WO2000070093A1 - Arrays with modified oligonucleotide and polynucleotide compositions - Google Patents

Abstract

The present invention provides arrays having associated modified oligonucleotides, methods of making such arrays, assays for using such arrays, and kits containing such arrays. In one embodiment, the arrays of the invention exhibit an increased binding affinity with complementary nucleic acids, and in particular with complementary RNA. In another embodiment, the associated nucleic acids of the array of the invention exhibit substantial acid resistance, allowing the arrays to be treated with low pH solutions. In another embodiment, the modified associated nucleic acids of the array of the invention exhibit substantial resistance to nuclease degradation.

Description

ARRAYS WITH MODIFIED OLIGONUCLEOTIDE AND POLYNUCLEOTIDE COMPOSITIONS

FIELD OF THE INVENTION The field of this invention is arra\ s having associated ohgonucleotides and/or polynucleotides. methods of producing such arrays, and uses thereof

BACKGROUND OF THE INVENTION

Arrays of binding agents, such as ohgonucleotides and polynucleotides, have become an increasmgly important tool in the biotechnology industry and related fields These arrays, m which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencmg, mutation analysis, and the like One important use of arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed m the cells being compared In methods of differential gene expression, arrays find use by serving as a substrate with associated binding fragments such as ohgonucleotides Nucleic acid sequences are obtamed from analogous cells, tissues or organs of a healthy and diseased organism, and hybridized to the immobilized set of binding fragments associated with the array Differences between the resultant hybridization patterns are then detected and related to differences in gene expression in the two sources

A variety of different array technologies have been developed in order to meet the growing need of the biotechnology industry Despite the wide variety of array technologies currently m preparation or available on the market, there is a contmued need to identify new array devices to meet the needs of specific applications Of particular mterest are arrays which provide increased binding affinity, because these allow the use of shorter binding fragments and fewer bound fragments can be used to obtain the results currently available with conventional technology Also of mterest is the development of an array capable of providing high throughput analysis of differential gene expression, where the array itself is reusable Such an array is needed for a number of reasons such as decreasmg experimental variability, confirmmg results, and for decreasing costs of such analysis

SUMMARY OF THE INVENTION The present mvention provides arrays having associated o gonucleotide and/or polynucleotides with modified structures (e g , V, 2', 3', 5' and/or modifying the πbose oxygen), methods of making such arrays, assays for using such arrays, and kits containing such arrays The modifications described herein provide numerous advantages, including a higher binding affinity for complementary nucleic acids, acid resistance and/or nuclease resistance The mvention comprises an array device comprised of a support surface and polymer molecules bound to the support surface The polymer molecules are not naturally occurring ohgonucleotides or polynucleotides, but rather

modified backbones with bases attached m the desired sequential positioning and the desired spacmg between the bases The backbone is preferabh modified to obtain improved results compared to natural ohgonucleotides or polynucleotides including (1) higher binding affinity, (2) greater acid resistance, (3) greater resistance to enzymatic degradation, and/or (4) overall better performance and reusability

In one embodiment, the modified associated ohgonucleotides and/or polynucleotides of the mvention provide additional binding

with respect to corresponding, unmodified ohgonucleotides having the same sequence The binding affinity is preferably mcreased by a modification at the 2' site of the sugar group e g a 2'-F or a 2'-OR modification such as 2'-0-methyl or 2'-0-methoxyethoxy Alternatively or in combmation. the bmding affimty can be mcreased by modification m the 3' linkage group, e g phosphoramidate linkages, or a modification replacmg the oxygen with a carbon

In another embodiment, the modified associated ohgonucleotides and/or polynucleotides of the array exhibit substantial acid resistance, allow mg the arrays to be treated with low pH solutions This allows the array to be exposed to low pH in order to remove any bound nucleic acids that are not modified, e g , bound test nucleic acids

It is thus an object of the present mvention to provide arrays havmg associated chemically modified ohgonucleotides and/or polynucleotides characterized by substantial acid resistance Such arrays may be exposed to low pH environments to facilitate clearance from the array of the test binding agents

In yet another embodiment, the modified associated ohgonucleotides and/or polynucleotides of the array exhibit substantial resistance to nuclease degradation These molecules preferably have an end-blocking group that confers nuclease resistance to the molecule, e g , a butanol or butyl group It is thus an object of the mvention to provide arrays having associated chemically modified ohgonucleotides and/or polynucleotides to confer substantial nuclease resistance Nucleases can be used to digest the test substrate binding agent, freeing the associated binding agents for further use The chemical modification may be on the 5' end for ohgonucleotides and/or polynucleotides attached to a substrate at the 3' end, or alternatively the chemical modification may be on the 3' end for ohgonucleotides and/or polynucleotides attached to a substrate at the 5' end The associated ohgonucleotides and/or polvnucleotides remain unaffected as to the binding capacity of the associated ohgonucleotides

These arrays also offer the significant advantage that the individual chip can be tested for efficacy and/or quality pπor to use with a test sample which is particularly helpful if the amount of test sample is limited or if the array is being used as a medical device and must comply with FDA quality control requirements

The present invention further pro\ ides an assay usmg the arrays of the mvention to determine physiological responses such as gene expression, where the response is determmed by the hybridization pattern of the array after exposure to test samples The test samples may be mRNA, cDNA, whole cell extracts, and the like

It is an advantage of the associated modified ohgonucleotides and/or polynucleotides of the arrays of the mvention that the chemical modifications enhance the chemical binding mteractions. e g , mcrease bmdmg affinity over standard Watson-Crick base paiπng with complementary ohgonucleotides and/or polynucleotides, particularly when binding to mRNA It is another advantage that the modified ohgonucleotides and or polynucleotides of the array may be synthesized to have approximately the same T_m, by varying the length of the nucleic acids m each composition

