the volume of the reaction chamber is 10 nanoliter-100 microliter ⁇ L whereas that of the feed chambers is about 100 nano liter- 1 mililiter.
  • One of the mechanisms for supplying nutrients from the feeding chambers to the reaction chamber and for removing byproducts from the reaction chamber to the feeding chambers is osmosis, a net flow resulting from the difference in chemical potential of solutes between two solutions separated by the membrane.
  • the degree of the potential difference is determined by their difference in solute activities, which are correlated with solute concentrations.
  • the osmotic pressure, FI resulting from a byproduct in the reaction chamber can be approximately calculated by Van't Hoff s Law assuming it is in the limit of infinite dilution (Levine, 1988).
  • C b is the concentration of the byproduct
  • R is gas constant
  • T is the temperature.
  • ⁇ P pg ⁇ /z [0207] where p is the density of solution and g is gravitational acceleration.
  • the volume flow rate in the channel, Q can be calculated using the equation below based on a fully developed flow in the channel.
  • D is the channel diameter
  • is viscosity
  • L is the distance between chambers.
  • ⁇ h 1 mm
  • D 50 ⁇ m
  • L 3 mm
  • water's density and viscosity the velocity is calculated to be 0.50 nL/s. At this speed, the amount of reagent in the feeding chamber will last for many hours, which is longer than the reaction time needed for completing protein synthesis in a microdevice.
  • Pattern recognition and database After establishing a baseline for toxin simulants and ricin, the response pattern of other toxins can be examined. In addition, a database of response patterns of known toxins can be established, so that comparisons of the pattern of a sample with the database can be made.
  • the pattern recognition algorithms and other features that have been developed for the electronic nose (Dutta et al., (2002) Bacteria classification using Cyranose 320 electronic nose. BiomedEng Online, 1, 4) are expected to be useful to the toxin sensor array.
  • the storage with cell-free reagents in the system of the invention is likely less challenging than the microbial sensors (Riedel et al., (2002) Adv Biochem Eng Biotechnol, 75, 81-118; Shetty et al, (2004) Biotechnology and Bioengineering, 88, 664-670) using live cells. Nevertheless, using biological agents such as enzymes is still a concern in terms of shelf-life. We believe, however, the concern can be addressed by formulation, evidenced from many commercial products such as glucose sensors (that use an enzyme) and pregnancy test kits (that use an antibody). Third, other issues related to real-world application could be addressed in a way similar to the electronic nose, which is commercially available and also based on sensor array concept as mentioned above (Dutta et al., 2002).
  • FIG. IA and IB shows an illustrative example of a sensor array for detecting toxins using IVT.
  • the device consists of an array of IVT wells; each well is designed to express one protein and thus functions as a sensor.
  • the number of IVT sensors can be as high as 96 or its integral multiples, in the format of traditional microplates and can be easily adapted to commercial plate readers.
  • Multiple wells form one set, in which the top row is for the positive controls to express each of 3 proteins, the second row for the negative controls, and the third and fourth rows are for the sample, allowing one repeat to enhance the precision.
  • Use of the positive and negative controls and comparison of the signal from the sample wells with those in the control wells reduce false positives and negatives.
  • the set expresses a group (three in this case) of pre-characterized proteins in different expression systems; the proteins and expression systems are judiciously selected so that protein synthesis in each well is inhibited or affected differentially by different type of toxins.
  • the unique response pattern (or signature) of a toxin due to different inhibitory effects can be registered and used as a tool for detection and identification.
  • New agents can be identified by comparing the response pattern with signatures of known agents in a pre-acquired database.
  • the rest of wells in the 96- well array can be designed to detect seven additional types of toxins if the 12-well set is proved to be enough for identification.
  • the first protein is Green Fluorescent Protein (GFP), a widely-used fluorescent molecule with known DNA sequence and crystal structure.
  • GFP Green Fluorescent Protein
  • Protein expression was carried out by using an expression vector as a DNA template, which consists of GFP coding sequence and the necessary regulatory elements including T7-RNA polymerase promoter, ribosome binding site, start codon, stop codon, and T7 terminator.
  • the expression vector was mixed with E. coli lysate and a reaction mix consisting of T7-RNA polymerase, nucleotides, amino acids, and other reagents. GFP product was confirmed by fluorescence spectrometry and Western blotting.
  • FIG. 3 A The result of Western blotting is shown in Figure 3 A.
  • a clear band in lane 4 indicates the presence of GFP in the expression product.
  • the molecular weight of GFP expressed is estimated ⁇ 31 KD.
  • Expressed GFP contains a stretch of additional six histidines (6xHis) at its C-terminal, causing its molecular weight slightly larger than recombinant GFP (rGFP) purchased commercially.
  • the negative control in the experiment contains all reagents except for the expression vector.
  • the second protein is chloramphenicol acetyl-transferase (CAT), an enzyme responsible for bacterial resistance to an antibiotic drug, chloramphenicol.
  • CAT was expressed in the same E. coli expression system; success of the protein expression was also confirmed using Western blot as shown in Figure 3B. According to pre-stained protein markers, the molecular weight of CAT expressed is estimated -26 KD.
  • the third protein is luciferase, an enzyme from firefly tails that catalyzes the production of light in the presence of luciferin, adenosine triphosphate (ATP), Mg 2+ , and oxygen. Synthesis of luciferase was carried out using rabbit reticulocyte expression system as described in the experimental section. Detection of the expression product was achieved by monitoring the intensity of luminescence after mixing 2 ⁇ L of the product with the assay reagent. As shown in Figure 3 C, the luminescence signal of the product is 5 orders of magnitude higher than that of the negative control.
  • proteins may also be produced using in vitro transcription/translation.
  • the examples include dihydrofolate reductase, interleukins, erythropoietin, and phosphoserine phosphatase. More than forty proteins with a variety of biological functions have been successfully synthesized using IVT.
  • TC tetracycline
  • CH cycloheximide
  • TC is an antibiotic substance produced by Streptomyces species. It acts only on prokaryotic cells and it blocks binding of aminoaceyl-transfer RNA to A-site of ribosomes.
  • CH acts specifically on eukaryotic cells and it inhibits the activity of peptidyl transferase, an enzyme needed in the translocation reaction on ribosomes.
  • Figures 4A-4G shows the effects of a series of concentrations of TC or CH on the expression yields of GFP, CAT, and luciferase synthesized in two protein expression systems.
  • Figures 4D and 4E exhibit similar disparity between TC and CH for CAT production in the E. coli expression system.
  • TC has inhibitory effect on CAT production and the degree of inhibition is proportional to the amount of TC in the sample
  • CH has a negligible effect on the yield of CAT production and the level of minor inhibition remained essentially same in the range of amount of TC we used.
  • comparison of Figure 4B and 4D indicates that although TC has inhibitory effect on both GFP and CAT production, the degree of inhibition per unit amount of TC differs between these two proteins, evident from the difference in the slopes of respective linear regression lines.
  • Example 5 Miniaturized IVT Array.
  • IVT and toxin detection were determined in a miniaturized well device.
  • the design of the experiments was a part of the 96-well array in Figure 2, in which a set of 3x4 wells is assigned for detecting one toxin at a time.
  • the first row of 3 wells was used as the positive control, expressing GFP and CAT vector in the E. coli expression system and luciferase vector in the rabbit reticulocyte expression system. These wells were free of toxins.
  • the second row of 3 wells was used as the negative control without DNA vectors added.
  • FIG. 5A shows the response pattern of the IVT sensor array when 25 ng of TC was used whereas the response pattern of the same IVT array for 17 ng of CH is illustrated in Figure 5B. Although there is slight difference between two sample repeats for rows 3 and 4 for each toxin, the response pattern is reproducible as expected. The significant difference in the response patterns between CH and TC clearly indicates that it is feasible to use IVT sensor array to detect and identify toxins.
  • GFP As an indicator for the detection of protein expression due to its green fluorescence.
  • GFP has been used for visualization, tracking, and quantification of a variety of proteins in cells after they are fused together.
  • An increase of fluorescence signal in an IVT well indicates the production of GFP or GFP-fused proteins.
  • Quantitative information may be obtained by comparing the fluorescence signals of sample wells and of reference wells, which include both positive and negative controls in the array device. Any variation or adverse effects are cancelled out between control and sample wells. The magnitude of the signal can be correlated to the amount of proteins produced in the device.
  • GFP fluorescence spectrometer
  • CAT and luciferase are to be expressed in the form of GFP fusions.
  • the expression vector containing a coding sequence for expressing an additional stretch of six histidines (6xHis) at the C-terminal of the protein of interest is to be designed.
  • Many proteins produced by recombinant techniques are designed to contain a 6xHis tag, so that they can be purified through interactions between 6xHis tags and Ni-nitrilotriacetate chromatographic columns. Both GFP and CAT proteins produced here contain 6xHis tag, even though this purification step was not needed.
  • a novel concept for toxin detection is presented based on toxin's inhibition of biological protein synthesis in the step of either DNA transcription or protein translation. This was demonstrated t by (1) in vitro expression of three proteins, including Green Fluorescent Protein, chloramphenicol acetyl-transferase, and luciferase; (2) confirming differential inhibitory effects of two toxin simulants, tetracycline and cycloheximide, on the expression yields of these proteins in either prokaryotic or eukaryotic expression system; (3) obtaining unique response pattern (or signature) of the 3x4 IVT array device for each toxin simulant. Such a sensor array is useful in the situations where one type of toxins is suspected.
  • micro fluidic elements offer a means to supply nutrients continuously and to remove byproducts of protein synthesis.
  • the experimental results in the bench-top scale suggest that high-yield protein expression can be attained in the flow of a feeding solution.
  • Removal of small molecule byproducts e.g., hydrolysis products of triphosphates
  • Microfluidic manipulation enhances protein expression yield and accordingly increase toxin detection sensitivity.
  • micro fluidics may also reduce the response time of the sensor array, which is limited by the time needed for protein production.
  • the device is also important for high-throughput screening of drug candidates or enzymes.
  • tetracycline is an antibiotic drug used clinically suggests that the IVT array device provides a nice platform for searching for the candidates that have maximum effects on prokaryotic microorganisms but least effects on eukaryotic cells.
  • such an IVT array device is useful for studying potential drug candidates through enzyme inhibition assays.
  • Example 6 Ricin Detection By Biological Signal Amplification in a Solution Array
  • the RTS 100 wheat germ CECF kit, RTS 500 E. coli kit, and the expression vector containing the gene of green fluorescent protein (GFP) were obtained from Roche Diagnostics GmbH (Mannheim, Germany).
  • T7 luciferase DNA vector, luciferase assay reagent, and nuclease-free water were acquired from Promega Corporation (Madison, WI).
  • Ricin and ricin B chain were purchased from Vector Labs (Burlingame, CA) while ricin A chain and 2-mecaptoethanol were from Sigma (St. Louis, MO).
  • the pitch (the distance between the hole centers) is 9 mm, following the microplate standards defined by the Society for Biomolecular Screening (SBS) and accepted by the American National Standards Institute.
  • the sheet with holes is further milled from the bottom side to create a flange using a CNC-mill (Flashcut 2100, Menlo Park, CA), resulting in a 1 mm-thick wall for the tray chamber.
  • the dialysis membrane was then glued using the epoxy to the bottom of each hole to form the tray chamber.
  • the bottom part, well was created by milling an array of 4 mm deep wells into a piece of a 6.3 mm-thick acrylic sheet.
  • the diameter of the wells is 7 mm; each well is concentric with the corresponding tray chamber when they are assembled.
  • Luciferase was synthesized using RTS 100 wheat germ expression kit.
  • the reaction solution for trays was prepared by mixing 15 ⁇ L wheat germ extract, 15 ⁇ L reaction mix (provided in the kit), 4 ⁇ L amino acids, 1 ⁇ L methionine and 15 ⁇ L of nuclease-free water containing 1 ⁇ g of luciferase vector.
  • the vector can be either circular plasmid vector or a linear vector created by polymerase chain reaction; both vectors were purchased from Promega. For each tray chamber, 8 ⁇ L of the reaction solution was used.
  • the feeding solution for wells was prepared by combining 900 ⁇ L feeding mix (provided in the kit), 80 ⁇ L amino acid and 20 ⁇ L methionine. In each well, 80 ⁇ L of the feeding solution was introduced.
  • the tray and well plates were assembled and then placed on a shaker (at room temperature) for a period of time (e.g., 0.5 hour).
  • the amount of luciferase synthesized was determined by mixing the expression product with luciferase assay reagents, followed by luminescence detection in a luminometer (Berthold, Germany), as described previously (Mei, Q.; Fredrickson, C. K.; Jin, S.; Fan, Z. H. Anal Chem. 2005, 77, 5494-5500).
  • Protein Expression Array As mentioned above, protein expression can be produced in a cell-free medium employing IVT. We fabricated an array device consisting of a mechanism for fluid manipulation; it also has a potential to implement protein synthesis in a high-throughput format due to miniaturization. Miniaturization also results in reduction of the reagent consumption by more than 2 orders of magnitude. As illustrated in Figures IA and IB, IYT was implemented in an array of units; each unit is for expression of one protein (e.g., luciferase). The units on the left of the array are for the positive controls (free of ricin), the units in the middle for the negative controls (no DNA vectors), and the units on the right are for samples.
  • IVT Protein Expression Array
  • Each unit in the device consists of a tray and a well ( Figure IB and Figure 2).
  • the tray chamber is for the IVT reaction; the well is concentric with the corresponding tray chamber and functions as a nutrient reservoir.
  • the well contains amino acids, adenosine triphosphate (ATP), and other reagents.
  • the tray contains the cell-free expression mixture extracted from wheat germs, as well as the same reagents in the well.
  • a dialysis membrane is glued to the bottom of the tray, connecting the tray and well and providing a means to supply nutrients and remove the reaction byproducts.
  • the incorporation of membrane allows for: (1) the flow of a nutrient-feeding solution leads to higher expression yield compared to static conditions, because protein synthesis does not terminate earlier due to fast depletion of the energy source (ATP); (2) removal of small molecular byproducts is also critical to high yield expression of proteins in a cell-free medium, because possible inhibition of protein synthesis by the byproducts (e.g., hydrolysis products of triphosphates) does not take place.
  • solution array offers maximal flexibility without compromising the binding activity of proteins.
  • the solution array does not possess heterogeneous solid-liquid attachment, eliminating the issues encountered in the protein arrays/chips (e.g. maintaining the conformation, thus biological activity, of proteins attached to a solid surface).
  • Biological Signal Amplification Ricin causes toxic effects by inactivating ribosomes and inhibiting protein synthesis in biological cells and then leading to cell death and tissue damage. We exploit its toxicity mechanism as the sensing scheme to detect ricin. This detection method possesses inherent biological signal amplification, as illustrated in Figure 7.
  • One copy of DNA is transcribed into one copy of messenger RNA. However, for each copy of RNA, thousands of copies of proteins can be produced. This is estimated by the amount of the DNA vector used and the amount of the corresponding proteins produced in IVT. The inhibitory effects of ricin exist on the production of every copy of protein, as illustrated in Figure 7.
  • the detection signal i.e., the difference between the sample and the positive control
  • the detection signal is accumulated, leading to an amplified signal.
  • the amplification of the inhibitory effects of ricin on protein synthesis is also evident from its toxicity on biological cells. A single ricin molecule that enters the cytosol can inactivate over 1,500 ribosomes per minute and kill the cell.
  • This signal amplification is similar, to some degree, to the enzyme-enabled signal amplification in ELISA.
  • Intrinsic to ELISA is the addition of reagents conjugated to enzymes; assays are then quantified by the build-up of colored products after the addition of substrates.
  • the signal amplification results from the enzyme that catalyzes many substrate molecules to detectable products.
  • Two widely-used enzymes are horseradish peroxidase and alkaline phosphatase, which transfer o-phenylene diamine and p-nitrophenylphosphate, respectively, and generate colored products. Therefore, we expect the detection method based on protein inhibition has comparable sensitivity with ELISA.
  • a number of potent biological toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin produced by Shigella dysenteriae, Shiga-like toxin by enteropathogenic E. coli, diphtheria toxin from Corynebacterium diphtheriae and exotoxin A of ' Pseudomonas aeruginosa.
  • the selectivity of our detection method can be achieved by the response pattern of a protein expression array.
  • a few proteins are judiciously selected so that their production yields are inhibited differentially by toxin simulants as illustrated previously.
  • luciferase can be synthesized in as short as 5 minutes, as shown in Figure 9 A and 9B. Similar to the. result in Figure 6 A and 6B when a longer expression time was used, we also observed the difference in the luciferase expression yield between the miniaturized device and a microcentrifuge tube when a short expression time ( ⁇ 30 minutes) was used. The result suggests that enough difference takes place in as short as 5-minute. A short IVT time is critical for those applications that need a quick response. We confirmed that we were able to detect ricin within 5 minutes, as shown Figure 9B, though the detection limit is higher than when a longer expression time is used.
  • Ricin is a dimer, in which B chain binds to cell surface, allowing A chain to penetrate the cell to inhibit protein synthesis.
  • the toxicity of ricin comes from the A chain.
  • IVT does not provoke dissociation of the A-B dimer
  • all experimental results discussed above are from ricin A chain.
  • Whole ricin (with A and B chains) is used, to determine the toxic effect in the IVT device. As shown in Figure 10, we observed the similar result in IVT device.
  • the A chain has highest toxicity
  • B chain has no detectable toxicity
  • whole ricin shows a toxicity level less than the A chain.
  • 2- mercaptoethanol-treated ricin is almost as effective as A chain due to reduction of disulfide bond between two chains.
  • the solution array in this invention enables high-throughput protein expression for proteomics applications. Completion of mapping the human genome has prompted strong interest in identifying the functions of newly discovered genes and the proteins encoded therein. To match high-throughput gene discovery, methods to produce a large number of proteins in parallel are needed. The current method of producing proteins in E. coli cells is difficult to be implemented in a high-throughput format (Gilbert, M.; Albala, J. S. Current Opinion in Chemical Biology 2002, 6, 102-105). The miniaturized, high yield, parallel in vitro protein expression array in this invention could be a solution.
  • Example 7 Cell Array.
  • the system comprises an array of microcompartments; each microcompartment functions as a surrogate cell, consisting of a reaction chamber, two feeding chambers, and a channel to connect them as shown in Figure 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs.
  • the feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis.
  • the reaction chamber contains cell-free expression system with other reagents as in the feeding chambers.
  • the channel connected to the chambers provides a means to supply nutrients and remove byproducts.
  • the selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at ⁇ 10 KDa, which at the same time allows entry of nutrients into the feeding chamber.
  • Dialysis membrane has been used in miniaturized devices for the different purpose (e.g., sampling).
  • the incorporation of membrane is important because of two facts: (1) the flow of a feeding solution leads to higher expression yield compared to static conditions, because protein synthesis does not terminate earlier due to fast depletion of energy sources (ATP and GTP); (2) removal of small molecule byproducts is also very critical to high yield expression of proteins in a cell-free medium, because possible inhibition of protein synthesis does not take place by the small molecular byproducts such as hydrolysis products of triphosphates.
  • the device possesses appropriate geometric configuration to achieve desired properties for in vitro protein expression.
  • the dimension of the microchannel is in the range of 5 ⁇ m to 10 mm, depending on the flow rate required for supplying nutrients and removing byproduct.
  • the feeding chambers are sufficiently large compared to the reaction chamber so that they function as reservoirs.' In the current design, the volume of the reaction chamber is 0.1-100 ⁇ L whereas that of the feed chambers is about 1 ⁇ L -5000 ⁇ L. AU calculations are as described above.
  • FIG. 6B shows the expression yield of GFP as a function of expression time, and the comparison between a microcentrifuge tube and the miniaturized device with flow manipulation. Expression of GFP was observed in both tube and device after 4 hours, but the yield remains the same with time when it was in the tube. In contrast, the yield increased continuously with time due to continuous supply of the nutrients and removal of byproducts when the reaction took place in the device. The production yield increased more than 14 fold in the device than in a microcentrifuge tube. The result suggests that we achieved the desired fluid manipulation in the device.
  • the essential elements such as 20 AA and 20 aminoacyl-tRNA synthetase can be considered as one component to reduce the work load. Mimicking the approach used for combinatorial chemical synthesis, the amount of these components can be systematically varied and their corresponding protein expression yields provide quantitative information about the interplay among the constituents. These results not only leads to an approach achieving high-yield, high-throughput protein expression, but also very useful for devising a panel of protein expression conditions for the identification of physiological targets.
  • Table 1. Major components for protein translation.
  • the surrogate cell array consists of multiple compartments to accommodate an array of protein expression.
  • An example of a format is a traditional 96-well microplate.
  • gene transcription and protein translation takes place as shown in the inset, Figure 11.
  • a subset of 24 surrogate cells (3 x 8) is chosen to produce one protein e.g., GFP ( Figure 11).
  • row A functions as the positive control by producing GFP in the E. coli expression system
  • row B is for the negative controls (no DNA vectors).
  • the positive and negative controls are used to facilitate quantification.
  • Rows C-H are for producing GFP using a mixture of reconstructed ingredients, in which all components are at the same concentration as in the cell lysate except for one component, elongation factor 2 (EF-2).
  • the mixtures are prepared in a series of concentrations of EF-2, for example, 0.01, 0.05, 0.1 , 0.5, 1 , and 5 ⁇ M for rows C, D, E, F, G, and H 5 respectively.
  • the variation in the EF-2 concentration results in quantitative information about the effects of EF-2 on the protein expression. For each sample, three repeats can be performed in columns 1-3 to enhance the precision.
  • AU expected signals are normalized against the positive controls (row A in Figure 11). No signal is expected in the negative controls (row B) due to lack of DNA vector.
  • the protein expression yield is expected to increase with the concentration of EF-2 from row C to H, since EF-2 is a catalyst responsible for the translocation of ribosome along mRNA. A larger concentration of EF-2 than the normal cellular value may result in higher expression yield in row H than the positive control.
  • the surrogate cell array is used to identify the physiological targets, through which Ochratoxin A (OTA) inhibits protein expression.
  • OTA Ochratoxin A
  • the surrogate cell array as a sensitive method to detect ricin or other toxins.
  • the methods to detect ricin at a low concentration include enzyme-linked immunosorbent assay (ELISA) and immunoassay using radioactive labeling.
  • ELISA and radioimmunoassay offer high sensitivity, they involve several labor-intensive and time-consuming steps, and the handling and disposal of radioisotopes are environmental challenges.
  • fluorescence-based multianalyte immunosensor that has a detection limit of 25 pg/ ⁇ L of ricin.
  • Another group exploited specific interaction between ricin and glycosphingolipids and developed a quartz crystal microbalance sensor with the detection limit of 5000 pg/ ⁇ L of ricin.
  • Another group depicted an immunoassay- based magnetoelastic sensor that shows a detection limit of 5 pg/ ⁇ L of ricin.
  • We expect lower detection limit by employing the inhibitory effect of ricin on protein expression as the sensing mechanism, because the signal output representing the inhibitory effect is amplified due to the accumulation of inhibitory effects on the production of each copy of proteins. And thousands of copies of proteins can be produced from each copy of messenger RNA.
  • a higher yield of protein expression in a continuous flow system also contributes to a larger detection signal.
  • OTA Ochratoxin A
  • Penicillium and Aspergillus Several major mechanisms have been shown to be involved in the toxicity of OTA, including inhibition of protein synthesis, though the exact mechanism is not well-understood.

Abstract

Detection of inhibition of biological synthesis of pre-characterized proteins indicates presence of a toxic analyte. The sensor array comprises an array of units; each unit is for expression of one protein and thus functions as one sensor. The sensor array expresses a group of pre-characterized proteins in different expression system, thus the response pattern (or signature) of an analyte due to different inhibitory effects is registered and used as a tool for detection and identification. New agents are identified by comparing the response pattern with signatures of known agents in a pre-collected database. The protein synthesis array device also functions as surrogate cells, which can be used for systematic and quantitative studies of the interplay among a large number of the constituents in a biological cell, for studying molecular responses to toxins, and for search for the therapeutic targets.

Description

MINIATURIZED IN VITRO PROTEIN EXPRESSION ARRAY
FIELD OF THE INVENTION [0001] Sensor arrays for detection of toxins, drug target discovery and screening.
BACKGROUND
[0002] Detection and identification of toxic agents are important for medical diagnostics, food/water safety testing, and biological warfare defense. Methods to detect them include immunoassays, sensors, mass spectrometry, and genetic analysis. Nucleic acid-based genetic analysis involves DNA amplification that offers high sensitivity and unambiguous identification. However, nucleic acid-based genetic analysis is not applicable to agents that contain no nucleic acids. One example of such agents is ricin, which is listed as a Category B bioterrorism agent according to the Centers for Disease Control and Prevention. Immunoassay is advantageous over other methods due to its simplicity and rapid analysis, but it requires an antibody that is specific to the agent of interest. Therefore, it can not be used for detecting unknown or new agents because of the fact that antibody is simply not available. [0003] With the increasing ability to modify and engineer potential warfare agents, the ability to detect agents that have not been identified previously becomes more important. There is thus a need in the art to develop assays for the detection of toxins and other biological agents.
[0004] In addition, a platform is needed for studying molecular responses to toxins. The quantitative information about the effects of a variety of constituents on the yield of protein expression can lead to identification of the physiological targets of a toxin, which is useful for development of therapeutic drugs.
SUMMARY
[0005] A sensor array is designed for detecting inhibition of biological synthesis of pre- characterized proteins which indicates presence of a toxic analyte. The sensor array comprises an array of units; each unit is for expression of one protein and thus functions as one sensor. The sensor array expresses a group of pre-characterized proteins in different expression system, thus the response pattern (or signature) of an analyte due to different inhibitory effects is registered and used as a tool for detection and identification. New agents are identified by comparing the response pattern with signatures of known agents in a pre- collected database. [0006] In a preferred embodiment, a sensor for detecting toxins, comprises a substrate; at least one well formed in or on said substrate; at least a first in vitro transcription and translation (IVT) unit disposed in said well, said first IVTs comprising a DNA template including a coding sequence which is transcribed into messenger RNA using an RNA polymerase; a eukaryotic or prokaryotic lysate providing ribosomes for protein translation by said messenger RNA, wherein said first IVT expresses a first protein, and a detector for detecting a signal related to a concentration of said first protein, wherein a level of said signal is reduced when a target toxin which inhibits expression of said first protein by said IVT is present as compared to when said target toxin is not present.
[0007] In another preferred embodiment, a sensor array for detecting multiple toxins, comprises a substrate; a plurality of wells formed in or on said substrate; a first and at least a second in vitro transcription and translation (IVT) unit disposed in a first and at least a second of said plurality of said wells, respectively, said first and second IVTs each comprising a DNA template including a coding sequence which is transcribed into messenger RNA using an RNA polymerase; a eukaryotic or prokaryotic lysate providing ribosomes for protein translation by said messenger RNA, wherein said first IVT expresses a first protein and said second IVT expresses a second protein different from said first protein, and a detector for detecting a signal related to a concentration of said proteins, wherein a level of said signal is reduced when a target toxin capable of inhibiting protein translation for said IVTs is present as compared to when said target toxin is not present.
[0008] In another preferred embodiment, a sensor array in which each sensor comprises at least a feeding chamber; a reaction chamber; a membrane for feeding nutrients and removing by products. Preferably, a channel connects a feeding chamber and a reaction chamber and a pumping mechanism is used for supplying nutrients. [0009] In another preferred embodiment, a pumping mechanism is used for removing byproducts and/or a pumping mechanism is used for supplying nutrients and removing byproducts. Preferably, a pumping mechanism comprises at least one of: osmosis, capillary force, pneumatic pump, syringe pump, electrokinetic pump, piezoelectric pump, acoustic, or other pumps.
[0010] In another preferred embodiment, microfluidic features and means are used. [0011] In another preferred embodiment, a method of identifying inhibitors of protein expression comprises expressing a nucleic acid sequence encoded by an expression vector in a reaction mixture wherein the nucleic acid is transcribed and translated in presence or absence of a candidate drug; measuring protein expression in the presence or absence of a candidate drag; and, identifying inhibitors of protein expression. Preferably, the reaction mixture comprises polymerases, nucleotides, amino acids.
[0012] In a preferred embodiment, a surrogate cell array comprises a microcompartment wherein each microcompartment comprises a reaction chamber, feeding chambers, and a channel or a cavity to connect each microcompartment. Preferably, the reaction chamber is a miniaturized reaction chamber having a diameter of about 0.1 mm to 30 mm.
[0013] hi another preferred embodiment, the feeding chamber comprises amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer.
[0014] In another preferred embodiment, the reaction chamber comprises a cell-free expression system, such as for example, in vitro transcription and translation (TVT).
[0015] hi another preferred embodiment, a channel or a cavity connected to the chambers comprises a dialysis membrane with the molecular weight cutoff of about 1 kDa to about 20
KDa. Preferably, the dialysis membrane has a molecular weight cut off of about 10 KDa.
[0016] hi another preferred embodiment, the channel or cavity diameter is about 5 μm up to 10 mm. Preferably, the feeding chamber is dimensionally proportioned to about 2 to 100 times larger than the dimensions of the reaction chamber.
[0017] In another preferred embodiment, a miniaturized array comprises a reaction chamber dimensionally proportioned to contain a volume of fluid 10 times less than a feeding chamber. Preferably, the reaction chamber is dimensionally proportioned to contain about
0.1 μl up to 100 μl of fluid and the feed chamber is dimensionally proportioned to contain about 1 μl up to 5000 μl of fluid.
[0018] In another preferred embodiment, the chambers comprising the surrogate cell array are made from at least one material selected from the group consisting of polypropylene, polycarbonate, polystyrene, vinyl, acrylic, plastics, metal and glass.
[0019] In another preferred embodiment, the surrogate cell array comprises a plurality of microcompartments and channels.
[0020] To assist in analyzing the sample, the new detection systems can include pattern recognition software. The software compares the target molecule binding pattern corresponding to the unknown sample with binding patterns corresponding to known compounds. From these comparisons, the software can determine the composition of the sample, or deduce information about the source of the sample. The systems can be used to detect the existence of characteristic compounds, or "molecular fingerprints," associated with certain chemicals or conditions. For example, the systems can be used for human drug testing by detecting the presence of drugs. The systems can also be used for pollution monitoring by detecting compounds characteristic of the discharge of certain toxic pollutants.
Numerous other applications are also possible.
[0021] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] There is shown in the drawings embodiments, which are presently preferred, it being understood, however, that the invention can be embodied in other forms without departing from the spirit or essential attributes thereof.
[0023] Figure IA is a schematic representation showing three dimensional view of a miniaturized, 2x3 solution array for ricin detection. The units were laid out according to the standards of 96-well plates (i.e., 9 mm pitch). Figure IB is a schematic representation of a cross-sectional view of one unit of the array in Figure IA.
[0024] Figure 2 is a schematic representation showing a surrogate cell array for in vitro protein expression. The layout is presented in the format of 96-well microplates. An exploded view of one cell is shown at the bottom, consisting of a reaction chamber, two feeding chambers, a channel connecting the chambers, and a dialysis membrane. The design is not to scale.
[0025] Figure 3 A shows Green Fluorescent Proteins (GFP) expression confirmed by Western blotting. Lane 1: pre-stained protein markers; 2: negative control (containing all reagents except for the DNA template); 3: recombinant GFP (rGFP) purchased; 4: GFP expressed. Expressed GFP contains a stretch of six histidines at its C-terminal, causing its molecular weight slightly larger than rGFP. Figure 3B shows CAT expression confirmed by Western blotting. Lane 1 : protein markers; 2: negative control; 3: CAT expressed. Figure 3 C shows luciferase expression confirmed by luminescence detection. Lane 1 : negative control; 2: luciferase expressed. The intensity of luminescence in the y axis is in log scale. [0026] Figure 4A shows the inhibitory effects of tetracycline (TC) on the expression yield of GFP in E. coli expression system. Lane 1 of Western blotting analysis: pre-stained protein markers; 2: expression with 3000 ng/μL of TC; 3: 300 ng/μL; 4: 30 ng/μL; 5: 3 ng/μL; 6: 0.3 ng/μL 7: positive control (no TC); 8: negative control; 9: rGFP purchased. Figures 4B- 4G shows the effects of TC (Figures 4B, 4D and 4F or cycloheximide (CH) (Figures 4C, 4E, 4G) on the expression yield of GFP (Figures 4B, 4C) in E. coli expression system, of CAT (Figures 4D, 4E) in E. coli expression system, and of luciferase in rabbit reticulocyte expression system. All x axes are the concentration (ng/μL) of toxin in log scale. Y axes are the amount of expressed protein either normalized to the positive control (Figures 4B-4E) or in log scale of luminescence signal (Figures 4F, 4G).
[0027] Figures 5 A and 5B shows the response pattern of the 3x4 IVT sensor array for two toxin simulants, tetracycline (TC, Figure 5A) and cycloheximide (CH, Figure 5B). The experiments were carried out in two of the 2x3 well devices. The signals for the positive control were from the first row of 3 wells in the device, in which GFP, CAT, and luciferase were expressed in their respective expression systems. These wells were free of toxins. The signals for the negative control were from the second row of 3 wells in the device, in which the expression vector was not added. The signals for the samples were from the remaining two rows of 3 wells in the device, in which either 17 ng of CH or 25 ng of TC was added into the protein expression system.
[0028] Figure 6 A is a graph showing normalized production yield of luciferase as a function of the expression time in the device or in a microcentrifuge tube. A linear DNA vector was used. Figure 6B is a graph showing GFP expression yield as a function of time. Expression took place in microcentrifuge tube (closed circles) or in a device with dialysis membrane (open circles). The error bars were obtained from 3 repeats [0029] Figure 7 is a schematic representation illustrating signal amplification as a result of the inhibitory effects of ricin on the production of every copy of protein. Thousands of copies of a protein can be expressed from each copy of RNA.
[0030] Figure 8 is a graph showing the calibration curve for ricin detection. Protein expression yield, indicated by luminescence, decreased with the concentration of ricin A chain (solid circles). However, the expression yield remained the same when the ricin A chain was heat denatured and its toxicity was deactivated (open circles). Experiments were carried out in the device and the expression time was 4 hours. The error bars are the standard deviation obtained from three repeat experiments. Lines are the best fit of linear regression of the experimental results.
[0031] Figure 9 A is a graph showing luciferase production as a function of the expression time in a miniaturized device or in a microcentrifuge tube. The expression yield is indicated by the luminescence. A circular DNA vector was used. Figure 9B is a graph showing protein expression yield, indicated by luminescence, decreased with the concentration of ricin A chain when the expression time was fixed at 5 minutes (solid circles). However, the expression yield remained the same when the ricin' s toxicity was deactivated by heat denature (open circles). [0032] Figure 10 is a graph showing the comparison among ricin A chain, B chain, whole ricin, and ricin treated with 2-mercaptoethanol. Luciferase expression yield, indicated by luminescence, is plotted with the concentration of each reagent. [0033] Figure 11 is a schematic illustration of a surrogate cell array for protein expression. The area shadowed with diagonal lines indicates a subset of 24 wells. The row A is for the positive controls, B for the negative controls, and C-H for the samples. The inset in the expanded view shows transcription and translation in each surrogate cell.
DETAILED DESCRIPTION
[0034] Detection and identification of toxins are important for medical diagnostics, food/water safety testing, and biological warfare defense. Methods to detect toxic agents include immunoassay, sensors, mass spectrometry, and genetic analysis. Nucleic acid-based genetic analysis involves DNA amplification that offers high sensitivity and unambiguous identification. However, it is not applicable to agents that contain no nucleic acids. One example of such agents is ricin, which is listed as a Category B bioterrorism agent according to the Centers for Disease Control and Prevention. Immunoassay is advantageous over other methods due to its simplicity and rapid analysis, but it requires an antibody that is specific to the agent of interest. Therefore, it can not be used for detecting unknown or new agents because of the fact that antibody is simply not available. With the increasing ability to modify and engineer potential warfare agents, the ability to detect agents that have not been identified previously becomes more important.
[0035] The array, described herein, is based on the mechanism of toxin actions. One of mechanisms toxins use to cause toxic effects is to inhibit protein synthesis in cells. For instance, ricin acts on the 28S ribosomal subunit and prevents the binding of elongation factor-2, a critical protein in the process of protein translation. This interaction inactivates ribosomes, inhibits protein synthesis, and leads to cell death. Similarly, a number of potent biological toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin, diptheria toxin and exotoxin A. Since each type of toxin has unique mechanism of toxicity, it is possible to exploit the mechanism of toxin actions for toxin detection. [0036] It is also well-recognized that high-yield protein expression of IVT can be attained in the flow of a feeding solution, but not under static conditions in a fixed volume (Spirin, A. S. et al., Science 1988, 242, 1162-1164; Spirin, A. S. Trends Biotechnol 2004, 22, 538-545). As a result, commercial bench-top instruments incorporate the principle of continuous flow with a magnetic stirrer (Betton, J. M. Curr Protein Pept Sci 2003, 4, 73-80; Martin, G. A. et al, Biotechniques 2001, 31, 948-950, 952-943). However, the bench-top instruments often employs milliliters of reagents (Betton, J. M. Curr Protein Pept Sd 2003, 4, 73-80) and it is difficult to achieve the high-throughput format as discussed by Angenendt et al. (Angenendt, P.; Nyarsik, L.; Szaflarski, W.; Glokier, J.; Nierhaus, K. H.; Lehrach, H.; Cahill, D. J.; Lueking, A. Anal. Chem. 2004, 76, 1844-1849).
Definitions
[0037] Prior to setting forth the invention the following definitions are provided:
[0038] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
[0039] As used herein, the terms "exon" and "intron" are art-understood terms referring to various portions of genomic gene sequences. "Exons" are those portions of a genomic gene sequence that encode protein. "Introns" are sequences of nucleotides found between exons in genomic gene sequences.
[0040] As used herein the terms "rare" or "low copy numbers" refer to nucleic acid molecules that are less than about 300 copies per cell. The terms "moderate" or "medium copy numbers" refer to nucleic acid molecules that are about 300-1,000 copies per cell. The terms "abundant" or "high copy numbers" refer to nucleic acid molecules that are about
1,000-3,000 copies per cell.
[0041] "Amplification" relates to the production of additional copies of a nucleic acid sequence.
[0042] The terms "nucleic acid molecule" or "polynucleotide" will be used interchangeably throughout the specification, unless otherwise specified. As used herein,
"nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides
(adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides
(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA--DNA, DNA-RNA and
RNA--RNA helices are possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. [0043] As used herein, the term "fragment or segment", as applied to a nucleic acid sequence, gene or polypeptide, will ordinarily be at least about 5 contiguous nucleic acid bases (for nucleic acid sequence or gene) or amino acids (for polypeptides), typically at least about 10 contiguous nucleic acid bases or amino acids, more typically at least about 20 contiguous nucleic acid bases or amino acids, usually at least about 30 contiguous nucleic acid bases or amino acids, preferably at least about 40 contiguous nucleic acid bases or amino acids, more preferably at least about 50 contiguous nucleic acid bases or amino acids, and even more preferably at least about 60 to 80 or more contiguous nucleic acid bases or amino acids in length. "Overlapping fragments" as used herein, refer to contiguous nucleic acid or peptide fragments which begin at the amino terminal end of a nucleic acid or protein and end at the carboxy terminal end of the nucleic acid or protein. Each nucleic acid or peptide fragment has at least about one contiguous nucleic acid or amino acid position in common with the next nucleic acid or peptide fragment, more preferably at least about three contiguous nucleic acid bases or amino acid positions in common, most preferably at least about ten contiguous nucleic acid bases amino acid positions in common. [0044] As used herein, the terms "oligonucleotide" or "primers" are used interchangeably throughout the specification and include linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorthiorate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, Hoδgsteen or reverse Hoδgsteen types of base pairing, or the like.