It is another advantage that modified ohgonucleotides and/or polynucleotides of the mvention hybπdize more tightly with complementary RNA sequences than natural DNA ohgonucleotides, allowing the use of shorter binding fragments (e g one or more ohgonucleotides in lieu of a complete cDNA)

It is an advantage of the associated modified ohgonucleotides and/or polynucleotides of the mvention that the acid stable modifications confer an improved stability on the modified ohgonucleotides and/or polynucleotides m an acidic environment (e g , as low as pH of 1 to 2) It is another advantage of the associated ohgonucleotides and/or polynucleotides of the mvention that they bmd with specificity to test nucleic acids

It is an object of the invention that the ohgonucleotides and or polynucleotides can be used m a variety of array applications, such as identification of new genes, determination of expression levels, diagnosis of disease, and the like These and other objects, advantages, and features of the invention will become apparent to those skilled m the art upon reading the details of the ohgonucleotides and/or polynucleotides and uses thereof as more fully descnbed below BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 -7 illustrate the chemical structure of exemplary modifications that result in acid stability

Figures 8-9 illustrate the chemical structure of end-blocked, acid stable molecules used in the invention

Figure 10 illustrates other potential modifications that may be used m the present invention

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is to be understood that this mvention is not limited to the particular methodology, support surfaces, matenals and modifications descnbed and as such may, of course, vary It is also to be understood that the terminology used here is for the purpose of descnbing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims

It must be noted that as used herem and m the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise Thus, for example, reference to "an oligonucleotide" may mclude a plurality of ohgonucleotide molecules and equivalents thereof known to those skilled m the art, and so forth

Unless defined otherwise, all technical and scientific terms used herem have the same meaning as commonly understood to one of ordinary skill m the art to which this mvention belongs Although any methods, devices and materials similar or equivalent to those descnbed herem can be used in the practice or testmg of the invention, the preferred methods, devices and matenals are now described

All publications mentioned are incorporated herem by reference for the purpose of descnbmg and disclosing, for example, matenals, constructs, and methodologies that are descnbed m the publications which might be used m connection with the presently descnbed mvention The publications discussed above and throughout the text are provided solely for their disclosure pnor to the filing date of the present application Nothing herem is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of pnor mvention

DEFINITIONS The terms "nucleic acid" and "nucleic acid molecule" as used interchangeably herem, refer to a molecule compnsed of one or more nucleotides, l e , nbonucleotides, deoxynbonucleotides, or both The term mcludes monomers and polymers of nbonucleotides and deoxynbonucleotides, with the nbonucleotides and/or deoxynbonucleotides bemg connected together, m the case of the polymers, via 5' to 3' linkages However, linkages may mclude any of the linkages known m the nucleic acid synthesis art mcludmg, for example, nucleic acids compnsmg 5' to 2' linkages The nucleotides used in the nucleic acid molecule may be naturally occu ing or may be synthetically produced analogues that are capable of forming base-pair relationships with naturally occurring base pairs. Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogues, aza and deaza purine analogues, and other heterocyclic base analogues, wherein one or more of the carbon and nitrogen atoms of the purine and pyrimidine rings have been substituted by heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.

The term "oligonucleotide" as used herein refers to a nucleic acid molecule comprising from about 2 to about 300 nucleotides. Ohgonucleotides for use in the present invention are preferably from 80-200, more preferably from 100-150 in length.

The term "polynucleotide" as used herein refers to nucleic acid molecules comprising a plurality of nucleotide monomers including but not limited to nucleic acid molecules comprising over 200 nucleotides.

The terms "modified oligonucleotide" and "modified polynucleotide" as used herein refers to ohgonucleotides and or polynucleotides with one or more chemical modifications at the molecular level of the natural molecular structures of all or any of the bases, sugar moieties, internucleoside phosphate linkages, as well as to molecules having added substituents, such as diamines, cholesterol or other lipophilic groups, or a combination of modifications at these sites. The internucleoside phosphate linkages can be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate and/or sulfone internucleotide linkages, or 3'-3', 5'-2' or 5'-5' linkages, and combinations of such similar linkages (to produce mixed backbone modified ohgonucleotides). The modifications can be internal (single or repeated) or at the end(s) of the oligonucleotide molecule, and can include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl, diamine compounds with varying numbers of carbon residues between amino groups and teπninal ribose, and deoxyribose and phosphate modifications which cleave or cross-link to the opposite chains or to associated enzymes or other proteins. Electrophilic groups such as ribose-dialdehyde could covalently link with an epsilon amino group of the lysyl-residue of such a protein. A nucleophilic group such as M-ethylmaleimide tethered to an oligomer could covalently attach to the 5' end of an mRNA or to another electrophilic site. The terms "modified ohgonucleotides" and "modified polynucleotides" also include ohgonucleotides and/or polynucleotides comprising modifications to the sugar moieties (e.g., 2'- substituted ribonucleotides or deoxyribonucleotide monomers), any of which are connected together via 5' to 3' linkages. Modified ohgonucleotides may also be comprised of PNA or morpholino modified backbones where target specificity of the sequence is maintained. A modified oligonucleotide of the invention (1) does not have the structure of a naturally occumng oligonucleotide and (2) will hybridize to a natural oligonucleotide mRNA or cDNA Further the modification preferably provides (3) higher binding affinity, (4) greater acid resistance, and (5) better stabilits agamst digestion with enzymes as compared to a natural oligonucleotide The term "oligonucleotide backbone" as used herein refers to the structure of the chemical moiety linking nucleotides m a molecule The invention preferably comprises a backbone which is different from a naturally occurring backbone and is further characterized b\ (1) holding bases m correct sequential order and (2) holdmg bases a correct distance between each other to allow a natural oligonucleotide to hybndize to it This ma\ mclude structures formed from any and all means of chemically linking nucleotides A modified backbone as used herein includes modifications (relative to natural linkages) to the chemical linkage between nucleotides, as well as other modifications that may be used to enhance stability and affimty such as modifications to the sugar structure For example an α-anomer of deoxynbose may be used, where the base is inverted with respect to the natural β-anomer In a preferred embodiment the 2'-H or 2'-OH of the sugar group (for RNA and DNA, respectively) may be altered to 2'-0-alkyl or 2'-0-alkvl-n(0-alkyl), which provides resistance to degradation without compnsmg affimty