[0045] The oligonucleotide may be composed of a single region or may be composed of several regions. For example, hinge regions comprising different lengths and base composition. The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of this invention "chimeric" compounds are oligonucleotides, which comprise two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above. [0046] The oligonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in preferred cases a length not exceeding about 100 carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
[0047] As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.
[0048] In the present context, the terms "nucleobase" covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered "non- naturally occurring" have subsequently been found in nature. Thus, "nucleobase" includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5- methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat No. 5,432,272. The term "nucleobase" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.
[0049] As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
[0050] "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429-4443, Toulme, J. J., Nature Biotechnology 19:17-18 (2001); Manoharan M., Biochemica et Biophysica Acta 1489:117-139(1999); Freier S., M., Nucleic Acid Research, 25:4429-4443 (1997), Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic Acid Drug Dev., 10:297-310 (2000), ); T-O, 3Λ-C-linked [3.2.0] bicycloarabinonucleosides (see e.g. N.K Christiensen., et al, J. Am. Chem. Soc, 120: 5458-5463 (1998). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like. [0051] The term "stability" in reference to duplex or triplex formation generally designates how tightly an antisense oligonucleotide binds to its intended target sequence; more particularly, "stability" designates the free energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability. Preferably, oligonucleotides are selected that have melting temperatures of at least 450C when measured in 100 mM NaCl, 0.1 mM EDTA and 10 niM phosphate buffer aqueous solution, pH 7.0 at a strand concentration of both the oligonucleotide and the target nucleic acid of 1.5 μM. Thus, when used under physiological conditions, duplex or triplex formation will be substantially favored over the state in which the antigen and its target are dissociated. It is understood that a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes. Preferably, modified oligonucleotides, e.g. comprising LNA units, of the invention form perfectly matched duplexes and/or triplexes with their target nucleic acids.
[0052] As used herein, the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O0C. for short probes (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. [0053] As used herein, the terms "probe" or "capture probe" are defined as a nucleic acid, capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e. A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, probes may be peptide nucleic acids (PNA) in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. [0054] The term "target nucleic acid" refers to a nucleic acid (often derived from a biological sample), to which the probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from context. [0055] The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence, but with only insubstantial hybridization to other sequences or to other sequences such that the difference may be identified. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 50C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
[0056] As used herein, the term "downstream" when used in reference to a direction along a nucleotide sequence means in the direction from the 5' to the 3' end. Similarly, the term "upstream" means in the direction from the 3' to the 5' end.
[0057] As used herein, the term "gene" means the gene and all currently known variants thereof and any further variants which may be elucidated.
[0058] As used herein, "variant" of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
[0059] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic", "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type target genes. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance. [0060] As used herein, the term "mRNA" means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated. [0061] The term, "complementary" means that two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence. Normally, the complementary sequence of the oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence. Preferably, alleles or variants thereof can be identified. A BLAST program also can be employed to assess such sequence identity.
[0062] The term "complementary sequence" as it refers to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule by the base-pairing rules. More particularly, the term or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% of the nucleotides of the other strand, usually at least about 98%, and more preferably from about 99 % to about 100%. Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software, for example the BLAST program.
[0063] The "percentage of sequence identity" or "sequence identity" is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the polynucleotide sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical subunit (e.g. nucleic acid base or amino acid residue) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Percentage sequence identity when calculated using the programs GAP or BESTFIT is calculated using default gap weights. [0064] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and WunschJ MoI. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sd. USA 85: 2444 (1988), by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA), or by inspection. In particular, methods for aligning sequences using the CLUSTAL program are well described byHiggins and Sharp in Gene, 73: 237-244 (1988) and in CABIOS 5: 151-153 (1989)). [0065] A "heterologous" component refers to a component that is introduced into or produced within a different entity from that in which it is naturally located. For example, a polynucleotide derived from one organism and introduced by genetic engineering techniques into a different organism is a heterologous polynucleotide which, if expressed, can encode a heterologous polypeptide. Similarly, a promoter or enhancer that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter or enhancer.
[0066] A "promoter," as used herein, refers to a polynucleotide sequence that controls transcription of a gene or coding sequence to which it is operably linked. A large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources, are well known in the art and are available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
[0067] An "enhancer," as used herein, refers to a polynucleotide sequence that enhances transcription of a gene or coding sequence to which it is operably linked. A large number of enhancers, from a variety of different sources are well known in the art and available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoter sequences (such as the commonly-used CMV promoter) also comprise enhancer sequences. [0068] "Operably linked" refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner. A promoter is operably linked to a coding sequence if the promoter controls transcription of the coding sequence. Although an operably linked promoter is generally located upstream of the coding sequence, it is not necessarily contiguous with it. An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. Operably linked enhancers can be located upstream, within or downstream of coding sequences. A polyadenylation sequence is operably linked to a coding sequence if it is located at the downstream end of the coding sequence such that transcription proceeds through the coding sequence into the polyadenylation sequence.
[0069] A "detectable marker gene" is a gene that allows cells carrying the gene to be specifically detected (e.g., distinguished from cells which do not carry the marker gene). A large variety of such marker genes are known in the art. Preferred examples thereof include detectable marker genes which encode proteins appearing on cellular surfaces, thereby facilitating simplified and rapid detection and/or cellular sorting. Byway of illustration, the lacZ gene encoding beta-galactosidase can be used as a detectable marker, allowing cells transduced with a vector carrying the lacL gene to be detected by staining.
[0070] A "selectable marker gene" is a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selective agent. By way of illustration, an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be positively selected for in the presence of the corresponding antibiotic. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. A variety of such marker genes have been described, including bifunctional (i.e. positive/negative) markers (see, e.g., WO
92/08796, published May 29, 1992, and WO 94/28143, published Dec. 8, 1994).
[0071] The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
[0072] "Substrate" or "probe substrate" refers to a solid phase onto which an adsorbent can be provided (e.g., by attachment, deposition, etc.).
[0073] "Eluant" or "washing solution" refers to an agent that can be used to mediate adsorption of a marker to an adsorbent. Eluants and washing solutions are also referred to as
"selectivity threshold modifiers." Eluants and washing solutions can be used to wash and remove unbound materials from the substrate surface.
[0074] "Detect" refers to identifying the presence, absence or amount of the object to be detected.
[0075] "Detectable moiety" or a "label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, 6xHis, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety in a sample.
[0076] "Energy absorbing molecule" or "EAM" refers to a molecule that absorbs energy from an ionization source in a mass spectrometer thereby aiding desorption of analyte from a surface. Depending on the size and nature of the analyte, the energy absorbing molecule can be optionally used. Energy absorbing molecules used in MALDI are frequently referred to as
"matrix." Cinnamic acid derivatives, sinapinic acid ("SPA"), cyano hydroxy cinnamic acid
("CHCA") and dihydroxybenzoic acid are frequently used as energy absorbing molecules in laser desorption of bioorganic molecules.
[0077] "Sample" is used herein in its broadest sense. A sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like. A sample also comprises any chemical compound; any environmental agent such as toxins, pollutants and the like, water samples, air samples, and soil samples.
[0078] "Substantially purified" refers to nucleic acid molecules or proteins that are removed from their natural environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90% free, from other components with which they are naturally associated.
[0079] "Substrate" refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
[0080] As used herein, "reaction mixture" refers to an amplification mixture, a DNA dependent DNA polymerase, an RNA polymerase, the triphosphate nucleotides and the triphosphate deoxynucleotides, a DNA dependent RNA polymerase, a cellular translation extract, the mixtures necessary for amplification, transcription and translation and optionally one or several substances permitting revelation of the activity of the reporter molecule. Significance
[0081] The potential impacts of the invention, especially on matters of national defense, include the following. First, the system can be used as a platform technology to develop therapeutic drug, identify targets for future use on biosensors and evaluate cellular responses to toxic/pathogenic insult at the molecular level. The systematic and quantitative study of the interplay among a large number of the constituents in the range outside the typical concentration in a biological cell can be used to quickly identify the target molecules of any toxins that inhibit protein expression. The target information will lead to the development of therapeutics and ultimately clinical diagnostic assays for use in hospital emergency rooms. Currently, no such capability exists. Second, the approach in this invention provides a unique approach to detect unknown or genetically modified agents. With the increasing ability to modify and engineer potential biological warfare agents, the ability to detect agents that have not been identified or fingerprinted has become of critical importance. In addition, the invention will lead to significant payoffs in civilian applications. The multiplexed surrogate cell array offers a means for high-throughput protein expression that is needed by the emerging proteomics research. As more and more new genes are being identified, there is a considerable need to determine the function and properties of the proteins encoded by these genes. To match high-throughput gene discovery, methods to produce a large number of proteins in parallel are needed. The invention will also contributes to basic sciences and researches by understanding nanoliter-scale transcription and translation, expanding the applications of miniaturization, and bringing forward biosensor array technology. [0082] The surrogate cell array system comprises an array of microcompartments; each microcompartment functions as a surrogate cell, consisting of a reaction chamber, two feeding chambers, and a channel to connect them as shown in Figures 1 and 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs. The feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis. The reaction chamber contains cell-free expression system with other reagents as in the feeding chambers. The channel connected to the chambers provides a means to supply nutrients and remove byproducts. The selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at between about 1 KDa up to 100 KDa, which at the same time allows entry of nutrients into the feeding chamber. Device Design.
[0083] A variety of distinct components are integrated into the microfluidic device to meet the requirement of protein expression. The requirements include nutrient supplies, byproduct removal, evaporation control, and fluid manipulation. The device comprises an array of units; each unit consists of a reaction chamber, two feeding chambers, and a channel to connect them as shown in Figure 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs. The feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis. The reaction chamber contains cell-free expression system with other reagents as in the feeding chambers. The channel connected to the chambers provides a means to supply nutrients and remove byproducts. The selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at ~10 KDa, which at the same time allows all small molecules in the feeding chamber passing by.
[0084] The incorporation of membrane is important because of two facts: (1) the flow of a feeding solution leads to higher expression yield compared to static conditions, because protein synthesis will not terminate earlier due to fast depletion of energy sources (ATP and GTP); (2) removal of small molecule byproducts is also very important for the high yield expression of proteins in a cell- free medium, inhibition of protein synthesis resulting from the small molecular byproducts such as hydrolysis products of triphosphates is eliminated. [0085] The device possesses appropriate geometric configuration to achieve desired properties for in vitro protein expression. The dimension of the microchannel is in the range of 1 micron to 1 centimeter, depending on the flow rate required for supplying nutrients and removing byproduct. The feeding chambers must be sufficiently large compared to the reaction chamber so that they function as reservoirs. In the current design, the volume of the reaction chamber is 10 nanoliter-100 microliter whereas that of the feed chambers is about 100 nanoliter-1 mililiter.
[0086] One of the mechanisms for supplying nutrients from the feeding chambers to the reaction chamber and for removing byproducts from the reaction chamber to the feeding chambers is osmosis, a net flow resulting from the difference in chemical potential of solutes between two solutions separated by the membrane. The degree of the potential difference is determined by their difference in solute activities, which are correlated with solute concentrations. For example, the osmotic pressure, ϋ, resulting from a byproduct in the reaction chamber can be approximately calculated by Van't Hoff s Law assuming it is in the limit of infinite dilution (Levine, 1988 Physical Chemistry. New York: McGraw-Hill Book Co.).
U = CtRT
[0087] where Cb is the concentration of the byproduct, R is gas constant, and T is the temperature. Another mechanism for manipulating fluids is the capillary force due to surface tension, especially when the dimension is very small.
[0088] The exchange rate of chemicals between two sides of the membrane increases with the concentration gradient along the membrane. The exchange stops when equilibrium reaches. To increase the exchange rate, an active flow in channel is needed to supply fresh solution underneath the membrane. One approach is to use hydrostatic pressure, which results from the difference in the solution level between the feeding chamber and reaction chamber. When the feeding chamber has slightly higher solution level than the reaction chamber, the pressure difference, ΔP, can be defined by the difference in height, Δh, as follows:
ΔP = pgΔ/z
[0089] where p is the density of solution and g is gravitational acceleration. The volume flow rate in the channel, Q, can be calculated using the equation below based on a fully developed flow in the channel.
Q = (πΔp£>4)/128μL
[0090] where D is the channel diameter, p is viscosity, and L is the distance between chambers. Using Δh = 1 mm, D = 50 μm, L= 3 mm, and water's density and viscosity, the velocity is calculated to be 0.50 nL/s. At this speed, the amount of reagent in the feeding chamber will last for many hours, which is longer than the reaction time needed for completing protein synthesis in a microdevice.
[0091] Other pumping mechanisms may also be used for supplying nutrients and removing byproducts, including pneumatic pumps, syringe pumps, electrokinetic pumps, piezoelectric pumps, acoustic and other pumps.
[0092] Mixing primarily results from the diffusion unless other means are to be used for creating flows. As discussed above, the size of the reaction chamber ranges from micron to centimeter-scale. The time needed for a molecule to diffuse 1 mm is in seconds, which is much shorter than the time used for protein synthesis. (The diffusion time was estimated by using a diffusion coefficient of 5 x 10'4 cm2/s and the law x2 = 2Dt, where x is the distance, D is the diffusion coefficient, and t is the time).
[0093] Clearly, the factors affecting the fluid manipulation, nutrient supply, and byproduct removal will have effects on the yield of protein expression, which directly influences the detection sensitivity.
[0094] As a proof of concept, in vitro protein expression was selected for detecting toxins that inhibit protein synthesis. One of mechanisms toxins use to cause toxic effects is to inhibit protein synthesis in cells. For instance, ricin acts on the 28S ribosomal subunit and prevents the binding of elongation factor-2, a critical protein in the process of protein translation. This interaction inactivates ribosomes, inhibits protein synthesis, and leads to cell death. Similarly, a number of potent biological toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin, diphtheria toxin and exotoxin A. Since each type of toxin has unique mechanism of toxicity, it is possible to exploit the mechanism of toxin actions for toxin detection.
[0095] While protein expression is commonly implemented using prokaryotic Escherichia coli (E. colϊ) cells, it has also been realized in a cell- free medium employing a process called in vitro transcription and translation (IVT). In IVT systems, a DNA template consisting of a coding sequence is transcribed into messenger RNA using RNA polymerases; either eukaryotic or prokaryotic lysate is then exploited for providing ribosomes and additional components necessary for protein translation. The transcription and translation steps are coupled together and take place in the same reaction mixtures. Due to the absence of cellular control mechanisms, IVT overcomes the limitations (e.g., cytotoxicity) experienced by cell-based recombinant protein production. IVT has been demonstrated for various applications, including protein chips synthesis of drug transporters and liigh- throughput screening it has also been implemented in miniaturized devices. [0096] As an illustrative example, not meant to limit or construe the invention in any way, the toxin detection using IVT, is illustrated in Figure 2. The device comprises an array of IVT units; each unit is for expression of one protein and thus functions as one sensor. The array expresses a group of pre-characterized proteins in different expression system; the proteins and expression systems are judiciously selected so that protein synthesis in each unit is inhibited or affected differently by different type of toxins. The response pattern (or signature) of a toxin due to different inhibitory effects is registered and used as a tool for detection and identification. New agents are identified by comparing the response pattern with signatures of known agents in a pre-acquired database. [0097] Three proteins were first synthesized in two types of expression systems. The first protein is Green Fluorescent Protein (GFP), a widely-used fluorescent molecule with known DNA sequence and crystal structure. Protein expression was carried out by combining DNA template, E. coli lysate, and a reaction mix consisting of T7-RNA polymerase, nucleotides, amino acids, and other reagents. GFP product was confirmed by fluorescence spectrometry and Western blotting. The result of Western blotting is shown in Figure 3 A. The second protein is chloramphenicol acetyl-transferase (CAT), an enzyme responsible for bacterial resistance to an antibiotic drug, chloramphenicol. CAT was expressed in the same E. coli expression system; success of the protein expression was confirmed using Western blotting as shown in Figure 3B. The third protein is luciferase, an enzyme from firefly tails that catalyzes the production of light in the reaction between luciferin and adenosine triphosphate (ATP). Synthesis of luciferase was carried out using rabbit reticulocyte expression system. Detection of the expression product was achieved by monitoring the intensity of luminescence after mixing with luciferin, ATP, and other reagents, as shown in Figure 3C. [0098] To illustrate toxin detection and to study molecular responses of surrogate cells to toxins, tetracycline (TC) and cycloheximide (CH) as toxin simulants were used to study their inhibitory effects on protein synthesis. TC is an antibiotic substance produced by Streptomyces species. It acts only on prokaryotic cells and it blocks binding of aminoaceyl- transfer RNA to A-site of ribosomes. CH acts specifically on eukarybtic cells and it inhibits the activity of peptidyl transferase, an enzyme needed in the translocation reaction on ribosomes. Figure 4 shows the expression yield of GFP, CAT, and luciferase when a series of concentrations of TC or CH were added in two protein expression systems. As illustrated in Figure 4A, GFP synthesis was completely inhibited when 3000 ng/μL of TC was used. Partial inhibition was observed when a series of lower concentrations (300 ng/μL to 0.3 ng/μL) of TC was added. This result suggests that a qualitative and quantitative relationship exists between the expression yield and toxin amount.
[0099] The comparison between TC and CH for GFP production in the E. coli expression system is shown in Figures 4B and 4C. The results clearly indicate that TC has inhibitory effect on GFP production whereas CH has a negligible effect. Figure 4D and 4Ε exhibit similar disparity between TC and CH for CAT production in the E. coli expression system. Although TC has inhibitory effect on both GFP and CAT production, the degree of inhibition is different between these two proteins, evident from the difference in the slopes of respective linear regression lines. The comparison between TC and CH for luciferase production in rabbit reticulocyte expression system is shown in Figure 4F and 4G. An opposite effect was observed; TC has a negligible inhibitory effect on the luciferase production in the eukaryotic expression system, whereas CH has a significant inhibitory effect. These results demonstrate that it is possible to have a set of proteins and expression systems to generate response patterns for detection of toxins.
[0100] Although Western blotting and luminescence detection was used to monitor protein production in this concept demonstration, it should be feasible to use a common method for toxin detection. One of such methods is to use GFP as an indicator for the detection of protein expression due to its green fluorescence. GFP has been used for visualization, tracking, and quantification of a variety of proteins in cells after they are fused together. An increase of fluorescence signal in an IVT unit indicates the production of GFP or GFP-fused proteins. Quantitative information may be obtained by comparing the fluorescence signals of sample units and of reference units, which include both positive and negative controls in the array device. Any variation or adverse effects will be cancelled out between control and sample units. The magnitude of the signal can be correlated to the amount of proteins produced in the device.
[0101] m addition to GFP, DNA templates containing a coding sequence (reporter gene) for expressing an additional stretch of six histidines (6xHis) at the C-terminal of the protein of interest can also be used. A variety of biological assays are available for detecting the amount of 6xFfis tag fused with a protein. In addition, luminescence detection can also be used by fusing luciferase with proteins of interest, as demonstrated in the examples which follow. Other examples include commercially available vectors with his tags, such as: E. coli (pET vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags, and the expression of recombinant protein can be evaluated by SDS-PAGE and Western blot analysis.
[0102] The reporter gene is a gene which can be transcribed and translated in vitro in the presence of sequences which regulate its expression. The protein that the reporter gene codes for can be detected by any technique known to a person skilled in the art. By way of example, the reporter gene can be the gene of the protein GFP (Green Fluorescent Protein), as described in the examples which follow, or that of the beta-lactamase (TEM-I). In the case of the GFP, it is the fluorescent emission which is measured. In the case of the beta- lactamase, it is the activity of this enzyme which is measured by incubating a fraction of the translation reaction in a buffer containing nitrocephine. Nitrocephine is a chromogenic beta- lactamine which has the property of changing color from yellow to red when it is hydrolyzed by a beta-lactamase. Any other reporter gene can be contemplated in the process of the invention, such as beta-galactosidase, beta-glucuronidase, luciferase, peroxidase or a microperoxidase, etc.
[0103] The reporter gene advantageously encodes for an enzyme. The specificity of the labeling of the target substance can be carried out by any direct or indirect method known to a person skilled in the art. For the direct method, it is understood that the target substance is directly combined with the gene and with the elements necessary for the expression of said reporter gene in vitro. It relates, for example, to a recombinant nucleic acid molecule where the target substance is a nucleic sequence included in said recombinant nucleic acid molecule equally including the reporter gene and the sequences necessary for its in vitro expression. [0104] For the indirect method, it is understood that the target substance is combined with a reporter gene and with the sequences necessary for its expression in vitro, by the intermediary of a specific ligand of the of the target substance. This ligand is combined with the reporter gene and with the elements necessary for its expression in vitro. It is therefore the contacting of this ligand with the target substance which permits the carrying out of the specific labeling of the target substance. It relates for example to an antibody labeled by the reporter gene and the sequences necessary for its expression in vitro which is capable of specifically recognizing a target substance composed of an antigen. A target/ligand couple substance is understood as for example: an antigen/antibody, a nucleic sequence/a nucleic sequence, a probe, a receptor/a receptor ligand, etc. The labeling of the specific ligand of the target substance can be as previously a direct or indirect labeling.
[0105] According to the indirect method of the invention, the combination of the reporter gene and a target sequence corresponding to a protein allows several embodiments. In effect, the bonding of a nucleic acid molecule composed of a reporter gene and of the sequences necessary for its expression in vitro, on a protein can be carried out by techniques known to a person skilled in the art making use of bonding compounds such as: streptavidine/biotin (Kipriyanov et al., (1995). Hum Antibodies Hybridomas 6 (3), 93-101); a peptide corresponding to polylysine (Avrameas et al., (1998). PNAS 95 (10), 5601-6; Curiel et al., (1992). Hum Gen Ther 3 (2), 147-54; Wu et al., (1991). J Biol Chem. 266 (22), 14338-42; Kwoh et al., (1999). Biochim Biophys Acta 1444 (2), 171-90; Wu et al., (1994). J Biol Chem. 269 (15), 11542-6; the p-azido-tetrafluoro-benzyl (Ciolina et all, (1999). Bioconjug Chem. 10 (D, 49-55).
[0106] hi a preferred embodiment, the device is constructed using solid substrates. Solid- state substrates for use can include any solid material. Preferred materials include but not limited to: plastic materials including polystyrene, polymethylmethacrylate (PMMA), polythylene terephthalate polycarbonate, polydimethylsiloxane (PDMS), poly(cyclic olefin), and a variety of copolymers. Other materials include: acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid substrates can have any useful form including, micro well plates, thin films or membranes, beads, bottles, dishes, fibers, optical fibers, woven fibers, chips, compact disks, shaped polymers, particles and microparticles.
[0107] In one preferred embodiment, an array comprises surrogate cells for in vitro protein expression. Each cell comprises elements for biological protein synthesis, nutrient supply, removal of inhibitory byproducts, and flow controls. In vitro protein expression is produced by implementing the coupled transcription/translation reactions in the surrogate cell.
[0108] In another preferred embodiment, a method of determining the interplay among the constituents required for protein expression is provided. Quantitative studies are performed by generating a panel of protein expression conditions by supplementing purified key components. The in vitro process allows for varying the components and their concentration ratios of the reconstituted components including initiation factors, elongation factors, release factors, and many enzymes. The effects of the amount of these components and their concentration ratios on the protein expression yield are determined. [0109] In another preferred embodiment, a method of identifying molecular responses of the surrogate cell array to toxins is provided. For example, orchratoxin A (OTA) — a mycotoxin produced by fungi that displays toxic effect on human and animals — for demonstrating the capability of the surrogate cell array, since its exact mechanism is not known. Using a panel of reconstituted protein expression system with varying levels of key components, the physiologic targets of OTA can be identified.
[0110] Toxic substances can be produced by an animal, plant or microbe. A mechanism toxins use to cause toxic effects is through inhibition of protein synthesis in cells. For instance, ricin — a bioterrorism agent — blocks protein synthesis by inactivating ribosomes. Similarly, a number of other potent toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin produced by Shigella dysenteriae, Shiga-like toxin by enteropathogenic E. coli, diphtheria toxin from Corynebacterium diphtheriae and exotoxin A of Pseudomonas aeruginosa. Other toxins possess different mechanisms of toxicity: staphylococcal enterotoxin B inflicts toxic effect through activation of inflammation (superantigen), botulinum inhibits the release of neurotransmitter, cholera toxin activates adenylcyclase activity, and perfringin toxin causes pore-formation leading to the leakage of the cell content. The surrogate cell array is modified to study different toxicity mechanisms, which can be used for toxin detection and identification based on the array response pattern as well as for searching for therapeutics.
[0111] The toxin used as an illustrative example, for demonstrating the capability of surrogate cell array is orchratoxin A (OTA), a mycotoxin produced by fungi. Hundreds of mycotoxins produced by fungi have been identified, many of them are potent cytotoxins that cause cell disruption and interfere with essential cellular processes. Commonly a single mycotoxin can cause more than one type of toxic effect. OTA is a mycotoxin produced by Penicillium and Aspergillus. Several major mechanisms have been shown as involved in the toxicity of OTA, including inhibition of protein synthesis. However, the exact mechanism is not known. The method uses the surrogate cell arrays to obtain quantitative information about the inhibitory effects of OTA on protein synthesis and identify its target molecules. [Ol 12] Artificial Cells: Biological cells can be altered by modern techniques such as cloning and mutation. However, genetic engineering comes with limitations in cost and efficiency, as well as possible toxicity of the engineered product to the cell. As a result, artificial cells or cell surrogates have been developed to overcome the limitations. Artificial cells offer an alternative method for studying the functions of a variety of molecules with biological significance, for understanding the regulation of cellular activities, and for searching for the synthetic and metabolic pathways. In addition, artificial polymer cells have been employed for encapsulating medicines for drug delivery and organ transplant. Similarly, liposome is one of popular methods for delivering therapeutics at targeted locations. Artificial cells or cell surrogates will have significant impacts on pharmacology, medical diagnostics, and the related biological fields.
[0113] Cell Array Fabrication: In a preferred embodiment, plastics such as for example, used in typical tissue culture, are used for fabricating the cell array device because of the following reasons. First, a wide range of plastics are available to be selected for a biological assay of interest. The compatibility between plastics and chemical/biological reagents is evidenced by the fact that many labwares such as microcentrifuge tubes and microplates are made of plastics. A variety of plastic materials including polystyrene, polymethylmethacrylate (PMMA), polycarbonate, polydimethylsiloxane (PDMS), and poly(cyclic olefin) have been investigated for microfabrication and microscale assays. Second, there is vast experience in manufacturing low-cost, high- volume plastic parts and the manufacturing process is well-developed. Plastic parts made by techniques such as injection molding or embossing can be quite inexpensive: the manufacturing cost of an injection- molded compact disc (CD). Therefore, plastic devices can be made so cheaply as to be disposable after a single use. This could have tremendous impact in applications where cross-contamination of sequential samples is of concern.
[0114] Devices are fabricated following the method we described previously (Mei, Q.; Fredrickson, C. K.; Jin, S.; Fan, Z. H. Anal Chem 2005, 77, 5494-5500) though they are modified and optimized . One modification is incorporation of dialysis membrane. Among dialysis membranes that are commercially available, a polycarbonate membrane with an appropriate pore sizes or the like, is used. Thermal lamination of polycarbonate with other plastic layers is routinely practiced in industry; thermally laminating the membrane with plastic substrates forms the array device.
Detection ofRicin
[0115] In a preferred embodiment, the apparatus detects biological agents such as toxins, nerve gases, bacterial agents and the like. An example of detection of ricin is shown in the examples which follow. The ricin detection method is based on ricin' s inhibitory effects on protein synthesis. Biological synthesis (expression) of a protein includes the steps of gene transcription and protein translation; and these reactions can be coupled into one-step operation and carried out in a cell-free medium. Ricin is known to inhibit protein synthesis by interaction with 28S ribosome RNA; the inhibitory effect is exploited as the sensing mechanism in this invention. For each copy of RNA, thousands of copies of proteins can be produced. As a result, the inhibitory effects of ricin are amplified, leading to a significantly enhanced detection signal (the difference between the positive control and samples). An array of protein expression units is developed to accommodate positive/negative controls and multiple samples. The array device contains a solution without any reagent captured on a solid surface, offering flexibility without comprising the activities of biomolecules. The miniaturized solution array possesses a mechanism to supply nutrients continuously and remove byproducts, leading to higher protein expression yields and thus larger detection signals (lower detection limit) when ricin is present. The production of green fluorescent protein and luciferase in the solution array is described in the examples which follow. A calibration curve has been obtained between the luciferase expression yield and the ricin concentration, showing a detection limit of 1 pg/μL of ricin. The array device is also demonstrated for measuring the toxicity level of ricin after physical or chemical treatment. [0116] Methods to detect ricin at a low concentration include enzyme-linked immunosorbent assay (ELISA) and immunoassay using radioactive labeling. Although offering high sensitivity, ELISA involves several labor-intensive and time-consuming steps. For radioimmunoassay, the handling and disposal of radioisotopes are environmental challenges.
[0117] We describe an approach that exploits the mechanism — by which ricin causes toxic effects — as the sensing scheme. Ricin kills people by blocking protein synthesis in cells of human body. Biological synthesis (expression) of a protein includes the steps of gene transcription and protein translation. While protein expression is commonly implemented using Escherichia coli cells, it has also been realized in a cell-free medium employing a process called in vitro transcription and translation (IVT). IVT couples the following reactions into one step: (1) DNA consisting of a coding sequence is transcribed into messenger RNA; and (2) RNA is then translated to proteins in a cell lysate (product of burst cells) that provides ribosomes and other necessary components. Due to the absence of cellular control mechanisms, IVT overcomes the limitations experienced by cell-based recombinant protein production, including poor expression yield, low solubility, cytotoxicity, or susceptibility to proteolysis. IVT has been demonstrated for various applications, including protein chips and drug screening.
[0118] IVT has also been implemented in micro fluidic devices. For instance, Nojima et al. synthesized an mRNA by flowing two reactants from two inlets and mixing them through a Y-shaped structure into one outlet (Nojima, T. et al., Bioprocess Engineering 2000, 22, 13- 17). The product was collected from the outlet and then analyzed off-the-device. Although the work showed the feasibility to implement cell-free transcription in a microfluidic device, the key drawback of this device is the use of excessive accessories including external pumps and valves, and the lack of integration, making it difficult to be implemented in a high- throughput format. IVT has also been demonstrated in the microplate format. Angenendt et al. accomplished protein synthesis in microfabricated nano-wells; (Angenendt, P. et al., Anal. Chem. 2004, 76, 1844-1849). One of the major downsides of these microplates is that they do not consist of any fluid manipulation. As a result, nutrients can not be refurbished and inhibitory byproducts can not be removed, significantly reducing protein expression yield (Spirin, A. S. et al., Science 1988, 242, 1162-1164). [0119] We demonstrated expression of three proteins in micro-wells and used the response pattern of an array for identification of two toxin simulants. The array device described in the examples which follow, consists of a mechanism for fluid manipulation. As a result, higher protein expression yields can be obtained, leading to larger detection signals (lower detection limit) when ricin is present. More importantly, this detection approach accomplishes signal amplification. For each copy of RNA, thousands of copies of proteins can be produced. The inhibitory effects of ricin on the production of each copy of protein are accumulated, leading to a significantly enhanced detection signal. In addition, ricin detection using the one-step operation of protein expression simplifies the detection procedure, compared to ELISA that consists of many steps of reagent application and washing. The detection of ricin can be achieved in as short as 5 minutes.
Expression Vectors
[0120] The device of the invention allows for the screening of inhibition of protein expression from any encoded nucleic acid sequence. The nucleic acids are preferably expressed in an expression vector. The vector comprising the desired nucleic acid sequence, preferably has at least one such nucleic acid sequence. Alternatively, the vector may comprise more than one such nucleic acid sequence, or combinations of allelic variants. [0121] Genetic constructs suitable for use include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers may be required for gene expression of the sequence of choice, variants or fragments thereof. It is necessary that these elements be operably linked to the sequence that encodes the desired proteins and that the regulatory elements are operable in the individual to whom they are administered. [0122] Initiation codons and stop codons are generally considered to be part of a nucleotide sequence that encodes the immunogenic target protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence.
[0123] A wide variety of expression vectors may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, nonchromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El5 pCRl, pBR322, pMal-G2, pET, pGEX (Smith et al., 1988, Gene 67: 31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNA' s, e.g., the numerous derivatives of λ phage, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
[0124] For example, in a baculo virus expression systems, both non-fusion transfer vectors, such as but not limited to pVL941 (BamHl cloning site; Summers), pVL1393 (BamHl, Smal, Xbal, EcoRl, Notl, Xmalll, BgIII, and Pstl cloning site; Invitrogen), ρVL1392 (BgIII, Pstl, Notl, Xmalll, EcoRI, Xbal, Smal, and BamHl cloning site; Summers and Invitrogen), and pBlueBacIII (BamHl, BgIII, Pstl, Ncol, and HindIII cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc700 (BamHl and Kpnl cloning site, in which the BamHl recognition site begins with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700, with different reading frames), pAc360 (BamHl cloning site 36 base pairs downstream of a polyhedron initiation codon; itivitrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamHl, BgIII, Pstl, Ncol, and HindIII cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen (220)) can be used.
[0125] Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g., any expression vector with a DHFR expression vector, or a DHFR/methotrexate co- amplification vector, such as pED (Pstl, Sail, Sbal, Smal, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, a glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (HindIII, Xbal, Smal, Sbal, EcoRI, and BcII cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech). In another embodiment, a vector that directs episomal expression under control of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamHl, Sfil, Xhol, Notl, Nhel, HindIII, Nhel, PvuII, and Kpnl cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHl, Sfil, Xhol, Notl, Nhel, HindIII, Nhel, PvuII, and Kpnl cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, HindIII, Notl, Xhol, Sfil, BamHl cloning site, inducible m'ethallothionein Ha gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamHl, Xhol, Notl, Hindlll, Nhel, and Kpnl cloning site, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (Kpnl, Nhel, Hindlll, Notl, Xhol, Sfil, and BamHI cloning site, RS V-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). Selectable mammalian expression vectors for use in the invention include pRc/CMV (Hindlll, BstXI, Notl, SbaL and Apal cloning site, G418 selection; Invitrogen), pRc/RSV (Hindlll, Spel, BstXI, Notl, Xbal cloning site, G418 selection; Invitrogen), and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra) for use according to the invention include but are not limited to pSCl 1 (Smal cloning site, TK- and β-gal selection), pMJ601 (Sail, Smal, AfII, Narl, BspMII, BamHI, Apal, Nhel, SacII, Kpnl, and Hindlll cloning site; TK- and β-gal selection), and pTKgptFIS (EcoRI, Pstl, Sail, Accl, Hindll, Sbal, BamHI, and Hpa cloning site, TK or XPRT selection).
[0126] Yeast expression systems can also be used according to the invention to express Eph polypeptides. For example, the non- fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXI, EcoRI, BstXI, BamHI, Sad, Kpnl, and Hindlll cloning sit; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXI, EcoRI, BamHI, Sad, Kpnl, and Hindlll cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the present invention. [0127] Promoters and polyadenylation signals: Examples of promoters useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metallothionein. [0128] Examples of polyadenylation signals useful to practice the present invention, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. For example, the SV40 polyadenylation signal is in pCEP4 plasmid (Invitrogen, San Diego Calif), referred to as the SV40 polyadenylation signal. [0129] In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
Identification of nucleic acid sequences for expression.
[0130] The invention is useful for identifying candidate drugs and to screen the effects of the drugs in protein expression for therapy. The nucleic acid sequences used for detecting protein expression in the presence or absence of a candidate drug can be any nucleic acid sequence that may be involved in a disease, for example, over expression of tumor genes. These genes can include any allelic variants, mutants and the like. Sequences of different nucleic acids are accessible from various public databases such as GenBank. [0131] As more genes or variants thereof, are identified, oligonucleotide sequences are generated, or fragments thereof, may be employed as probes in the purification, isolation and detection of genes with similar sequences. Assays may be used for determining the sequence of an unknown nucleic acid; single nucleotide polymorphism (SNP) analysis; analysis of gene expression patterns from a particular species, tissue, cell type, etc.; gene identification; etc.
[0132] Other methods to determine the contributions of individual genes and or variants thereof, and their expression products. Genes or variants, thereof, can be isolated. Techniques are available to inactivate or alter any genetic region to any mutation desired by using targeted homologous recombination to insert specific changes into chromosomal variants. One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening individual clones (Kim et al., Nucleic Acids Res. 16:8887-8903 (1988); Kim et al, Gene 103:227-233 (1991)). Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly (Sedivy et al., Proc. Natl. Acad. Sd. USA 86:227-231 (1989)). One of the most general approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes for which no direct selection of the alteration exists (Mansour et al., Nature 336:348-352: (1988); Capecchi, Science 244:1288-1292, (1989); Capecchi, Trends in Genet. 5:70-76 (1989)). The PNS method is more efficient for targeting genes that are not expressed at high levels because the marker gene has its own promoter. Nonhomologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV- TK) gene and selecting against its nonhomologous insertion with the herpes drugs such as gancyclovir (GANC) or FIAU (l-(2-deoxy 2-fluoro-B-D-arabmofluranosyl)-5-iodouracii). By this counter-selection, the number of homologous recombinants in the surviving transformants can be enriched. Such transformants can be correlated with phenotypes. [0133] With respect to the cloning of allelic variants of the mammalian genes such as human, and homologues from other species {e.g., mouse), isolated gene sequences of interest may be labeled and used to screen a cDNA library constructed from mRNA obtained from cells or tissues (e.g., stem cells, brain tissues) derived from the organism (e.g., mouse) of interest. The hybridization conditions used should be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived.
[0134] Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions, as described in detail the Examples which follow. Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N. Y.; and Ausubel, et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. [0135] Another preferred method for identification of potential nucleic acids for screening of candidate drugs includes combining PCR with SAGE. Serial Analysis of Gene Expression (SAGE), is based on the identification of and characterization of partial, defined sequences of transcripts corresponding to gene segments. These defined transcript sequence "tags" are markers for genes which are expressed in a cell, a tissue, or an extract, for example.
[0136] SAGE is based on several principles. First, a short nucleotide sequence tag (9 to 10 bp) contains sufficient information content to uniquely identify a transcript provided it is isolated from a defined position within the transcript. For example, a sequence as short as 9 bp can distinguish about 262,144 transcripts given a random nucleotide distribution at the tag site, whereas estimates suggest that the human genome encodes about 80,000 to 200,000 transcripts (Fields, et al., Nature Genetics, 7:345 1994). The size of the tag can be shorter for lower eukaryotes or prokaryotes, for example, where the number of transcripts encoded by the genome is lower. For example, a tag as short as 6-7 bp may be sufficient for distinguishing transcripts in yeast. [0137] Second, random dimerization of tags allows a procedure for reducing bias (caused by amplification and/or cloning). Third, concatenation of these short sequence tags allows the efficient analysis of transcripts in a serial manner by sequencing multiple tags within a single vector or clone. As with serial communication by computers, wherein information is transmitted as a continuous string of data, serial analysis of the sequence tags requires a means to establish the register and boundaries of each tag. The concept of deriving a defined tag from a sequence in accordance with the present invention is useful in matching tags of samples to a sequence database. In the preferred embodiment, a computer method is used to match a sample sequence with known sequences.