The term "end-blocked" as used herem refers to an oligonucleotide with a chemical modification at the molecular level that prevents the degradation of selected nucleotides, e g , by nuclease action This chemical modification is positioned such that it protects the integral portion of the oligonucleotide, for example the region of the oligonucleotide that is targeted for hybndization (I e , the test sequence of the oligonucleotide) An end block may be a 3' end block or a 5' end block For example, a 3' end block may be at the 3 '-most position of the molecule, or it may be internal to the 3' ends, provided it is 3' of the mtegral sequences of the oligonucleotide

The term "substantially nuclease resistant" refers to ohgonucleotides that are resistant to nuclease degradation as compared to naturally occurring or unmodified ohgonucleotides Modified ohgonucleotides of the mvention are at least 1 25 times more resistant to nuclease degradation than their unmodified counterpart, more preferably at least 2 times more resistant, even more preferably at least 5 times more resistant, and most preferably at least 10 times more resistant than their unmodified counterpart Such substantially nuclease resistant ohgonucleotides mclude, but are not limited to, ohgonucleotides with modified backbones such as phosphorothioates, methylphosphonates, ethylphosphotnesters, 2'-0-methylphosphorothιoates, 2'-0-methyl-p-ethoxy nbonucleotides, 2'-0- alkyls, 2'-0-alkyl-n(0-alkyl), 3'-0-alkλls. 3'-0-alkyl-n(0-alkyl), 3'-0-methyl nbonucleotides, 2'- fluoros, 2'-deoxy-erythropentofuranosyls 2'-0-methyl nbonucleosides, methyl carbamates, methyl carbonates, inverted bases (e g . inverted T's), or chimenc versions of these backbones The term "substantially acid resistant" as used herein refers to ohgonucleotides that are resistant to acid degradation as compared to unmodified ohgonucleotides Typically, the relative acid resistance of an oligonucleotide will be measured by companng the percent degradation of a resistant oligonucleotide with the percent degradation of its unmodified counterpart (1 e , a correspondmg oligonucleotide with "normar backbone bases and phosphodiester linkages) An oligonucleotide that is acid resistant is preferably at least 1 5 times more resistant to acid degradation, at least 2 times more resistant, even more preferably at least 5 times more resistant, and most preferably at least 10 times more resistant than their unmodified counterpart

The term "alkyl" as used herem refers to a branched or unbranched saturated hyrdrocarbon chain containing 1-6 carbon atoms, such as methyl, ethyl, propyl, tert-butyl, n-hexyl and the like

The term "array type" refers to the type of gene represented on the array by the associated test ohgonucleotides, where the type of gene that is represented on the anay is dependent on the intended purpose of the array, e g , to monitor expression of key human genes, to monitor expression of known oncogenes, etc I e , the use for which the anay is designed As such, all of the test ohgonucleotides on a given array conespond to the same type or category or group of genes Genes are considered to be of the same type if they share some common linking characteristics, such as species of ongm, e g , human, mouse, rat, etc , tissue or cell type of ongm, e g , muscle, neural, dermal, organ, etc , disease state, e g , cancer, functions, e g , protem kinases, tumor supressors and the like, participation m the same normal biological process, e g , apoptosis. signal transduction, cell cycle regulation, proliferation, differentiation etc . and the like For example, one anay type is a "cancer array" in which each of the "unique" associated test ohgonucleotides conespond to a gene associated with a cancer disease state Likewise, a "human array" may be an array of test ohgonucleotides correspondmg to umque tightly regulated human genes Similarly, an "apoptosis array" may be an array type in which the associated test ohgonucleotides conespond to umque genes associated with apoptosis

The terms "associated oligonucleotide," "associated polynucleotide" and "substrate oligonucleotide" and the like refer to the oligonucleotide or polynucleotide composition that makes up each of the samples associated to the array Thus, the term "associated oligonucleotide" mcludes oligonucleotide compositions of umque sequences and/or control or calibrating sequences (e g , ohgonucleotides correspondmg to housekeepmg genes) The oligonucleotide and/or polynucleotide compositions are preferably compnsed of smgle stranded nucleic acid, where all of the nucleic acids m a sample composition may be identical to each other Alternatively, there may be nucleic acids having two or more sequences m each composition, for example two different ohgonucleotides that are separate but complementary to each other THE INVENTION IN GENERAL Arrays having associated ohgonucleotides and/or polynucleotides with modified backbone structures, such as ohgonucleotides with 2'-0-alkyl and 2'-0-alkyl-n(0-alkyl) sugar moieties, changes m the nbose oxygen, 5' linkage modifications and/or 3' linkage modifications, are provided Modified ohgonucleotides and polynucleotides of the r ention also may be acid resistant and/or exonuclease resistant to further decrease the sensitivity of the oligonucleotide molecule In one embodiment, an exonuclease resistant block is added to the 3' or the 5' end of the oligonucleotide or polynucleotide depending on the attachment of the nucleic acid to the substrate The resultmg modified ohgonucleotides and/or polynucleotides of the invention bmd tightly to their RNA or DNA targets Modified ohgonucleotides and/or polynucleotides of the invention preferably have an mcreased bmdmg affinity over their non-modified RNA or DNA counterparts This bmdmg affimty can be determmed usmg T_m assays such as those descnbed in L L Cummins et al , Nucleic Acids Research 23 2019-2024 (1995) Typically, the T_m of an oligonucleotide binding to RNA will increase approximately 1 °C for each 2'-0-methyl substitution in a molecule, and the T_m increases even more for 2'-0-propyl and 2'-F substitutions Thus, m one embodiment, the T_ra of the modified oligonucleotide bound to RNA is 2-15 °C. and even more preferably 8-10°C higher than the correspondmg non-modified DNA oligonucleotide