[0138] The tags used herein, uniquely identify genes. This is due to their length, and their specific location (3') in a gene from which they are drawn. The full length genes can be identified by matching the tag to a gene data base member, or by using the tag sequences as probes to physically isolate previously unidentified genes from cDNA libraries. The methods by which genes are isolated from libraries using DNA probes are well known in the art. See, for example, Veculescu et al., Science 270: 484 (1995), and Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, N. Y.). Once a gene or transcript has been identified, either by matching to a data base entry, or by physically hybridizing to a cDNA molecule, the position of the hybridizing or matching region in the transcript can be determined. If the tag sequence is not in the 3' end, immediately adjacent to the restriction enzyme used to generate the SAGE tags, then a spurious match may have been made. Confirmation of the identity of a SAGE tag can be made by comparing transcription levels of the tag to that of the identified gene in certain cell types.
[0139] Analysis of gene expression is not limited to the above method but can include any method known in the art. All of these principles may be applied independently, in combination, or in combination with other known methods of sequence identification. [0140] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et al, FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al, Proc. Natl. Acad. ScL U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al, FEBS Lett., 2000, 480, 2-16; Jungblut, et al, Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al, FEBS Lett., 2000, 480, 2-16; Larsson, et al, J. BiotechnoL, 2000, 80, 143-57), subtractive RNA fingerprinting (SuRP) (Fuchs, et al, Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol, 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al, J. Cell Biochem. Suppl, 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
[0141] In another preferred embodiment, Expressed Sequenced Tags (ESTs), can also be used to identify nucleic acid molecules which are over expressed in a tumor cell. ESTs from a variety of databases can be identified. For example, preferred databases include, for example, Online Mendelian Inheritance in Man (OMIM), the Cancer Genome Anatomy Project (CGAP), GenBank, EMBL, PIR, SWISS-PROT, and the like. OMIM, which is a database of genetic mutations associated with disease, was developed, in part, for the National Center for Biotechnology Information (NCBI). OMEVI can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/Omim/. CGAP, which is an interdisciplinary program to establish the information and technological tools required to decipher the molecular anatomy of a cancer cell. CGAP can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/ncicgap/. Some of these databases may contain complete or partial nucleotide sequences. In addition, alternative transcript forms can also be selected from private genetic databases. Alternatively, nucleic acid molecules can be selected from available publications or can be determined especially for use in connection with the present invention.
[0142] Alternative transcript forms can be generated from individual ESTs which are within each of the databases by computer software which generates contiguous sequences. In another embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is determined by assembling a plurality of overlapping ESTs. The EST database (dbEST), which is known and available to those skilled in the art, comprises approximately one million different human mRNA sequences comprising from about 500 to 1000 nucleotides, and various numbers of ESTs from a number of different organisms. dbEST can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/dbEST/index.html. These sequences are derived from a cloning strategy that uses cDNA expression clones for genome sequencing. ESTs have applications in the discovery of new genes, mapping of genomes, and identification of coding regions in genomic sequences. Another important feature of EST sequence information that is becoming rapidly available is tissue-specific gene expression data. This can be extremely useful in targeting selective gene(s) for therapeutic intervention. Since EST sequences are relatively short, they must be assembled in order to provide a complete sequence. Because every available clone is sequenced, it results in a number of overlapping regions being reported in the database. The end result is the elicitation of alternative transcript forms from, for example, normal cells and tumor cells.
[0143] Assembly of overlapping ESTs extended along both the 5' and 3' directions results in a full-length "virtual transcript." The resultant virtual transcript may represent an already characterized nucleic acid or may be a novel nucleic acid with no known biological function. The Institute for Genomic Research (TIGR) Human Genome Index (HGI) database, which is known and available to those skilled in the art, contains a list of human transcripts. TIGR can be accessed through the world wide web of the Internet, at, for example, tigr.org. Transcripts can be generated in this manner using TIGR- Assembler, an engine to build virtual transcripts and which is known and available to those skilled in the art. TIGR- Assembler is a tool for assembling large sets of overlapping sequence data such as ESTs, BACs, or small genomes, and can be used to assemble eukaryotic or prokaryotic sequences. TIGR-Assembler is described in, for example, Sutton, et ah, Genome Science & Tech., 1995, 1, 9-19, which is incorporated herein by reference in its entirety, and can be accessed through the file transfer program of the Internet, at, for example, tigr.org/pub/software/TIGR. assembler. Li addition, GLAXO-MRC, which is known and available to those skilled in the art, is another protocol for constructing virtual transcripts. PHRAP is used for sequence assembly within Find Neighbors and Assemble EST Blast. PHRAP can be accessed through the world wide web of the Internet, at, for example, chimera.biotech. washington.edu/uwgc/tools/phrap.htm. Identification of ESTs and generation of contiguous ESTs to form full length RNA molecules is described in detail in U.S. application Ser. No. 09/076,440, which is incorporated herein by reference in its entirety.
[0144] hi another preferred embodiment, alternative transcript information could be also retrieved from other gene databases, such as for example, LOCUSLINK, Alternative Splicing Database (ASD), and ASAP database.
Modified Primers
[0145] In another preferred embodiment, primers used in the reaction mixture can comprise modified nucleobases. [0146] The term "succeeding monomer" relates to the neighboring monomer in the 5'- terminal direction and the "preceding monomer" relates to the neighboring monomer in the 3 '-terminal direction.
[0147] Monomers are referred to as being "complementary" if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, pseudoisocytosine with G, etc.
[0148] Preferred oligonucleotides of the invention also may have at least one non- modified nucleic acid located either at or within a distance of no more than three bases from the mismatch position(s) of a complementary oligonucleotide, such as at a distance of two bases from the mismatch position, e.g. at a distance of one base from the mismatch position, e.g. at the mismatch position.
[0149] In accordance with the invention, any desired primer may be used. For example, primers for use in the disclosed amplification method can be oligonucleotides having sequence complementary to the target sequence. This sequence is referred to as the complementary portion of the primer. The complementary portion of a primer can be any length that supports specific and stable hybridization between the primer and the target sequence under the reaction conditions. Generally, for reactions at 37°C, this can be, for example about 5 to about 35 nucleotides long or about 16 to about 24 nucleotides long. If whole genome amplification is desired, the primers can be from about 5 to about 60 nucleotides long, and in particular, can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 nucleotides long.
[0150] In a preferred embodiment, target sequences that are to be amplified and used in the screening of candidate drugs, are of unknown sequence. For example, nucleic acid isolated from a sample for which the sequence is from an individual or any organism. In such cases, primers may be random, or of degenerate sequence (that is, use of a collection of primers having a variety of sequences), primer hybridization need not be specific. In such cases the primers need only be effective in priming synthesis. For example, in whole genome amplification specificity of priming is not essential since the goal generally is to amplify all sequences equally. Sets of random or degenerate primers can comprise primers of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 nucleotides long or more. Primers six nucleotides long are referred to as hexamer primers. For example, preferred primers for whole genome amplification are random hexamer primers. That is, random hexamer primers where every possible six nucleotide sequence is represented in the set of primers. Similarly, sets of random primers of other, particular lengths, or of a mixture of lengths preferably comprise every possible sequence the length of the primer, or, in particular, the length of the complementary portion of the primer. Use of random primers is described in U.S. Pat. Nos. 5,043,272 and 6,214,587 the contents of which are hereby incorporated by reference in their entirety.
[0151] In another preferred embodiment, the primers, can have one or more modified nucleotides. Such primers are referred to herein as modified primers. Modified primers have several advantages. First, some forms of modified primers, such as RNA/2'-O-methyl RNA chimeric primers, have a higher melting temperature (Tm) than DNA primers. This increases the stability of primer hybridization and will increase strand invasion by the primers. This will lead to more efficient priming. Also, since the primers are made of RNA, they will be exonuclease resistant. Such primers, if tagged with minor groove binders at their 5' end, will also have better strand invasion of the template dsDNA. In addition, RNA primers can also be very useful for amplification of nucleic acid molecules from biological samples such as cells or tissue. Since the biological samples contain endogenous RNA, this RNA can be degraded with RNase to generate a pool of random oligomers, which can then be used to prime the polymerase for amplification of the DNA. This eliminates any need to add primers to the reaction. Alternatively, DNase digestion of biological samples can generate a pool of DNA oligonucleotide primers for RNA dependent DNA amplification. [0152] Chimeric primers can also be used. Chimeric primers are primers having at least two types of nucleotides, such as both deoxyribonucleotides and ribonucleotides, ribonucleotides and modified nucleotides, or two different types of modified nucleotides. One form of chimeric primer is peptide nucleic acid/nucleic acid primers (PNA/NAP). For example, 5'-PNA-DNA-3' or 5'-PNA-RNA-3' primers may be used for more efficient strand invasion and polymerization invasion. The DNA and RNA portions of such primers can have random or degenerate sequences. Other forms of chimeric primers are, for example, 5'-(2'-O- Methyl)RNA-RNA-3' or 5'-(2'-O-Methyl)RNA-DNA-3'.
[0153] Many modified nucleotides (nucleotide analogs) are known and can be used in oligonucleotides. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and 2- aminoadenin-9-yl. A modified base includes but is not limited to locked nucleic acids (LNA), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other aikyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, Englisch et al., Angewaήdte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289- 302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Other modified bases are those that function as universal bases. Universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases substitute for the normal bases but have no bias in base pairing. That is, universal bases can base pair with any other base. Primers composed, either in whole or in part, of nucleotides with universal bases are useful for reducing or eliminating amplification bias against repeated sequences in a target sample. This would be useful, for example, where a loss of sequence complexity in the amplified products is undesirable. Base modifications often can be combined with for example a sugar modification, such as 2'-O-methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, which detail and describe a range of base modifications. Each of these patents is herein incorporated by reference. [0154] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxyribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to ClO, alkyl or C2 to ClO alkenyl and alkynyl. 2' sugar modifications also include but are not limited to ~0[(CH2)n0]m CH3, --O(CH2)nOCH3, -0(CHa)nNHi --O(CH2)nCH3, "-0(CH2Jn-ONH2, and -O(CH2)nON[(CH2)nCH3)] 2, where n and m are from 1 to about 10.
[0155] Other modifications at the 2' position include but are not limited to: Cl to ClO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures such as U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.
[0156] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkages between two nucleotides can be through a 3 '-5' linkage or a 2'-5' linkage, and the linkage can comprise inverted polarity such as 3 -5' to 5 -3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;' 5,476,925;' 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference. [0157] It is understood that nucleotide analogs need only comprise a single modification, but may also comprise multiple modifipations within one of the moieties or between different moieties.
[0158] Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize and hybridize to complementary nucleic acids in a Watson-Crick or Hoδgsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
[0159] Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
[0160] It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al, Science 254:1497-1500 (1991)). [0161] Primers can comprise nucleotides and can be made up of different types of nucleotides or the same type of nucleotides. For example, one or more of the nucleotides in a primer can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 10% to about 50% of the nucleotides can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 50% or more of the nucleotides can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; or all of the nucleotides are ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides. The nucleotides can comprise bases (that is, the base portion of the nucleotide) and can (and normally will) comprise different types of bases. For example, one or more of the bases can be universal bases, such as 3-nitropyrrole or 5- nitroindole; about 10% to about 50% of the bases can be universal bases; about 50% or more of the bases can be universal bases; or all of the bases can be universal bases. [0162] In another preferred embodiment, primers with complementary sequences to target nucleic acids are preferred. Primers may also comprise additional sequence at the 5' end of the primer that is not complementary to the target sequence. This sequence is referred to as the non-complementary portion of the primer. The non-complementary portion of the primer, if present, serves to facilitate strand displacement during DNA replication. The non- complementary portion of the primer can also include a functional sequence such as a promoter for an RNA polymerase. The non-complementary portion of a primer may be any length, but is generally about 1 to 100 nucleotides long, and preferably about 4 to 8 nucleotides long. The use of a non-complementary portion is not preferred when random or partially random primers are used for example, in whole genome amplification. [0163] The non-complementary portion of a primer can include sequences to be used to further manipulate or analyze amplified sequences. An example of such a sequence is a detection tag, which is a specific nucleotide sequence present in the non-complementary portion of a primer. Detection tags have sequences complementary to detection probes. Detection tags can be detected using their cognate detection probes. Detection tags become incorporated at the ends of amplified strands. The result is amplified DNA having detection tag sequences that are complementary to the complementary portion of detection probes. If present, there may be one, two, three, or more than three detection tags on a primer. It is preferred that a primer have one, two, three or four detection tags. Most preferably, a primer will have one detection tag. Generally, it is preferred that a primer have 10 detection tags or less. There is no fundamental limit to the number of detection tags that can be present on a primer except trie size of the primer. When there are multiple detection tags, they may have the same sequence or they may have different sequences, with each different sequence complementary to a different detection probe. It is preferred that a primer comprise detection tags that have the same sequence such that they are all complementary to a single detection probe. For some multiplex detection methods, it is preferable that primers comprise up to six detection tags and that the detection tag portions have different sequences such that each of the detection tag portions is complementary to a different detection probe. A similar effect can be achieved by using a set of primers where each has a single different detection tag. The detection tags can each be any length that supports specific and stable hybridization between v the detection tags and the detection probe. For this purpose, a length of about 10 to about 35 nucleotides is preferred, with a detection tag portion about 15 to about 20 nucleotides long being most preferred.
Detection labels for detecting nucleic acid sequences for expression and use in the device [0164] The oligomer can comprise a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand that facilitates the direct of indirect detection of the oligomer or the immobilization of the oligomer onto a solid support. Such groups are typically attached to the oligonucleotide when it is intended as a probe for in situ hybridization, in Southern hybridization, Dot blot hybridization, reverse Dot blot hybridization, or in Northern hybridization.
[0165] When the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand includes a spacer, the spacer may suitably comprise a chemically cleavable group.
[0166] In the present context, the term "photochemically active groups" covers compounds which are able to undergo chemical reactions upon irradiation with light. Illustrative examples of functional groups hereof are quinones, especially 6-methyl-l,4- naphtoquinone, anthraquinone, naphtoquinone, and 1,4-dimethyl-anthraquinone, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds. [0167] In the present context "thermochemically reactive group" is defined as a functional group which is able to undergo thermochemically-induced covalent bond formation with other groups. Illustrative examples of functional parts thermochemically reactive groups are carboxylic acids, carboxylic acid esters such as activated esters, carboxylic acid halides such as acid fluorides, acid chlorides, acid bromide, and acid iodides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.
[0168] In the present context, the term "chelating group" means a molecule that contains more than one binding site and frequently binds to another molecule, atom or ion through more than one binding site at the same time. Examples of functional parts of chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA), aminophosphonic acid, etc.
[0169] In the present context, the term "reporter group" means a group which is detectable either by itself or as a part of an detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g. light or X-rays, of a certain wavelength, and which subsequently reemits the energy absorbed as radiation of longer wavelength; illustrative examples are dansyl (5-dimethylamino)-l-naphthalenesulfonyl), DOXYL (N-oxyl-4,4- dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylρyrrolidine), TEMPO (N-oxyl- 2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems, Inc.), erythrosine, coumaric acid, umbelliferone, Texas red, rhodamine, tetram ethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-l-diazole (NBD), pyrene, fluorescein, Europium, Ruthenium, Samarium, and other rare earth metals), radioisotopic labels, chemiluminescence labels (labels that are detectable via the emission of light during a chemical reaction), spin labels (a free radical {e.g. substituted organic nitroxides) or other paramagnetic probes {e.g. Cu2+, Mg2+) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy), enzymes (such as peroxidases, alkaline phosphatases, β~galactosidases, and glycose oxidases), antigens, antibodies, haptens (groups which are able to combine with an antibody, but which cannot initiate an immune response by itself, such as peptides and steroid hormones), carrier systems for cell membrane penetration such as: fatty acid residues, steroid moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folic acid peptides for specific receptors, groups for mediating endocytose, epidermal growth factor (EGF), bradykinin, and platelet derived growth factor (PDGF). Especially interesting examples are biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium, Cy5, Cy3, etc. [0170] In the present context "ligand" is a molecule, such as an antibody, hormone, or drug, that binds to a receptor. A ligand can comprise a molecule, ion, or atom that is bonded to the central metal atom of a coordination compound. Ligands can comprise functional groups such as: aromatic groups (such as benzene, pyridine, naphthalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, Ci-C2O alkyl groups optionally interrupted or terminated with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulfur atoms, optionally containing aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-α-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides, oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids, and also "affinity ligands", i.e. functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- and oligosaccharides, and other biomolecules.
[0171] It should be understood that the above-mentioned specific examples under DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the "active/functional" part of the groups in question. For the person skilled in the art it is furthermore clear that DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are typically represented in the form M-K- where M is the "active/functional" part of the group in question and where K is a spacer through which the "active/functional" part is attached to the 5- or 6-membered ring. Thus, it should be understood that the group B, in the case where B is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, has the form M-K-, where M is the "active/functional" part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand, respectively, and where K is an optional spacer comprising 1-50 atoms, preferably 1-30 atoms, in particular 1-15 atoms, between the 5- or 6-membered ring and the "active/functional" part. [0172] In the present context, the term "spacer" means a thermochemically and photochemically non-active distance-making group and is used to join two or more different moieties of the types defined above. Spacers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length (e.g. see Hermanson et. al., "Immobilized Affinity Ligand Techniques", Academic Press, San Diego, California (1992), p. 137-ff). Generally, the length of the spacers are less than or about 400 A, in some applications preferably less than 100 A. The spacer, thus, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulfur atoms. Thus, the spacer K may comprise one or more amide, ester, amino, ether, and/or thioether functionalities, and optionally aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-α-alanine, polyglycine, polylysine, and peptides in general, oligosaccharides, oligo/polyphosphates. Moreover the spacer may consist of combined units thereof. The length of the spacer may vary, taking into consideration the desired or necessary positioning and spatial orientation of the "active/functional" part of the group in question in relation to the 5- or 6-membered ring. In particularly interesting embodiments, the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulphide groups cleavable under reductive conditions, peptide fragments cleavable by peptidases, etc. [0173] To further aid in detection and quantitation of nucleic acids amplified using the disclosed method, detection labels can be directly incorporated into amplified nucleic acids or can be coupled to detection molecules. As used herein, a detection label is any molecule that can be associated with amplified nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. Many such labels for incorporation into nucleic acids or coupling to nucleic acid probes are known to those of skill in the art. Examples of detection labels suitable for use in the disclosed method are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands.
[0174] Examples of suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY™, Cascade Blue™, Oregon Green™, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dye™, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Examples of other specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy Fl, Brilliant Sulpho flavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1- Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH--CH3, Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 1OGF, Genacryl Pink 3 G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rliodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodarmine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodanine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC. [0175] Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection. Other examples of fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5l-dichloro-6-carboxyrhodamine (JOE), T- chloro-5'-fluoro-7',8'-fused phenyl- l^-dichloro-ό-carboxyfiuorescein (NED), and 2'-chloro- 7'-phenyl-l,4-dichloro-6-carboxyfiuorescein (VIC). Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ.; Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland, Ohio. [0176] Labeled nucleotides are a preferred form of detection label since they can be directly incorporated into the amplification products during synthesis. Examples of detection labels that can be incorporated into amplified nucleic acids include nucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke, Mutation Research 290:217-230 (1993)), amino allyldeoxyuridine (Henegariu et ah, Nature Biotechnology 18:345-348 (2000)), 5-methylcytosine (Sano et al, Biochim. Biophys. Acta 951:157-165 (1988)), bromouridine (Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al, Proc. Natl. Acad. ScL USA 78:6633 (1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal. Biochem. 205:359-364 (1992)). Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotide analog detection label for DNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma- Aldrich Co). Other preferred nucleotide analogs for incorporation of detection label into DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular Biochemicals). A preferred nucleotide analog for incorporation of detection label into RNA is biotin- 16-UTP (biotin- 16-uridine-5'- triphosphate, Roche Molecular Biochemicals). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling. Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labeled probes. [0177] Detection labels that are incorporated into amplified nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art. For example, biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[l,2,- dioxetane-3-2'-(5'-chloro)tricyclo[3.3.1.1.sup.3,7 ]decane]-4-yl)phenyl phosphate; Tropix, Inc.). Labels can also be enzymes, such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases, that can be detected, for example, with chemical signal amplification or by using a substrate to the enzyme which produces light (for example, a chemiluminescent 1,2-dioxetane substrate) or fluorescent signal.