The modified ohgonucleotides and or polynucleotides of the array may be synthesized to have approximately the same T_m. by varying the length of the nucleic acids m each composition Thus, an oligonucleotide with an A-T rich sequence would be designed to be longer than an oligonucleotide with a G-C nch sequence to provide approximate!, the same T_m The T_m of each of the compositions on an array can be held relatively constant by providing lengths of ohgonucleotides and polynucleotides based on the bmdmg affimty of the base sequence

Acid stable associated ohgonucleotides and/or polynucleotides of the mvention are stable when exposed to a pH of 1-2, while their bmdmg partners are not This allows an array hav g associated acid stable ohgonucleotides and/or polynucleotides to be exposed to a first sample, treated with an acidic solution applied m any of several possible protocols to free the array from the first bmdmg partner, and reused with a second sample Direct companson of two different samples of bmdmg partners usmg a smgle array has the advantage of limiting potential experimental vanation present when comparmg multiple arrays Performing the experiment with the same sample on the same array allows a confirmation of the results obtained m the first instance, thus effectively confirming results without havmg vanation m the array composition

Similarly, associated end-blocked ohgonucleotides and/or polynucleotides display a resistance to nucleases, allowing the arrays to be exposed to DNA nucleases to free the array from a sample of binding partners An array of the invention having nuclease resistant associated ohgonucleotides can be treated with an appropπate nuclease and reused with a different or the same sample

The arrays of the present invention encompass associated ohgonucleotides chemically modified to be acid stable from a pH of 0 01 to 7 0. and more preferably acid stable in a pH of 1 0 to 4 0, allowing such molecules to retain their structural mtegnties in acidic environments Although any 2'-modιfied oligonucleotide may be used in the present invention, m a preferred embodiment the ohgonucleotides of the invention are

and 2'-0-alkyl-n(0-alkyl) ohgonucleotides which, unlike unsubstituted phosphodiester or phosphorothioate DNA or RNA, exhibit significant acid resistance m solutions with pH as low as 0-1 even at 37 °C Acid stability of this first component coupled with the mtroduction of 3' and/or 5' acid stable, exonuclease resistant ends, confers several unique properties on 2'-0-alkyl and 2'-0-alk5 l-n(O-alkyl) ohgonucleotides These low toxicity, highly specific, acid stable, end-blocked 2'-0-alkyl and 2'-0-alkyl-n(0-alkyl) ohgonucleotides represent a novel and improved oligonucleotide structure for use m array technologies

Typically, the relative nuclease resistance of a oligonucleotide can be measured by comparmg the percent digestion of a resistant oligonucleotide with the percent digestion of its unmodified counterpart (1 e , a correspondmg oligonucleotide with "normal" backbone, bases, and phosphodiester linkage) Percent degradation may be determined by usmg analytical HPLC to assess the loss of full length ohgonucleotides, or by any other suitable methods (e g , by visualizing the products on a sequencing gel using staining, autoradiography fluorescence, etc , or measuring a shift in optical density) Degradation is generally measured as a function of time

Comparison between unmodified and modified ohgonucleotides can be made by ratiomg the percentage of mtact modified oligonucleotide to the percentage of mtact unmodified oligonucleotide For example, if, after 15 minutes of exposure to a nuclease, 25% (I e , 75% degraded) of an unmodified oligonucleotide is mtact, and 50% (I e , 50% degraded) of a modified oligonucleotide is mtact, the modified oligonucleotide is said to be 2 times (50% divided by 25%) more resistant to nuclease degradation than is the unmodified oligonucleotide Generally, a substantially nuclease resistant oligonucleotide will be at least about 1 25 times more resistant to nuclease degradation than an unmodified oligonucleotide with a correspondmg sequence, typically at least about 1 5 times more resistant, preferably about 1 75 times more resistant, and more preferably at least about 10 times more resistant after 15 minutes of nuclease exposure

Percent acid degradation may be determmed by usmg analytical HPLC to assess the loss of full length ohgonucleotides, or by any other suitable methods (e g , by visualizing the products on a sequencing gel usmg staining, autoradiography, fluorescence, etc , or measuring a shift m optical density) Degradation is generally measured as a function of time Comparison between unmodified and modified ohgonucleotides can be made by ratioing the percentage of mtact modified oligonucleotide to the percentage of intact unmodified oligonucleotide For example, if. after 30 minutes of exposure to a low pH environment, 25% (l e , 75% degraded) of an unmodified oligonucleotide is mtact, and 50% (I e . 50% degraded) of a modified oligonucleotide is mtact, the modified oligonucleotide is said to be 2 times (50% divided by 25%) more resistant to nuclease degradation than is the unmodified oligonucleotide Generally, substantially "acid resistant" ohgonucleotides will be at least about 1 25 times more resistant to acid degradation than an unmodified oligonucleotide with a conespondmg sequence, typically at least about 1 5 times more resistant, preferably about 1 75 more resistant, more preferably at least 5 times more resistant and even more preferably at least about 10 times more resistant after 30 mmutes of exposure at 37° C to a pH of about 1 5 to about 4 5