[0178] Molecules that combine two or more of these detection labels are also considered detection labels. Any of the known detection labels can be used with probes, tags, and method to label and detect nucleic acid amplified using the disclosed method. Methods for detecting and measuring signals generated by detection labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection label coupled to the antibody. As used herein, detection molecules are molecules which interact with amplified nucleic acid and to which one or more detection labels are coupled.
[0179] The methods and/or compositions disclosed herein, can be used to generate amplification libraries for screening and the information derived from such a library can be utilized during the device production. For example, oligonucleotide libraries may be employed as probes in the purification, isolation and detection of for instance pathogenic organisms such as viral, bacteria and fungi etc. Oligonucleotides also may be used as generic tools for the purification, isolation, amplification and detection of nucleic acids from groups of related species such as for instance rRNA from gram-positive or gram negative bacteria, fungi, mammalian cells etc.
[0180] Nucleic Acid Fingerprints: Identified nucleic acid sequences can be used to produce replicated strands that serve as a nucleic acid fingerprint of a complex sample of nucleic acid. Such a nucleic acid fingerprint can be compared with other, similarly prepared nucleic acid fingerprints of other nucleic acid samples to allow convenient detection of differences between the samples. The nucleic acid fingerprints can be used both for detection of related nucleic acid samples and comparison of nucleic acid samples. For example, the presence or identity of specific genes can be detected by producing a nucleic acid fingerprint of the test organism and comparing the resulting nucleic acid fingerprint with reference nucleic acid fingerprints prepared from known organisms. Changes and differences in gene expression patterns can also be detected by preparing nucleic acid fingerprints of mRNA from different cell samples and comparing the nucleic acid fingerprints. The replicated strands can also be used to produce a set of probes or primers that is specific for the source of a nucleic acid sample. The replicated strands can also be used as a library of nucleic acid sequences present in a sample. Nucleic acid fingerprints can be made up of, or derived from, for example, whole genome amplification of a sample such that the entire relevant nucleic acid content of the sample is substantially represented, or from multiple strand displacement amplification of selected target sequences within a sample.
[0181] Nucleic acid fingerprints can be stored or archived for later use. For example, replicated strands produced in the disclosed method can be physically stored, either in solution, frozen, or attached or adhered to a solid-state substrate such as an array. Storage in an array is useful for providing an archived probe set derived from the nucleic acids in any sample of interest. As another example, informational content of, or derived from, nucleic acid fingerprints can also be stored. Such information can be stored, for example, in or as computer readable media. Examples of informational content of nucleic acid fingerprints include nucleic acid sequence information (complete or partial); differential nucleic acid sequence information such as sequences present in one sample but not another; hybridization patterns of replicated strands to, for example, nucleic acid arrays, sets, chips, or other replicated strands. Numerous other data that is or can be derived from nucleic acid fingerprints and replicated strands produced in the disclosed method can also be collected, used, saved, stored, and/or archived.
[0182] Nucleic acid fingerprints can also comprise or be made up of other information derived from the information generated in the disclosed method, and can be combined with information obtained or generated from any other source. The informational nature of nucleic acid fingerprints produced using the disclosed method lends itself to combination and/or analysis using known bioinformatics systems and methods. [0183] Nucleic acid fingerprints of nucleic acid samples can be compared to a similar nucleic acid fingerprint derived from any other sample to detect similarities and differences in the samples (which is indicative of similarities and differences in the nucleic acids in the samples). For example, a nucleic acid fingerprint of a first nucleic acid sample can be compared to a nucleic acid fingerprint of a sample from the same type of organism as the first nucleic acid sample, a sample from the same type of tissue as the first nucleic acid sample, a sample from the same organism as the first nucleic acid sample, a sample obtained from the same source but at time different from that of the first nucleic acid sample, a sample from an organism different from that of the first nucleic acid sample, a sample from a type of tissue different from that of the first nucleic acid sample, a sample from a strain of organism different from that of the first nucleic acid sample, a sample from a species of organism different from that of the first nucleic acid sample, or a sample from a type of organism different from that of the first nucleic acid sample.
[0184] The same type of tissue is tissue of the same type such as liver tissue, muscle tissue, or skin (which may be from the same or a different organism or type of organism). The same organism refers to the same individual, animal, or cell. For example, two samples taken from a patient are from the same organism. The same source is similar but broader, referring to samples from, for example, the. same organism, the same tissue from the same organism, the same DNA molecule, or the same DNA library. Samples from the same source that are to be compared can be collected at different times (thus allowing for potential changes over time to be detected). This is especially useful when the effect of a treatment or change in condition is to be assessed. Samples from the same source that have undergone different treatments can also be collected and compared using the disclosed method. A different organism refers to a different individual organism, such as a different patient, a different individual animal, different mono-cellular or multi-cellular organisms . Different organism includes a different organism of the same type or organisms of different types. A different type of organism refers to organisms of different types such as a dog and cat, a human and a mouse, or bacteria such as E. coli and Salmonella. A different type of tissue refers to tissues of different types such as liver and kidney, or skin and brain. A different strain or species of organism refers to organisms differing in their species or strain designation as those terms are understood in the art.
Evaluation of Expression Patterns
[0185] Expression patterns can be evaluated by qualitative and/or quantitative measures.
Certain techniques for evaluating protein expression (protein products) yield data that are predominantly qualitative in nature. That is, the methods detect differences in expression that classify expression into distinct modes without providing significant information regarding quantitative aspects of expression. For example, a technique can be described as a qualitative technique if it detects the presence or absence of expression of a protein, i.e., as measured by detectable labels.
[0186] hi contrast, some methods provide data that characterize expression in a quantitative manner. That is, the methods relate expression on a numerical scale, e.g., a scale of 0-5, a scale of 1-10, a scale of +-+++, from grade 1 to grade 5, a grade from a to z, or the like. It will be understood that the numerical, and symbolic examples provided are arbitrary, and that any graduated scale (or any symbolic representation of a graduated scale) can be employed in the context of the present invention to describe quantitative differences in nucleotide sequence expression. Typically, such methods yield information corresponding to a relative increase or decrease in expression.
[0187] Any method that yields either quantitative or qualitative expression data is suitable for evaluating expression of protein in the presence or absence of a candidate drug. In some cases, e.g., when multiple methods are employed to determine expression patterns for a plurality of candidate drugs, the recovered data, e.g., the expression profile is a combination of quantitative and qualitative data.
Analysis of Expression Profiles
[0188] hi order to facilitate ready access, e.g., for comparison, review, recovery, and/or modification, the expression profiles are typically recorded in a database. Most typically, the database is a relational database accessible by a computational device, although other formats, e.g., manually accessible indexed files of expression profiles as photographs, analogue or digital imaging readouts, spreadsheets, etc. can be used. Regardless of whether the expression patterns initially recorded are analog or digital in nature and/or whether they represent quantitative or qualitative differences in expression, the expression patterns, expression profiles (collective expression patterns), and molecular signatures (correlated expression patterns) are stored digitally and accessed via a database. Typically, the database is compiled and maintained at a central facility, with access being available locally and/or remotely.
[0189] As additional samples are obtained, and their expression profiles determined and correlated with relevant subject data, the ensuing molecular signatures are likewise recorded in the database. However, rather than each subsequent addition being added in an essentially passive manner in which the data from one sample has little relation to data from a second (prior or subsequent) sample, the algorithms optionally additionally query additional samples against the existing database to further refine the association between a molecular signature and disease criterion. Furthermore, the data set comprising the one (or more) molecular signatures is optionally queried against an expanding set of additional or other disease criteria. [0190] The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. [0191] All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.
EXAMPLES
Reagents and Materials.
[0192] The RTS 100 E. coli HY kit, two expression vectors containing the genes encoding Green Fluorescent Protein (GFP) and chloramphenicol acetyl-transferase (CAT), and anti-6xHis were obtained form Roche Diagnostics GmbH (Mannheim, Germany). TNT Quick Coupled Transcription/Translation system, T7 luciferase DNA vector, luciferase assay reagent, and nuclease- free water were from Promega Corporation (Madison, WI). Acrylamide-bisacrylamide (electrophoretic grade, 5% C), tetramethylethylenediamine, sodium dodecyl sulfate (SDS), ammonium persulfate, tris(hydroxymethyl)aminomethane (Tris), glycine, sodium chloride, glycerol, bromophenol blue, β-mercaptoethanol, Tween-20, tetracycline, and cycloheximide were purchased from Fisher Scientific (Atlanta, GA). Polyvinylidene difluoride (PVDF) membranes (0.2 μm), and filter papers were from Bio-Rad Laboratories (Hercule, CA). Molecular weight standards, biotinylated secondary antibody and streptavidin-alkaline phosphatase were from Amersham Biosciences (Piscataway, NJ) while recombinant Green Fluorescent Protein (rGFP) and rabbit anti-GFP polyclonal antibody were from BD Biosciences (Palo Alto, CA). The phosphatase staining solution (Bromo-chloro-indoryl phosphate/Nitro Blue Tetrazolium, BCIP/NBT) was obtained from KPL (Gaithersburg, MD). Protein Expression.
[0193] For the prokaryotic expression system, 50 μL of RTS 100 reaction solution was composed of 12 μL E. coli lysate, 10 μL reaction mix (proprietary composition, supplied in the kit by the manufacturer), 12 μL amino acids without methionine, 1 μL methionine, 5 μL reconstitution buffer (proprietary composition, supplied in the kit by the manufacturer), and 10 μL nuclease-free water containing 1 μg GFP or CAT vector. The reaction solution was then incubated in a microcentrifuge tube at 3O0C for 4 hours. For GFP, the reaction solution was stored at 4 0C for additional 24 hours for the maturation of GFP. [0194] For the eukaryotic expression system, rabbit reticulocyte lysate was used. The reaction mix of 50 μL for luciferase synthesis was prepared by combining 40 μL TNT Quick master mix (proprietary composition, supplied in the kit by the manufacturer), 1 μL methionine, and 9 μL nuclease-free water containing 1 μg luciferase vector. Incubation was performed in a microcentrifuge tube at 30 °C for 1.5 hours.
[0195] For the toxin inhibition assay in a microcentrifuge tube, a stock solution of tetracycline and that of cycloheximide were prepared at 15 μg/μL and 10 μg/μL, respectively. A series of amounts of tetracycline or cycloheximide were added into protein expression mixture. The concentrations of toxins used are listed in the figures or text. To save reagents and match with miniaturized devices, 8 μL of prokaryotic or eukaryotic expression solution was used, making the total volume of each inhibition assay at 10 μL. For each set of experiments, a positive control (without inhibitor) and a negative control (without the expression vector) have also been included.
[0196] When the protein expression and toxin inhibition assays were implemented in the miniaturized device, the volume of the reaction mixture was reduced to 6.5 μL. Firms with pressure-sensitive adhesive, so-called "PCR tape" (3M, Minneapolis, MN), were used to seal the wells to prevent evaporation.
Device Fabrication.
[0197] A miniaturized device with an array of 2x3 wells was designed and fabricated for demonstrating toxin detection. A part of the device, is shown in Figure IA and IB, form an array of 3x4 wells, which is the format used for demonstrating toxin detection as discussed below. The device was made from acrylic (Lucite International, Cordova, TN) and the wells were created by a milling machine (Flashcut CNC, Menlo Park, CA). The distances between wells (center to center) are 9 mm, matching the standards for 96-well microplates defined by the Society for Biomolecular Screening and accepted by the American National Standards Institute; this arrangement insures compatibility with a variety of commercial fluid dispensing systems and plate readers. The diameter and depth of each well are 2.7 mm and 2.3 mm, respectively, providing the total well volume of -13 μL. This is about 25 times smaller than the wells in conventional 96-well microplates. The decrease in the well size will significantly reduce reagent consumption for high-throughput assays. The size of the well is also in agreement with our goal to integrated microfiuidic components. After fabrication, the device was sterilized by exposing to UV light for 30 minutes that ensured the consistency of the protein expression.
Detection.
[0198] Either Western blot or luminescence was used for measuring the yield of protein expression, depending on the property of the proteins expressed. Detection of expressed GFP and CAT was achieved using Western blot. A reaction product solution of 1 μL was mixed with 15 μL of gel-loading sample buffer, which contains 50 niM Tiis-HCl, pH 6.8, 2% W/V SDS, 0.01% W/V bromophenol blue, 10% V/V glycerol, and 5% V/V 2-mercaρtoethanol. The mixture was then separated in a 15% SDS-polyacrylamide gel in the Mini-Protean III Cell system (BioRad). After electrophoresis, the gel was removed from the glass plates and then equilibrated in the transfer buffer, which is comprised of 48 mM Tris, 39 mM glycine, and 20% V/V methanol. PVDF membrane was pre-soaked with methanol, followed by soaking in the transfer buffer for 1 hour. The Mini Trans-Blot system (BioRad) was set up with pre-wetted fiber pad, filter paper, gel, and PVDF membrane according to the instruction from the manufacturer. The cassette and ice-cooling unit were placed in the tank that was filled with the transfer buffer.
[0199] After blotting, the PVDF membrane were removed from the trans-blot apparatus and blocked with 5% w/v non-fat dried milk in Tris-buffered saline (TBS) solution (with 0.05% Tween-20) for 1 hour at room temperature. After being washed three times (5 minutes each time) with TBS solution, the membrane was incubated for 1 hour at room temperature with 1 μg/mL anti-GFP polyclonal antibody for GFP product or 0.3 μg/mL anti-6xHis monoclonal antibody for CAT product, respectively. At the end of conjugation, the membrane was washed three times and then incubated with 1.5 μg/mL biotinylated secondary antibody at room temperature for 1 hour. Upon completion of the incubation, the membrane was rinsed again with TBS solution for three times, followed by incubation at ambient temperature for 30 minutes with a solution of streptavidin-alkaline phosphatase (1:2000 dilution from the stock solution). After being washed, the membrane was immersed in chromogenic substrate (BCIP/NBT) for 3 minutes, followed by rinsing with water (to stop reaction). Images of protein bands were acquired with a color laser scanner (Canon); proteins bands were quantified using Image J from the National Institute of Health (Image J is developed on Mac OS X using its built in editor and Java compiler, plus the BBEdit editor and the Ant build tool. The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA. http://rsb.info.nih.gov/ij).
[0200] Detection of luciferase expressed by IVT was achieved by SIRIUS luminometer from Berthold (Pforzheim, Germany). The luminometer was programmed to have a two- second delay, followed by a five-second measurement of luciferase activity. The expression product of 2 μL was added to a luminometer tube containing 40 μL of luciferase assay reagent and mixed evenly. The tube was then placed in the luminometer; and the data were acquired.
Example 1: Device Design.
[0201] A variety of distinct components are integrated into the micro fluidic device to meet the requirement of protein expression. The requirements include nutrient supplies, byproduct removal, evaporation control, and fluid manipulation. The device comprises an array of units; each unit consists of a reaction chamber, two feeding chambers, and a channel to connect them as shown hi Figure 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs. The feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis. The reaction chamber contains cell-free expression system with other reagents as in the feeding chambers. The channel connected to the chambers provides a means to supply nutrients and remove byproducts. The selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at -10 KDa, which at the same time allows all small molecules in the feeding chamber passing by.
[0202] The incorporation of membrane is important because of two facts: (1) the flow of a feeding solution leads to higher expression yield compared to static conditions, because protein synthesis will not terminate earlier due to fast depletion of energy sources (ATP and GTP); (2) removal of small molecule byproducts is also very important for the high yield expression of proteins in a cell-free medium. Inhibition of protein synthesis due to the small molecular byproducts such as hydrolysis products of triphosphates is eliminated. [0203] The device possesses appropriate geometric configuration to achieve desired properties for in vitro protein expression. The dimension of the microchannel is in the range of 1 micron to 1 centimeter, depending on the flow rate required for supplying nutrients and removing byproduct. The feeding chambers must be sufficiently large compared to the reaction chamber so that they function as reservoirs. In the current design, the volume of the reaction chamber is 10 nanoliter-100 microliter μL whereas that of the feed chambers is about 100 nano liter- 1 mililiter.
[0204] One of the mechanisms for supplying nutrients from the feeding chambers to the reaction chamber and for removing byproducts from the reaction chamber to the feeding chambers is osmosis, a net flow resulting from the difference in chemical potential of solutes between two solutions separated by the membrane. The degree of the potential difference is determined by their difference in solute activities, which are correlated with solute concentrations. For example, the osmotic pressure, FI, resulting from a byproduct in the reaction chamber can be approximately calculated by Van't Hoff s Law assuming it is in the limit of infinite dilution (Levine, 1988).
U = CbRT
[0205] where Cb is the concentration of the byproduct, R is gas constant, and T is the temperature. The capillary force due to surface tension is expected to play a minor role due to similar surface property in chambers.
[0206] The exchange rate of chemicals between two sides of the membrane increases with the concentration gradient along the membrane. The exchange stops when equilibrium reaches. To increase the exchange rate, an active flow in channel is needed to supply fresh solution underneath the membrane. One approach is to use hydrostatic pressure, which results from the difference in the solution level between the feeding chamber and reaction chamber. When the feeding chamber has slightly higher solution level than the reaction chamber, the pressure difference, ΔP, can be defined by the difference in height, Δh, as follows:
ΔP = pgΔ/z [0207] where p is the density of solution and g is gravitational acceleration. The volume flow rate in the channel, Q, can be calculated using the equation below based on a fully developed flow in the channel.
Q = (πΔpD4)/128μL
[0208] where D is the channel diameter, μ is viscosity, and L is the distance between chambers. Using Δh = 1 mm, D = 50 μm, L= 3 mm, and water's density and viscosity, the velocity is calculated to be 0.50 nL/s. At this speed, the amount of reagent in the feeding chamber will last for many hours, which is longer than the reaction time needed for completing protein synthesis in a microdevice.
[0209] Mixing primarily results from the diffusion unless other means are to be used for creating flows. As discussed above, the size of the reaction chamber is in mm-scale. The time needed for a molecule to diffuse 1 mm is in seconds, which is much shorter than the time used for protein synthesis. (The diffusion time was estimated by using a diffusion coefficient of 5 x 10"4 cm2/s and the law x2 = 2Dt, where x is the distance, D is the diffusion coefficient, and t is the time).
[0210] Clearly, the factors affecting the fluid manipulation, nutrient supply, and byproduct removal will have effects on the yield of protein expression, which directly influences the detection sensitivity. Computational techniques are employed to model fluid manipulation for the optimum conditions. A commercial simulation software package CFO- ACE is used to model the fluids in the device. It is envisioned that geometric variables (e.g., length and height of each channel), material variables (e.g., fluid viscosity), and operating variables (e.g., displacement volume and flow rate) are to be included in modeling. Information from both simulation and actual testing is utilized to illuminate possible improvement and trade-off among the various geometric, physical, and operational variables. The data is fed back to the device design to optimize the protein expression. [0211] Long-Term Goals: These include development of a portable plastic microfluidic device that is capable of detecting both known and unknown toxins. After demonstration of the sensor array based on inhibition of protein synthesis, the array device is expanded to detect toxins with different modes of action. In addition to protein synthesis inhibition, there are other unique mechanisms of toxicity. Examples include those toxins affecting nerve system (e.g., botulinum inhibits the release of neurotransmitter) and those affecting gastrointestinal tract (e.g., cholera toxin activates the adenylate cyclase enzyme in cells of the intestinal mucosa). Accordingly, based on the mechanisms of toxicity, appropriate array format will be developed for the detection and identification of these types of toxins. [0212] Pattern recognition and database: After establishing a baseline for toxin simulants and ricin, the response pattern of other toxins can be examined. In addition, a database of response patterns of known toxins can be established, so that comparisons of the pattern of a sample with the database can be made. The pattern recognition algorithms and other features that have been developed for the electronic nose (Dutta et al., (2002) Bacteria classification using Cyranose 320 electronic nose. BiomedEng Online, 1, 4) are expected to be useful to the toxin sensor array.