In a preferred embodiment, the end-blocked ohgonucleotides of the compositions and methods of the invention are substantially nuclease resistant, substantially acid resistant, and preferably, both substantially nuclease resistant and substantially acid resistant This embodiment mcludes ohgonucleotides completely or partially denvatized by one or more linkages from the group compnsed of phosphorothioate linkages, 2'-0-methyl-phosphodιesters, 2'-0-alkyl, 2'-0-ethyl, 2'-0-propyl, 2'-0- butyl, 2'-0-alkyl-n(0-alkyl). 2'-methoxyethoxy, 2'-fluoro, 2'-deoxy-erythropentofuranosyl, 3'-0- methyl, p-isopropyl ohgonucleotides, phosphodiester, 2'-0(CH₂CH₂0)_xCH₃, butyne, phosphotnester, phosphoramidate, propargyl. siloxane, carbonate, carboxymethylester, methoxyethoxy, acetamidate, carbamate, thioether, bridged phosphoramidate. bridged methylene phosphonate, methylphosphonate, phosphorodithioate, bridged phosphorothioate and/or sulfone internucleotide linkages, or 3 '-3' or 5 '-5' or 5'-2' linkages, and combmations of such similar linkages (to produce mixed backbone modified ohgonucleotides), and any other backbone modifications

Exemplary modifications that result in acid stability can be seen m Figures 1-6 End-blocked acid stable molecules are illustrated in Figures 7-8 Other modifications that may be of use m the present mvention are illustrated See "The Medicmal Chemistry of Ohgonucleotides" m Medical Intelligence Unit Therapeutic Applications of Ohgonucleotides (1995) pp 85-108, and Mesmaeker et al , Acc Chem Res , 28 366-374 (1995)

This embodiment also mcludes other modifications that render the ohgonucleotides and/or polynucleotides substantially resistant to nuclease activity Methods of rendering an oligonucleotide nuclease resistant mclude, but are not limited to, covalently modifying the purme or pynmidme bases that compnse the oligonucleotide For example, bases may be methylated, hydroxymethylated, or otherwise substituted (e g , glycosylated) such that the ohgonucleotides compnsmg the modified bases are rendered substantially nuclease resistant In a preferred embodiment, the oligonucleotide and/or polynucleotide will have a backbone substantially resistant to acid degradation, exonuclease digestion, and endonuclease digestion In the most preferred embodiment an oligonucleotide is uniformly modified with 2'-0-alkyl or 2'-0-alkyl- n(O-alkyl) groups. 1 e , every base of the oligonucleotide is a 2'-0-alkyl or 2'-0-alkyl-n(0-alkyl) modified base

In another embodiment, the associated ohgonucleotides and/or polynucleotides of the current invention are used for diagnostic purposes For example, ohgonucleotides of the current invention may be used to detect complementary ohgonucleotides by contacting an oligonucleotide of the invention with an oligonucleotide sample under conditions that allow for the hybridization of the oligonucleotide of the invention to any complementary oligonucleotide present in the sample, and detecting such hybndization

Ohgonucleotides with a range of nuclease-resistant backbones were evaluated As a result, a preferred embodiment of the present invention is an end-blocked oligonucleotide with the chemical backbone structure

RNA-butanol-3 ' or 2'-0-alkyl-0-alkyl A particularly preferred embodiment of the present mvention is an oligonucleotide with the chemical backbone structure of 5'-butanol-2'-0-methyl RNA-butanol-3', 5'-butanol-2'-0-alkyl-0-alkyl RNA-butanol-3' or 2'-0-alkyl-0-alkyl RNA The end-blocking group on one end of the oligonucleotide may not be needed, dependmg on the manner of association with the substrate, as will be apparent to one skilled in the art upon readmg the present disclosure

ASSOCIATED OLIGONUCLEOTIDE AND POLYNUCLEOTIDE COMPOSITIONS OF THE ARRAYS

Each associated oligonucleotide and/or polynucleotide composition of the pattern present on the surface of the substrate is preferably made up of a set of unique nucleic acids, and preferably a umque oligonucleotide composition By '^"umque composition" is meant a collection or population of smgle stranded ohgonucleotides capable of participating in a hybridization event under appropnate hybndization conditions, where each of the individual ohgonucleotides may be the same — have the same nucleotide sequence ~ or different sequences, for example the oligonucleotide composition may consist of two different ohgonucleotides that are complementary to each other (I e , the two different ohgonucleotides are complementary but physically separated so as to be smgle stranded, l e , not hybridized to each other) In many embodiments, the oligonucleotide compositions will compnse two complementary, smgle stranded ohgonucleotides

In those compositions having umque ohgonucleotides, the sequence of the ohgonucleotides are chosen in view of the type and the mtended use of the array on which they are present The umque ohgonucleotides are preferably chosen so that each distmct umque oligonucleotide does not cross- hybridize with any other distinct umque oligonucleotide on the array. 1 e . the oligonucleotide will not cross-hybridize to any other oligonucleotide compositions that correspond to a different gene falling within the broad category or type of genes represented on the anay under appropnate conditions As such, the nucleotide sequence of each umque oligonucleotide of a composition will have less than 90% homology, usually less than 85 % homology and more usually less than 80%_o homology with any other different associated oligonucleotide composition of the array, where homolog-s is determmed by sequence analysis comparison using the FASTA program using default settings The sequence of umque associated ohgonucleotides m the compositions are not conserved sequences found m a number of different genes (at least two), where a conserved sequence is defined as a stretch of from about 4 to about 80 nucleotides which have at least about 90% sequence identity, where sequence identity is measured as above The associated oligonucleotide will generally have a length of from about 80 to about 300 nucleotides, usually from 100 to about 200 nucleotides The length of the nucleic acid can be chosen to best provide bmdmg to the test sequence

Although m a preferred embodiment the associated modified oligonucleotide composition will not cross-hybndize with any other associated ohgonucleotides on the anay under standard hybndization conditions, associated ohgonucleotides and hybridization conditions can be altered to allow bmdmg to multiple associated oligonucleotide compositions For example, m deteirnining the relatedness of a sample to ohgonucleotides representing different members of a class of protems, the oligonucleotide sequences may be more similar and/or less stringent hybndization conditions may be used

CHEMICAL MODIFICATIONS OF OLIGONUCLEOTIDES AND/OR POLYNUCLEOTIDES OF THE INVENTION The ohgonucleotides and/or polynucleotides of the mvention may contain any modification that confers on the molecules greater bmdmg with other nucleic acids, that mcreases the acid stability and or mcreases the nuclease stability of the molecule This mcludes ohgonucleotides and/or polynucleotides completely denvatized

phosphorothioate linkages, 2'-0-methylphosphodιesters, 2'- O-alkyl, 2'-0-alkyl-n(0-alkyl), 2'-fluoro. 2'-deoxy-erythropentofuranosyl, 3'-0-methylphosphodιesters, p-ethoxy ohgonucleotides, p-isopropyl ohgonucleotides, phosphoramidates, phosphoroamidites, chimenc linkages, carbonates, arrnnes, formacetals, silyls and siloxys, sulfonates, hydrocarbon, amides, ureas and any other backbone modifications, as well as other modifications, which render the ohgonucleotides and/or polynucleotides substantially resistant to endogenous nuclease activity The nucleotides m each oligonucleotide and/or polynucleotide may each contain the same modifications, may contain combmations of these modifications, or may combme these modifications with phosphodiester linkages Additional methods of rendering ohgonucleotides and/or polynucleotides nuclease resistant include, but are not limited to, covalently modifymg the purine or pynmidme bases that compnse the oligonucleotide For example bases may be methylated, h\ droxymethylated, or otherwise substituted (e g , glycosylated) such that the ohgonucleotides and/or polynucleotides comprising the modified bases are rendered substantially acid and nuclease resistant

The ring structure of the nbose group of the nucleotides in the modified oligonucleotide and/or polynucleotide may also have an oxygen in the ring structure substituted with N-H, N-R, S and/or methylene

Although 2'-0-alkyl substituted ohgonucleotides and/or polynucleotides exhibit marked acid stability and endonuclease resistance,

are sensitive to 3' exonucleases In order to enhance the exonuclease resistance of 2'-0-alkyl substituted ohgonucleotides and/or polynucleotides, the 3' or 5' and 3' ends of the nboohgonucleotide sequence are preferably attached to an exonuclease blocking function For example, one or more phosphorothioate nucleotides can be placed at either end of the oligonbonucleotide Additionally, one or more inverted bases can be placed on either end of the ohgoribonucleotide, or one or more alkyls. e g butanol-substituted nucleotides or chemical groups, can be placed on one or more ends of the oligonbonucleotide Accordmgly, a prefened embodiment of the present mvention is an oligonucleotide compnsmg a oligonucleotide havmg the following structure

A-B-C wherem "B" is a 2'-0-alkyl or 2'-0-alkyl-n(0-alkyl) oligonbonucleotide between about 2 and about 300 bases m length, and "A" and "C" are respective 5' and 3' end blocking groups (e g , one or more phosphorothioate nucleotides (but typically fewer than six), inverted base linkages, or alkyl, alkenyl, or alkynl groups or substituted nucleotides or 2'-0-alkyl-n(0-alkyl)) A partial list of blockmg groups mcludes inverted bases, dideoxynucleotides. methylphosphates, alkyl groups, aryl groups, cordycepm, cytosine arabanoside, 2'-methoxy, ethoxy nucleotides, phosphoramidates, a peptide linkage, dinitrophenyl group, 2'- or 3'-0-methyl bases with phosphorothioate linkages, 3'-0-methyl bases, fluorescent, cholesterol, biotin, acndine. rhodamine, psoralen, glyceryl, methyl phosphonates, butanol, butyl, hexanol, and 3'-0-alkyls An enzyme-resistant butanol preferably has the structure OH- CH₂CH₂CH₂CH₂ (4-hydroxybutyl) which is also referred to as a C4 spacer

OLIGONUCLEOTIDE AND POLYNUCLEOTIDE SYNTHESIS

Ohgonucleotides can be synthesized on commercially purchased DNA synthesizers from <luM to >lmM scales usmg standard phosphoramidite chemistry and methods that are well known in the art, such as, for example, those disclosed in Stec et al , J Am Chem Soc 106 6077-6089 (1984), Stec et al , J. Org Chem 50(20) 3908-3913 (1985), Stec et al , J Chromatog 326 263-280 (1985), LaPlanche et al , Nuc Acid Res 14(22) 9081-9093 (1986), and Fasman, Practical Handbook of Biochemistry and Molecular Biology. 1989, CRC Press, Boca Raton, FL, herem incorporated by reference Ohgonucleotides can be deprotected following phosphoramidite manufacturer's protocols Unpuπfied ohgonucleotides may be dried down under vacuum or precipitated and then dried Sodium salts of ohgonucleotides can be prepared usmg the commercially available DNA-Mate (Barkosigan Inc ) reagents or conventional techniques such as a commercially available exchange resin, e g , Dowex, or by addition of sodium salts follow ed by precipitation, diafiltration, or gel filtration, etc Ohgonucleotides to be purified can be chromatographed on commercially available reverse phase or ion exchange media, e g , Waters Protein Pak Pharmacia's Source Q, etc Peak fractions can be combined and the samples desalted and concentrated by means of reverse phase chromatography on poly(styrene-divinylbenzene) based columns like Hamilton's PRP. or Polymer Labs PLRP Alternatively, ethanol precipitation diafiltration, or gel filtration may be used followed by lyophihzation or solvent evaporation under \ acuum in commercially available instrumentation such as Savant's Speed Vac Optionally, small amounts of the ohgonucleotides may be electrophoretically punfied usmg polyacrylamide gels