[0213] Real-world applications: There are many elements related to actual use of the sensor in the field, including air sampling and delivery, interface to liquid/solid samples, and approach to deal with sample mixtures. Several logics are pointed out as follows. First, some of toxin samples are liquids (e.g., water) while others are in gas (aerosols). Collection of aerosols and then subjecting the sample into fluid-based testing is currently practiced in commercial instruments for biodefense (Belgrader et ah, (2001) Anal Chem, 73, 286-289). As a result, we expect the similar approach can be used for the device of this invention. Second, the storage with cell-free reagents in the system of the invention is likely less challenging than the microbial sensors (Riedel et al., (2002) Adv Biochem Eng Biotechnol, 75, 81-118; Shetty et al, (2004) Biotechnology and Bioengineering, 88, 664-670) using live cells. Nevertheless, using biological agents such as enzymes is still a concern in terms of shelf-life. We believe, however, the concern can be addressed by formulation, evidenced from many commercial products such as glucose sensors (that use an enzyme) and pregnancy test kits (that use an antibody). Third, other issues related to real-world application could be addressed in a way similar to the electronic nose, which is commercially available and also based on sensor array concept as mentioned above (Dutta et al., 2002).
Example 2: Toxin Detection Scheme.
[0214] As discussed above, detection of unknown or engineered agents is important due to the ease of the recombinant technology. Multiplexed sensor array is a unique approach to obtain the fingerprint of a new agent. Figures IA and IB shows an illustrative example of a sensor array for detecting toxins using IVT. The device consists of an array of IVT wells; each well is designed to express one protein and thus functions as a sensor. The number of IVT sensors can be as high as 96 or its integral multiples, in the format of traditional microplates and can be easily adapted to commercial plate readers. Multiple wells (e.g., 3x4 wells circumscribed within the dashed lines and shadowed with diagonal lines) form one set, in which the top row is for the positive controls to express each of 3 proteins, the second row for the negative controls, and the third and fourth rows are for the sample, allowing one repeat to enhance the precision. Use of the positive and negative controls and comparison of the signal from the sample wells with those in the control wells reduce false positives and negatives. The set expresses a group (three in this case) of pre-characterized proteins in different expression systems; the proteins and expression systems are judiciously selected so that protein synthesis in each well is inhibited or affected differentially by different type of toxins. Therefore the unique response pattern (or signature) of a toxin due to different inhibitory effects can be registered and used as a tool for detection and identification. New agents can be identified by comparing the response pattern with signatures of known agents in a pre-acquired database. In the particular format illustrated, the rest of wells in the 96- well array can be designed to detect seven additional types of toxins if the 12-well set is proved to be enough for identification.
Example 3: Protein Expression.
[0215] To demonstrate toxin detection by protein expression, three proteins were synthesized in two types of expression systems. The first protein is Green Fluorescent Protein (GFP), a widely-used fluorescent molecule with known DNA sequence and crystal structure. Protein expression was carried out by using an expression vector as a DNA template, which consists of GFP coding sequence and the necessary regulatory elements including T7-RNA polymerase promoter, ribosome binding site, start codon, stop codon, and T7 terminator. The expression vector was mixed with E. coli lysate and a reaction mix consisting of T7-RNA polymerase, nucleotides, amino acids, and other reagents. GFP product was confirmed by fluorescence spectrometry and Western blotting. The result of Western blotting is shown in Figure 3 A. A clear band in lane 4 indicates the presence of GFP in the expression product. According to pre-stained protein markers, the molecular weight of GFP expressed is estimated ~31 KD. Expressed GFP contains a stretch of additional six histidines (6xHis) at its C-terminal, causing its molecular weight slightly larger than recombinant GFP (rGFP) purchased commercially. The negative control in the experiment contains all reagents except for the expression vector.
[0216] The second protein is chloramphenicol acetyl-transferase (CAT), an enzyme responsible for bacterial resistance to an antibiotic drug, chloramphenicol. CAT was expressed in the same E. coli expression system; success of the protein expression was also confirmed using Western blot as shown in Figure 3B. According to pre-stained protein markers, the molecular weight of CAT expressed is estimated -26 KD. The third protein is luciferase, an enzyme from firefly tails that catalyzes the production of light in the presence of luciferin, adenosine triphosphate (ATP), Mg2+, and oxygen. Synthesis of luciferase was carried out using rabbit reticulocyte expression system as described in the experimental section. Detection of the expression product was achieved by monitoring the intensity of luminescence after mixing 2 μL of the product with the assay reagent. As shown in Figure 3 C, the luminescence signal of the product is 5 orders of magnitude higher than that of the negative control.
[0217] Many other proteins may also be produced using in vitro transcription/translation. The examples include dihydrofolate reductase, interleukins, erythropoietin, and phosphoserine phosphatase. More than forty proteins with a variety of biological functions have been successfully synthesized using IVT.
Example 4: Inhibitory Effects of Toxins on Protein Synthesis.
[0218] To illustrate the detection of toxins, tetracycline (TC) and cycloheximide (CH) were used as toxin simulants to study their inhibitory effects on protein expression. TC is an antibiotic substance produced by Streptomyces species. It acts only on prokaryotic cells and it blocks binding of aminoaceyl-transfer RNA to A-site of ribosomes. CH acts specifically on eukaryotic cells and it inhibits the activity of peptidyl transferase, an enzyme needed in the translocation reaction on ribosomes. Figures 4A-4G shows the effects of a series of concentrations of TC or CH on the expression yields of GFP, CAT, and luciferase synthesized in two protein expression systems. As illustrated in Figure 4 A, GFP synthesis was completely inhibited when 3000 ng/μL of TC was used. Partial inhibition was observed when a series of lower concentrations (300 ng/μL to 0.3 ng/μL) of TC was added. The experiment also included the positive control, in which no inhibitor (TC) was added. The negative control contained no expression vector, thus representing the background signal. These results suggest that a qualitative and quantitative relationship exists between the expression yield and toxin amount, a critical figure of merit for a sensor. [0219] The comparison between TC and CH for GFP production in the E. coli expression system is shown in Figures 4A and 4C. The expression yield for each protein was normalized against the expression yield of the positive control (without toxin), so that it is easier to compare the toxic effects. The results clearly indicate that TC has inhibitory effect on GFP production and the degree of inhibition is proportional to the amount of TC in the sample, whereas CH has a negligible effect on the yield of GFP production and the level of minor inhibition remained the same in the range of amount of TC we used. This result suggests that the differential inhibitory effects of a toxin on the expression of different proteins are possibly used for toxin detection.
[0220] Figures 4D and 4E exhibit similar disparity between TC and CH for CAT production in the E. coli expression system. Again, TC has inhibitory effect on CAT production and the degree of inhibition is proportional to the amount of TC in the sample, whereas CH has a negligible effect on the yield of CAT production and the level of minor inhibition remained essentially same in the range of amount of TC we used. Furthermore, comparison of Figure 4B and 4D indicates that although TC has inhibitory effect on both GFP and CAT production, the degree of inhibition per unit amount of TC differs between these two proteins, evident from the difference in the slopes of respective linear regression lines. These results further suggest that each toxin's differential inhibitory effects on expression of different protein can be used as a signature for toxin detection and identification.
[0221] The comparison between TC and CH for luciferase production in rabbit reticulocyte expression system is shown in Figure 4F and 4G. An opposite effect was observed; TC has a negligible effect on the luciferase production in the eukaryotic expression system, whereas CH has a significant inhibitory effect and the degree of inhibition is proportional to the amount of CH present in the sample. The result indicates that different expression systems can be used to expand the variability, so that a unique response pattern can be obtained for toxin detection by using a set of proteins produced in different expression systems. Another example of the protein expression systems is wheat germ extract. [0222] These results are significant because they indicate not only the feasibility for toxin detection, but also the possibility of using IVT assay for high-throughput screening of drug candidates. We confirmed differential inhibitory effects of antibiotic substances such as TC and CH on protein expression in vitro, in a way very similar to their effects on protein expression in vivo. Therefore, an IVT array device may provide a great platform for searching for the best drug candidates.
Example 5: Miniaturized IVT Array.
[0223] After the feasibility of toxin detection using protein expression is demonstrated in microcentrifuge tubes, IVT and toxin detection were determined in a miniaturized well device. The design of the experiments was a part of the 96-well array in Figure 2, in which a set of 3x4 wells is assigned for detecting one toxin at a time. The first row of 3 wells was used as the positive control, expressing GFP and CAT vector in the E. coli expression system and luciferase vector in the rabbit reticulocyte expression system. These wells were free of toxins. The second row of 3 wells was used as the negative control without DNA vectors added. The third and forth rows of 3 wells were added with a certain amount of a toxin stimulant, either CH or TC, into the protein expression system. Figure 5A shows the response pattern of the IVT sensor array when 25 ng of TC was used whereas the response pattern of the same IVT array for 17 ng of CH is illustrated in Figure 5B. Although there is slight difference between two sample repeats for rows 3 and 4 for each toxin, the response pattern is reproducible as expected. The significant difference in the response patterns between CH and TC clearly indicates that it is feasible to use IVT sensor array to detect and identify toxins.
[0224] Although Western blot and luminescence detection was used to monitor protein production, it is feasible to use a uniformed method for the detection. One of such methods is to use GFP as an indicator for the detection of protein expression due to its green fluorescence. GFP has been used for visualization, tracking, and quantification of a variety of proteins in cells after they are fused together. An increase of fluorescence signal in an IVT well indicates the production of GFP or GFP-fused proteins. Quantitative information may be obtained by comparing the fluorescence signals of sample wells and of reference wells, which include both positive and negative controls in the array device. Any variation or adverse effects are cancelled out between control and sample wells. The magnitude of the signal can be correlated to the amount of proteins produced in the device. Indeed, a fluorescence spectrometer was used to confirm the production of GFP expressed as mentioned above. CAT and luciferase are to be expressed in the form of GFP fusions. [0225] In addition to GFP, the expression vector containing a coding sequence for expressing an additional stretch of six histidines (6xHis) at the C-terminal of the protein of interest is to be designed. Many proteins produced by recombinant techniques are designed to contain a 6xHis tag, so that they can be purified through interactions between 6xHis tags and Ni-nitrilotriacetate chromatographic columns. Both GFP and CAT proteins produced here contain 6xHis tag, even though this purification step was not needed. A variety of biological assays are available for detecting the amount of proteins fused with a 6xHis tag. There are many other tags that may be fused with proteins as reviewed in the literature. In addition, luminescence detection can also be used by fusing luciferase with proteins of interest, as demonstrated in the present report.
[0226] A novel concept for toxin detection is presented based on toxin's inhibition of biological protein synthesis in the step of either DNA transcription or protein translation. This was demonstrated t by (1) in vitro expression of three proteins, including Green Fluorescent Protein, chloramphenicol acetyl-transferase, and luciferase; (2) confirming differential inhibitory effects of two toxin simulants, tetracycline and cycloheximide, on the expression yields of these proteins in either prokaryotic or eukaryotic expression system; (3) obtaining unique response pattern (or signature) of the 3x4 IVT array device for each toxin simulant. Such a sensor array is useful in the situations where one type of toxins is suspected.
[0227] Development of an IVT device that comprises micro fluidic elements, offers a means to supply nutrients continuously and to remove byproducts of protein synthesis. The experimental results in the bench-top scale suggest that high-yield protein expression can be attained in the flow of a feeding solution. Removal of small molecule byproducts (e.g., hydrolysis products of triphosphates) prevents protein expression from possible inhibition, leading to higher expression yield in a cell-free medium. Microfluidic manipulation enhances protein expression yield and accordingly increase toxin detection sensitivity. [0228] In addition, micro fluidics may also reduce the response time of the sensor array, which is limited by the time needed for protein production. Currently, both GFP and CAT require 4 hours to produce a detectable amount of proteins whereas luciferase needs 1.5 hours. Extremely high surface-to-volume ratio in a microfluidic device allows for efficient interactions, leading to rapid chemical reactions. Currently, there is no other viable alternative approach to the detection of new toxins.
[0229] The device is also important for high-throughput screening of drug candidates or enzymes. The fact that tetracycline is an antibiotic drug used clinically suggests that the IVT array device provides a nice platform for searching for the candidates that have maximum effects on prokaryotic microorganisms but least effects on eukaryotic cells. In addition, such an IVT array device is useful for studying potential drug candidates through enzyme inhibition assays.
Example 6: Ricin Detection By Biological Signal Amplification in a Solution Array [0230] Reagents and Materials: The RTS 100 wheat germ CECF kit, RTS 500 E. coli kit, and the expression vector containing the gene of green fluorescent protein (GFP) were obtained from Roche Diagnostics GmbH (Mannheim, Germany). T7 luciferase DNA vector, luciferase assay reagent, and nuclease-free water were acquired from Promega Corporation (Madison, WI). Ricin and ricin B chain were purchased from Vector Labs (Burlingame, CA) while ricin A chain and 2-mecaptoethanol were from Sigma (St. Louis, MO). Acrylic sheets with thickness ot'0.25 inches (6.3 mm) and 0.10 inch (2.5 mm) were from Lucite-ES (Lucite International, Inc., Cordova, TN). The dialysis membrane with the molecular weight cutoff of 8 KDa was obtained from Spectrum Labs (Rancho Dominguez, CA), while a biocompatible epoxy (353ND-T) was bought from Epoxy Technologies (Billerica, MA). [0231] Device Fabrication. The design of the array device is shown in Figures IA and IB; it consists of two parts. The top part, tray, was fabricated by drilling an array of holes in a 2.5 mm-thick acrylic sheet. The diameter of the hole is 3 mm. The pitch (the distance between the hole centers) is 9 mm, following the microplate standards defined by the Society for Biomolecular Screening (SBS) and accepted by the American National Standards Institute. The sheet with holes is further milled from the bottom side to create a flange using a CNC-mill (Flashcut 2100, Menlo Park, CA), resulting in a 1 mm-thick wall for the tray chamber. The dialysis membrane was then glued using the epoxy to the bottom of each hole to form the tray chamber. The bottom part, well, was created by milling an array of 4 mm deep wells into a piece of a 6.3 mm-thick acrylic sheet. The diameter of the wells is 7 mm; each well is concentric with the corresponding tray chamber when they are assembled. Both tray and well plates were sterilized by exposing to a UV light for 30 minutes. [0232] Protein Expression: Luciferase was synthesized using RTS 100 wheat germ expression kit. The reaction solution for trays was prepared by mixing 15 μL wheat germ extract, 15 μL reaction mix (provided in the kit), 4 μL amino acids, 1 μL methionine and 15 μL of nuclease-free water containing 1 μg of luciferase vector. The vector can be either circular plasmid vector or a linear vector created by polymerase chain reaction; both vectors were purchased from Promega. For each tray chamber, 8 μL of the reaction solution was used. The feeding solution for wells was prepared by combining 900 μL feeding mix (provided in the kit), 80 μL amino acid and 20 μL methionine. In each well, 80 μL of the feeding solution was introduced. The tray and well plates were assembled and then placed on a shaker (at room temperature) for a period of time (e.g., 0.5 hour). The amount of luciferase synthesized was determined by mixing the expression product with luciferase assay reagents, followed by luminescence detection in a luminometer (Berthold, Germany), as described previously (Mei, Q.; Fredrickson, C. K.; Jin, S.; Fan, Z. H. Anal Chem. 2005, 77, 5494-5500). When luciferase was synthesized in a microcentrifuge tube, the same reaction solution (8 μL) was used without the feeding solution. Expression of green fluorescent protein (GFP) was carried out in the E. coli expression mixture; the expression yield was quantified by Western blotting (Mei, Q.; et al, Anal Chem 2005, 77, 5494-5500). [0233] Ricin Detection: A series of concentrations of ricin A chain solutions, ranging from 0.001 ng/μL to 0.02 ng/μL, were prepared from a stock solution of 1 μg/μL. To demonstrate ricin detection, 6 μL of the reaction solution (discussed above) is pipetted into the tray, followed by 2 μL of ricin samples. The volume of the feeding solution remained at 80 μL. For the positive controls in the same device, 2 μL of water was added. The negative controls contain no luciferase vector, providing with the background signal. To achieve lower detection limit, 4-hr protein expression was used, though ricin detection can be achieved in as short as 5 minutes.
[0234] The same protocol was used in studying the toxicity level of various forms of ricin, including whole ricin, ricin A chain, ricin B chain, heat-denatured ricin A chain, and whole ricin treated with 2-mercaptoethanol. Denature of Ricin A chain was achieved by heating the samples at 95°C for 5 minutes. Reduction of disulfide bond of two chains in ricin was carried out by mixing ricin with 50 mM 2-mercaptoethanol, followed by the incubation of the mixture at 52°C for 10 minutes.
[0235] Protein Expression Array: As mentioned above, protein expression can be produced in a cell-free medium employing IVT. We fabricated an array device consisting of a mechanism for fluid manipulation; it also has a potential to implement protein synthesis in a high-throughput format due to miniaturization. Miniaturization also results in reduction of the reagent consumption by more than 2 orders of magnitude. As illustrated in Figures IA and IB, IYT was implemented in an array of units; each unit is for expression of one protein (e.g., luciferase). The units on the left of the array are for the positive controls (free of ricin), the units in the middle for the negative controls (no DNA vectors), and the units on the right are for samples. Two rows of each unit are for repeat experiments to enhance the precision. The positive and negative controls are used for quantification as well as for reducing false positives and negatives for toxin detection as discussed later on. The solution array is designed to conform with 96-well microplates, ensuring compatibility with a variety of commercial fluid dispensing systems and commercial plate readers for detection. [0236] Each unit in the device consists of a tray and a well (Figure IB and Figure 2). The tray chamber is for the IVT reaction; the well is concentric with the corresponding tray chamber and functions as a nutrient reservoir. The well contains amino acids, adenosine triphosphate (ATP), and other reagents. The tray contains the cell-free expression mixture extracted from wheat germs, as well as the same reagents in the well. A dialysis membrane is glued to the bottom of the tray, connecting the tray and well and providing a means to supply nutrients and remove the reaction byproducts. The incorporation of membrane allows for: (1) the flow of a nutrient-feeding solution leads to higher expression yield compared to static conditions, because protein synthesis does not terminate earlier due to fast depletion of the energy source (ATP); (2) removal of small molecular byproducts is also critical to high yield expression of proteins in a cell-free medium, because possible inhibition of protein synthesis by the byproducts (e.g., hydrolysis products of triphosphates) does not take place. [0237] Continuous supply of nutrients and removal of small molecular byproducts are achieved by osmosis, which results from the concentration difference of chemicals between two sides of the membrane. In addition, the flow to supply fresh solution from the well to the tray is augmented by a hydrostatic pressure, which is caused by the difference in the solution level between the tray and well. When the well has slightly higher solution level than the tray (~1 mm), the pressure difference resulting from the height difference drives nutrients from the well into the tray.
[0238] The result in Figure 6A confirms that the device design is proper. When IVT was implemented in a microcentrifuge tube, luciferase was synthesized in the first 0.5 hr and then the reactions ceased, hi contrast, when it was in the device with continuous feeding of nutrients and removal of byproducts, luciferase was continuously produced up to 4 hr. The yield of luciferase production in the miniaturized device is about 2.6 fold higher than that of microcentrifuge tube. A similar result Figure 6B was obtained for another protein, green fluorescence protein (GFP), and the production yield increased more than 14 fold in the device than in a microcentrifuge tube. These results suggest that we achieved the desired fluid manipulation in the device.