Lyophilized or dried-down preparations of ohgonucleotides can be dissolved in pyrogen-free, stenle, physiological saline (I e , 0 85 %> salme) sterile Sigma water, and filtered through a 0 45 micron Ge nan filter (or a stenle 0 2 micron pyrogen-free filter) The descnbed ohgonucleotides may be partially or fully substituted with any of a broad vanety of chemical groups or linkages mcludmg, but not limited to phosphoramidates, phosphorothioates, alkyl phosphorates, 2'-0-methyls, 2'-modιfied RNAs, morphohno groups, phosphate esters, propyne groups, or chimencs of any combmation of the above groups or other linkages (or analogs thereof)

A vanety of standard methods can be used to punfy the presently descnbed ohgonucleotides In bnef, the ohgonucleotides of the present mvention can be punfied by chromatography on commercially available reverse phase (for example, see the RAININ Instrument Co , Inc instruction manual for the DYNAMAX®-300A, Pure-DNA reverse-phase columns, 1989, or current updates thereof, herein incorporated by reference) or ion exchange media such as Waters' Protem Pak or

Pharmacia's Source Q (see generally, Wanen and Vella, 1994, "Analysis and Purification of Synthetic Nucleic Acids by High-Performance Liquid Chromatography", m Methods in Molecular Biology, vol 26, Protocols for Nucleic Acid Conjugates. S Agrawal, Ed , Humana Press, Inc , Totowa, NJ, Aharon et al , 1993, J Chrom 698 293-301. and Milhpore Technical Bulletin, 1992, Ant sense DNA Synthesis, Purification, and Analysis) Peak fractions can be combmed and the samples concentrated and desalted via alcohol (ethanol, butanol. isopropanol, and isomers and mixtures thereof, etc ) precipitation, reverse phase chromatography. diafiltration, or gel filtration

The modified polynucleotides and ohgonucleotides that are associated on the array may also be produced used established techniques such as polymerase cham reaction (PCR) and reverse transcπption (RT) These methods are similar to those currently known m the art (see e g , PCR Strategies Michael A Innis (Editor), et al (1995) and PCR Introduction to Biotechmques Series, C R Newton, A Graham (1997)), and preferabh the enzymes used to produce the polynucleotides or ohgonucleotides are optimized for incorporation of modified nucleotide monomers Methods of identifying which enzymes are best suited for incorporation of nucleotide monomers with specific modifications (e g , which enzymes will best incorporate 2'-modιfied dNTPs) are well known in the art, and thus one skilled m the art would be able to identify enzymes for use with the present invention based upon this disclosure For example, the process directed evolution can be used to unveil mechanisms of both thermal adaptation and incorporation efficiency, and is an effective and efficient approach to identifying optimal enzyme actmts Multiple generations of random mutagenesis, recombmation and high throughput can be used to create a polymerase that both mcorporates modified nucleotide monomers, e g , 2'-0-methyl substituted dNTPs. and remains thermostable at higher temperatures See e g , Zhao H, et al 12 47-53 (1999)

Other methods of altering catalytic activity include site-directed mutagenesis, codon-level mutagenesis and methods of incorporating deletions or insertions into available enzymes Genomic sequencing programs may also reveal conseπ ed regions m the enzyme structure and regions of vanabi ty between enzymes from closely related species, thus identifying regions of an enzyme that may be altered without affecting the desired activity It would be well withm the skill of one m the art to use such techniques to identify an enzyme ith optimal performance for producing the modified polynucleotides and ohgonucleotides of the invention Techmques for identification of specific enzymes for production of polynucleotides for association on the arrays of the invention are descnbed m Schmidt-Dannert C, et al , Trends Bwtechnol 17 135-6 (1999), Moreno-Hagelsieb G, et al , Bwl Res 29 127-40 (1996), Colacmo F, et al , Bwtechnol Genet Eng Rev 14 211-77 (1997), Soberon X Nat Bwtechnol 17 539-40 (1999), Arnold FH, et al , Ann N YAcadSci 870 400-3 (1999), and Joo H, et al , Nature 399 670-3 (1999), each of which are mcorporated herem by reference to describe such techmques and enzyme design

An oligonucleotide or polynucleotide is considered pure when it has been isolated so as to be substantially free of, inter aha, incomplete products produced dunng the synthesis of the desired oligonucleotide or polynucleotide Preferabh . a punfied oligonucleotide or polynucleotide will also be substantially free of contaminants which may hinder or otherwise mask the bmdmg activity of the molecule

ARRAY CONSTRUCTION

The arrays of the subject mvention have a plurality of associated modified ohgonucleotides and/or polynucleotides stably associated with a surface of a solid support, e g , covalently attached to the surface with or without a linker molecule Each associated sample on the array comprises a modified oligonucleotide composition, of known identity, usually of known sequence, as described m greater detail below Any conceivable substrate may be employed m the invention

In the arrays of the invention, the modified oligonucleotide compositions are stably associated with the surface of a solid support, where the support may be a flexible or ngid solid support By "stably associated ' is meant that the sample of associated modified ohgonucleotides and/or polynucleotides maintain their position relative to the solid support under hvbndization and washing conditions As such, the samples can be non-covalently or covalently stably associated with the support surface Examples of non-covalent association mclude non-specific adsorption, bmdmg based on electrostatic mteractions (e g , ion pair mteractions), hydrophobic interactions, hydrogen bondmg mteractions, specific bmdmg through a specific binding pair member covalently attached to the support surface, and the like Examples of covalent bindmg mclude covalent bonds formed between the ohgonucleotides and a functional group present on the surface of the rigid support (e g , -OH), where the functional group may be naturalK occurring or present as a member of an mtroduced linking group, as descnbed m greater detail below