[0239] One point worthy of note is the potential advantages of the solution array over ELISA or conventional protein arrays/chips. The solution array offers maximal flexibility without compromising the binding activity of proteins. One may choose different proteins to express to achieve the optimum yield and/or use different assays for detection. In addition, the solution array does not possess heterogeneous solid-liquid attachment, eliminating the issues encountered in the protein arrays/chips (e.g. maintaining the conformation, thus biological activity, of proteins attached to a solid surface).
[0240] Biological Signal Amplification: Ricin causes toxic effects by inactivating ribosomes and inhibiting protein synthesis in biological cells and then leading to cell death and tissue damage. We exploit its toxicity mechanism as the sensing scheme to detect ricin. This detection method possesses inherent biological signal amplification, as illustrated in Figure 7. One copy of DNA is transcribed into one copy of messenger RNA. However, for each copy of RNA, thousands of copies of proteins can be produced. This is estimated by the amount of the DNA vector used and the amount of the corresponding proteins produced in IVT. The inhibitory effects of ricin exist on the production of every copy of protein, as illustrated in Figure 7. As a result, the detection signal (i.e., the difference between the sample and the positive control) is accumulated, leading to an amplified signal. [0241] The amplification of the inhibitory effects of ricin on protein synthesis is also evident from its toxicity on biological cells. A single ricin molecule that enters the cytosol can inactivate over 1,500 ribosomes per minute and kill the cell.
[0242] This signal amplification is similar, to some degree, to the enzyme-enabled signal amplification in ELISA. Intrinsic to ELISA is the addition of reagents conjugated to enzymes; assays are then quantified by the build-up of colored products after the addition of substrates. The signal amplification results from the enzyme that catalyzes many substrate molecules to detectable products. Two widely-used enzymes are horseradish peroxidase and alkaline phosphatase, which transfer o-phenylene diamine and p-nitrophenylphosphate, respectively, and generate colored products. Therefore, we expect the detection method based on protein inhibition has comparable sensitivity with ELISA.
[0243] A number of potent biological toxins exert their toxic effects through inhibition of protein synthesis, including Shiga toxin produced by Shigella dysenteriae, Shiga-like toxin by enteropathogenic E. coli, diphtheria toxin from Corynebacterium diphtheriae and exotoxin A of ' Pseudomonas aeruginosa. However, the selectivity of our detection method can be achieved by the response pattern of a protein expression array. A few proteins are judiciously selected so that their production yields are inhibited differentially by toxin simulants as illustrated previously. As a result, the differential inhibitory effects of a toxin on expression of different proteins can be used as a signature for toxin detection and identification. [0244] Ricin Detection: To demonstrate the detection of ricin, we studied its inhibitory effects on luciferase expression by adding a series of concentrations of ricin A chain into the IVT reactions in the array device. The calibration curve is shown in Figure 8, in which the expression yield of luciferase (indicated by the luminescence) was plotted as a function of ricin concentration. The error bar of each data point indicates the standard deviation that was obtained from three repeat experiments. A linear relationship exists from 1 to 20 pg/μL. The detection limit is at least 1 pg/μL, since the signal-to-noise at this concentration is 4.1, which is slightly larger than the standard (typically 3).
[0245] Since the detection signal is dependent on the accumulation of inhibitory effects of ricin on protein production, longer protein expression leads to lower detection limit. The result in Figure 8 was obtained after 4 hours of protein expression, at which protein expression yield reaches a plateau as indicated in Figure 6A.
[0246] However, luciferase can be synthesized in as short as 5 minutes, as shown in Figure 9 A and 9B. Similar to the. result in Figure 6 A and 6B when a longer expression time was used, we also observed the difference in the luciferase expression yield between the miniaturized device and a microcentrifuge tube when a short expression time (<30 minutes) was used. The result suggests that enough difference takes place in as short as 5-minute. A short IVT time is critical for those applications that need a quick response. We confirmed that we were able to detect ricin within 5 minutes, as shown Figure 9B, though the detection limit is higher than when a longer expression time is used.
[0247] Toxicity Level of Ricin: Ricin is a dimer, in which B chain binds to cell surface, allowing A chain to penetrate the cell to inhibit protein synthesis. In other words, the toxicity of ricin comes from the A chain. Since IVT does not provoke dissociation of the A-B dimer, all experimental results discussed above are from ricin A chain. Whole ricin (with A and B chains) is used, to determine the toxic effect in the IVT device. As shown in Figure 10, we observed the similar result in IVT device. The A chain has highest toxicity, B chain has no detectable toxicity, and whole ricin shows a toxicity level less than the A chain. However, 2- mercaptoethanol-treated ricin is almost as effective as A chain due to reduction of disulfide bond between two chains.
[0248] We also studied the effects of heat-denature on the ricin. We observed that there was a negligible inhibitory effect on luciferase expression when ricin A chain was heat- denatured and its biological activity was disabled (Figures 8 and 9B). In other words, there was no significant difference between the positive controls (free of ricin) and the one with the denatured ricin A chain. We can infer from these results that the IVT method can detect the toxicity level of ricin after physical and/or chemical treatments.
[0249] We have developed a novel ricin detection method based on its inhibitory effects on protein expression. Signal amplification is achieved due to the fact that there is an inhibitory effect on production of every copy of protein; thousands of copies of proteins are generated from each copy of RNA. The protein expression was carried out in an array format for quantification and reduction of false positives/negatives. The miniaturized device consisted of the trays and wells separated by a dialysis membrane, which supplied nutrients continuously and removed byproducts. We demonstrated higher expression yield in the miniaturized device than in a conventional microcentrifuge tube, leading to a lower detection limit. The advantages of the solution array over ELISA include simpler one-step reaction, faster detection, flexibility in the array selection, and elimination of issues related to maintaining protein conformation on a solid surface.
[0250] The solution array in this invention enables high-throughput protein expression for proteomics applications. Completion of mapping the human genome has prompted strong interest in identifying the functions of newly discovered genes and the proteins encoded therein. To match high-throughput gene discovery, methods to produce a large number of proteins in parallel are needed. The current method of producing proteins in E. coli cells is difficult to be implemented in a high-throughput format (Gilbert, M.; Albala, J. S. Current Opinion in Chemical Biology 2002, 6, 102-105). The miniaturized, high yield, parallel in vitro protein expression array in this invention could be a solution.
Example 7: Cell Array.
[0251] A variety of distinct components are integrated into the array system to meet the requirement of protein expression. The requirements include nutrient supplies, byproduct removal, and fluid manipulation. The system comprises an array of microcompartments; each microcompartment functions as a surrogate cell, consisting of a reaction chamber, two feeding chambers, and a channel to connect them as shown in Figure 2. Both gene transcription and protein translation take place in the miniaturized reaction chamber while the feeding chambers function as nutrient reservoirs. The feeding chambers contain amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer. ATP and GTP are the primary energy sources required for protein synthesis. The reaction chamber contains cell-free expression system with other reagents as in the feeding chambers. The channel connected to the chambers provides a means to supply nutrients and remove byproducts. The selective removal of small molecule byproducts is accomplished by using a dialysis membrane with the molecular weight cutoff at ~10 KDa, which at the same time allows entry of nutrients into the feeding chamber.
[0252] Dialysis membrane has been used in miniaturized devices for the different purpose (e.g., sampling). The incorporation of membrane is important because of two facts: (1) the flow of a feeding solution leads to higher expression yield compared to static conditions, because protein synthesis does not terminate earlier due to fast depletion of energy sources (ATP and GTP); (2) removal of small molecule byproducts is also very critical to high yield expression of proteins in a cell-free medium, because possible inhibition of protein synthesis does not take place by the small molecular byproducts such as hydrolysis products of triphosphates. [0253] The device possesses appropriate geometric configuration to achieve desired properties for in vitro protein expression. The dimension of the microchannel is in the range of 5 μm to 10 mm, depending on the flow rate required for supplying nutrients and removing byproduct. The feeding chambers are sufficiently large compared to the reaction chamber so that they function as reservoirs.' In the current design, the volume of the reaction chamber is 0.1-100 μL whereas that of the feed chambers is about 1 μL -5000 μL. AU calculations are as described above.
[0254] An experiment, expressing GFP in a device with a dialysis membrane, confirms the concept. Figure 6B shows the expression yield of GFP as a function of expression time, and the comparison between a microcentrifuge tube and the miniaturized device with flow manipulation. Expression of GFP was observed in both tube and device after 4 hours, but the yield remains the same with time when it was in the tube. In contrast, the yield increased continuously with time due to continuous supply of the nutrients and removal of byproducts when the reaction took place in the device. The production yield increased more than 14 fold in the device than in a microcentrifuge tube. The result suggests that we achieved the desired fluid manipulation in the device.
[0255] Interplay among the Expression Constituents: A panel of protein expression conditions is generated by supplementing purified key components. It has been demonstrated that protein translation can be reconstructed from its purified ingredients. The major components, including more than 30 catalysts/cofactors, are list in Table 1. Some catalysts/factors can be overexpressed and purified from E. coli cells in recombinant forms while other components and large ribosomes can be purified directly from eukaryotic cells such as mammalian tissue culture cells or plant tissue culture cells. [0256] Protein expression is determined in the array by constructing a combination of different components to quantify the effects of different ratios of constituents on the yield of protein expression. The essential elements such as 20 AA and 20 aminoacyl-tRNA synthetase can be considered as one component to reduce the work load. Mimicking the approach used for combinatorial chemical synthesis, the amount of these components can be systematically varied and their corresponding protein expression yields provide quantitative information about the interplay among the constituents. These results not only leads to an approach achieving high-yield, high-throughput protein expression, but also very useful for devising a panel of protein expression conditions for the identification of physiological targets. Table 1. Major components for protein translation.
Figure imgf000072_0001
[0257] The surrogate cell array consists of multiple compartments to accommodate an array of protein expression. An example of a format is a traditional 96-well microplate. In each surrogate cell represented by a circle, gene transcription and protein translation takes place as shown in the inset, Figure 11. To illustrate the experimental design, a subset of 24 surrogate cells (3 x 8) is chosen to produce one protein e.g., GFP (Figure 11). In the area shadowed with diagonal lines (cells Al to H3), row A functions as the positive control by producing GFP in the E. coli expression system; row B is for the negative controls (no DNA vectors). The positive and negative controls are used to facilitate quantification. Rows C-H are for producing GFP using a mixture of reconstructed ingredients, in which all components are at the same concentration as in the cell lysate except for one component, elongation factor 2 (EF-2). The mixtures are prepared in a series of concentrations of EF-2, for example, 0.01, 0.05, 0.1 , 0.5, 1 , and 5 μM for rows C, D, E, F, G, and H5 respectively. The variation in the EF-2 concentration results in quantitative information about the effects of EF-2 on the protein expression. For each sample, three repeats can be performed in columns 1-3 to enhance the precision. A total of four subsets of such an arrangement (3 x 8 cells) can be made in this 96- cell array, thus four different catalysts/cofactors may be studied simultaneously. By the combinatorial method, all possible outcomes can be obtained by systematically varying all of the protein expression constituents. The results will provide quantitative information leading to high- yield protein expression and the condition for the identification of physiological targets.
[0258] AU expected signals are normalized against the positive controls (row A in Figure 11). No signal is expected in the negative controls (row B) due to lack of DNA vector. The protein expression yield is expected to increase with the concentration of EF-2 from row C to H, since EF-2 is a catalyst responsible for the translocation of ribosome along mRNA. A larger concentration of EF-2 than the normal cellular value may result in higher expression yield in row H than the positive control.
[0259] Molecular Responses of the Surrogate Cell Array to Toxins: The surrogate cell array is used to identify the physiological targets, through which Ochratoxin A (OTA) inhibits protein expression.
[0260] Method Verification: Ricin and exotoxin A are used for verification since it is known that ricin breaks down 23 s rRNA and exotoxin A inactivates elongation factor-2 (EF- 2). Similar to the experimental design illustrated in Figure 11, we can add a series of concentrations of exotoxin A into the expression mixture. Protein expression is expected to be inhibited. However, this inhibition should be partially or fully recovered when an additional amount of EF-2 is added to the expression mixture. The additional EF-2 will bind with exotoxin A while allowing the original EF-2 performs translocation of ribosomes. If EF-2 is not the physiological target of exotoxin A, no recovery should be observed when an additional amount of EF-2 is added. This method can be used to quickly identify the target molecules of any toxins that inhibit protein expression.
[0261] In addition, we use the surrogate cell array as a sensitive method to detect ricin or other toxins. The methods to detect ricin at a low concentration include enzyme-linked immunosorbent assay (ELISA) and immunoassay using radioactive labeling. Although ELISA and radioimmunoassay offer high sensitivity, they involve several labor-intensive and time-consuming steps, and the handling and disposal of radioisotopes are environmental challenges. Recently, it has been reported a fluorescence-based multianalyte immunosensor that has a detection limit of 25 pg/μL of ricin. Another group exploited specific interaction between ricin and glycosphingolipids and developed a quartz crystal microbalance sensor with the detection limit of 5000 pg/μL of ricin. Another group depicted an immunoassay- based magnetoelastic sensor that shows a detection limit of 5 pg/μL of ricin. We expect lower detection limit by employing the inhibitory effect of ricin on protein expression as the sensing mechanism, because the signal output representing the inhibitory effect is amplified due to the accumulation of inhibitory effects on the production of each copy of proteins. And thousands of copies of proteins can be produced from each copy of messenger RNA. A higher yield of protein expression in a continuous flow system also contributes to a larger detection signal. Compared to ELISA, the detection method is also simpler and faster. [0262] Target Identification: The surrogate cell array is used to identify the physiological targets of agents such as, Ochratoxin A (OTA). OTA is a mycotoxin produced by Penicillium and Aspergillus. Several major mechanisms have been shown to be involved in the toxicity of OTA, including inhibition of protein synthesis, though the exact mechanism is not well-understood. Using an array of protein expression conditions, each with increasing amount of a key transcription/translation component as illustrated in Figure 11, the inhibitory effect of OTA on the protein expression can be tested. Any component that diminishes the toxic effect of OTA will imply a likely substrate of the toxin.
Other Embodiments
[0263] While the above specification contains many specifics, these should not be construed as limitations on the scope of the invention, but rather as examples of preferred embodiments thereof. Many other variations are possible. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal equivalents.

Claims

What is claimed is:
1. A sensor for detecting toxins, comprising: a substrate; at least one well formed in or on said substrate; at least a first in vitro transcription and translation (TVT) unit disposed in said well, said IVT comprising a DNA template including a coding sequence which is transcribed into messenger RNA using an RNA polymerase; and a eukaryotic or prokaryotic lysate providing ribosomes for protein translation by said messenger RNA, wherein said FVT expresses a protein.
2. The sensor of claim 1, further comprising a detector for detecting a signal related to a concentration of said protein, wherein a level of said signal is reduced when a target toxin which inhibits expression of said protein by said IVT is present as compared to when said target toxin is not present.
3. A sensor array for simultaneously detecting multiple toxins, comprising: a substrate; a plurality of wells formed in or on said substrate; and a first and at least a second in vitro transcription and translation (FVT) unit disposed in a first and at least a second of said plurality of said wells, respectively, said first and second IVTs each comprising a DNA template including a coding sequence which is transcribed into messenger RNA using an RNA polymerase; and a eukaryotic or prokaryotic lysate providing ribosomes for protein translation by said messenger RNA, wherein said first IVT expresses a first protein and said second IVT expresses a second protein different from said first protein.
4. The sensor array of claim 3, further comprising of a detector for detecting a signal related to a concentration of said proteins, wherein a level of said signal is reduced when a target toxin capable of inhibiting protein translation for said IVTs is present as compared to when said target toxin is not present in said wells.
5. A method for detecting toxins, comprising: expressing a nucleic acid sequence encoded by an expression vector in a reaction mixture, wherein said nucleic acid is transcribed and translated in presence of a sample suspected of including at least one toxin, wherein at least one protein is expressed; measuring a level of said protein expression, and, identifying the presence or absence of said toxin based on said level, wherein said toxin inhibits expression of said protein.
6. A method of identifying inhibitors of protein expression, comprising: expressing a nucleic acid sequence encoded by an expression vector in a reaction mixture wherein said nucleic acid is transcribed and translated in presence or absence of a candidate drug wherein at least one protein is expressed; measuring a level of said protein expression, and, identifying candidate drugs that are inhibitors of protein expression.
7. The method of claim 6, wherein said reaction mixture comprises polymerases, nucleotides, amino acids.
8. A sensor array, comprising: a plurality of sensors, said sensors each comprising: at least a feeding chamber; a reaction chamber, and a a membrane for feeding nutrients and removing by products.
9. The sensor array of claim 8, further comprising a channel connecting said feeding chamber and said reaction chamber.
10. The sensor array of claim 8, further comprising a pump for supplying said nutrients.
11. The sensor array of claim 8, further comprising a pump for removing byproducts.
12. The sensor array of claim 8, further comprising a pump for supplying said nutrients and removing byproducts.
13. The method of claim 12, wherein said pump is an osmotic pump, electroosmotic pump, capillary force pump, pneumatic pump, syringe pump, electrokinetic pump, piezoelectric pump, or acoustic pump.
14. The sensor array of claim 8, wherein micro fluidic features and means are used.
15. A method of identifying antibiotic drug candidates, comprising: expressing a nucleic acid sequence encoded by an expression vector in a reaction mixture wherein said nucleic acid is transcribed and translated in presence or absence of a candidate drug wherein at least one protein is expressed; measuring a level of said protein expression, and, identifying candidate drugs that are inhibitors of protein expression.
16. A surrogate cell array comprising: a microcompartment wherein each microcompartment comprises a reaction chamber, feeding chambers, and a channel or a cavity to connect each microcompartment.
17. The surrogate cell array of claim 16, wherein the reaction chamber is a miniaturized reaction chamber having a diameter of about 0.1 mm to 30 mm.
18. The surrogate cell array of claim 16, wherein the feeding chamber comprises amino acids, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and buffer.
19. The surrogate cell array of claim 16, wherein the reaction chamber comprises a cell- free expression system.
20. The surrogate cell array of claim 16, wherein a channel or a cavity connected to the chambers comprises a dialysis membrane with the molecular weight cutoff of about 1 kDa to about 20 KDa.
21. The surrogate cell array of claim 20, wherein the dialysis membrane has a molecular weight cut off of about 10 KDa.
22. The surrogate cell array of claim 16, wherein the channel or cavity dimensions are about 5 μm up to 10 mm.
23. The surrogate cell array of claim 16, wherein the feeding chamber is dimensionally proportioned to about 2 to 100 times larger than the dimensions of the reaction chamber.
24. The surrogate cell array of claim 23, wherein a miniaturized array comprises a reaction chamber dimensionally proportioned to contain a volume of fluid 10 times less than a feed chamber.
25. The surrogate cell array of claim 23 , wherein a miniaturized array comprises a reaction chamber dimensionally proportioned to contain about 0.1 μl up to 100 μl of fluid and the feed chamber is dimensionally proportioned to contain about 1 μl up to 5000 μl of fluid.
26. The surrogate cell array of claim 16, wherein the chambers are made from at least one material selected from the group consisting of polypropylene, polycarbonate, polystyrene, vinyl, acrylic, plastics, metal and glass.
27. The surrogate cell array of claim 16, wherein the array comprises a plurality of microcompartments and channels.
28. A method of identifying drug targets, comprising: expressing a nucleic acid sequence encoded by an expression vector in a reaction mixture wherein said nucleic acid is transcribed and translated in presence or absence of one or multiple drug target wherein at least one protein is expressed; measuring a level of said protein expression, and, identifying drug targets that show no or little inhibition of protein expression.
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