As mentioned above, the array is present on either a flexible or ngid substrate A flexible substrate is capable of being bent, folded or similarly manipulated without breakage Examples of solid matenals which are flexible solid supports with respect to the present mvention include membranes, e g , nylon, flexible plastic films, and the like By "rigid" is meant that the support is solid and does not readily bend, l e , the support is not flexible As such, the rigid substrates of the subject arrays are sufficient to provide physical support and structure to the associated ohgonucleotides and/or polynucleotides present thereon under the assay conditions in which the array is employed, particularly under high throughput handling conditions Furthermore, when the rigid supports of the subject mvention are bent, they are prone to breakage The substrate may be biological, nonbiological, organic, inorganic, or a combmation of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillanes, pads, slices, films, plates, slides, etc The substrate may have any convenient shape, such as a disc, square, sphere, circle, etc

The substrate is preferably flat but may take on a vanety of alternative surface configurations For example, the substrate may contam raised or depressed regions on which the synthesis takes place The substrate and its surface preferably form a ngid support on which to carry out the reactions descnbed herem The substrate and its surface are also chosen to provide appropnate light-absorbing characteristics For instance, the substrate may be a polymenzed Langmuir Blodgett film, functiona zed glass, Si, Ge, GaAs, GaP, Sι0₂, SPN₄, modified silicon, or any one of a wide vanety of gels or polymers such as (poly)tetrafluoroethvlene, (poly)vιnylιdenedιfluonde, polystyrene, polycarbonate or combinations thereof Other substrate matenals will be readily apparent to those of skill m the art upon review of this disclosure

In a prefened embodiment the substrate is flat glass or single-crystal silicon with surface relief features of less than 10 angstroms Accordmg to some embodiments, the surface of the substrate is etched using well known techniques to provide for desired surface features For example, by way of the formation of trenches, v-grooves, mesa structures, or the like, the synthesis regions may be more closely placed within the focus point of impinging light, be provided with reflective "mirror" structures for maximization of light collection from fluorescent sources, etc

Surfaces on the solid substrate will usually, though not always, be composed of the same matenal as the substrate Alternatively, the surface may be composed of any of a wide variety of matenals, for example, polymers, plastics, resms polvsacchandes, silica or silica-based matenals, carbon, metals, morgamc glasses, membranes or any of the above-listed substrate matenals In some embodiments the surface may provide for the use of caged binding members which are attached firmly to the surface of the substrate Preferably, the surface will contain reactive groups, which could be carboxyl, ammo, hydroxyl, or the like Most preferably, the surface will be optically transparent and will have surface Sι--OH functionalities, such as are found on silica surfaces

The surface of the substrate is preferably provided with a layer of linker molecules, although it will be understood that the linker molecules are not required elements of the mvention The linker molecules are preferably of sufficient length to permit modified ohgonucleotides and/or polynucleotides of the mvention and on a substrate to hybridize to natural nucleic acid molecules and to interact freely with molecules exposed to the substrate The linker molecules should be 6-50 atoms long to provide sufficient exposure The linker molecules may also be, for example, aryl acetylene, ethyiene glycol ohgomers containing 2-10 monomer umts, diamines, diacids. amino acids, or combmations thereof Other linker molecules which can bmd to modified ohgonucleotides of the invention may be used in light of this disclosure

The linker molecules can be attached to the substrate via carbon-carbon bonds usmg, for example, (poly)tnf_uorochloroethylene surfaces, or preferably, by siloxane bonds (usmg, for example, glass or silicon oxide surfaces) Siloxane bonds with the surface of the substrate may be formed m one embodiment via reactions of linker molecules bearing tnchlorosilyl groups The linker molecules may optionally be attached m an ordered array, 1 e , as parts of the head groups m a polymenzed Langmuir Blodgett film In alternative embodiments, the linker molecules are adsorbed to the surface of the substrate

In one embodiment of the present mvention, the linker molecules and modified nucleotides used herem are provided with a functional group to which is bound a protective group Preferably, the protective group is on the distal or terminal end of the linker molecule opposite the substrate The protective group may be either a negative protective group (1 e the protective group renders the linker molecules less reactive with a monomer upon exposure) or a positive protective group (1 e , the protective group renders the linker molecules more reactive with a monomer upon exposure) In the case of negative protective groups an additional step of reactivation will be required In some embodiments, this will be done by heating The protective group on the linker molecules may be selected from a wide variety of positive hght-reactive groups preferably mcludmg mtro aromatic compounds such as o-nitrobenzyl derivatives or benzylsulfonyl In a preferred embodiment, 6-mtroveratryloxycarbonyl (NVOC), 2-nιtrobenzyloxycarbonyl (NBOC) or α,α-dιmethyl-dιmethoxybenzyloxycarbon\l (DDZ) is used Photoremovable protective groups are descnbed m, for example, Patchornik, J Am Chem Soc (1970) 92 6333 and Amit et al , J Org Chem (1974) 39 192, both of which are incorporated herem by reference

The substrate, the region for attachment of an individual oligonucleotide group could be of any size or shape For example, squares, ellipsoids rectangles triangles, circles, or portions thereof, along with irregular geometric shapes, may be utilized Duplicate synthesis regions may also be applied to a smgle substrate for purposes of redundancy The regions on the substrate can have a surface area of between about 1 cm² and 10 ¹⁰ cm² Preferabh. the regions have areas of less than about 10 ^x to 10⁷ cm², more preferably less than 10³ to 10⁶ cm², and even more preferably less than 10⁵ cm²

A smgle substrate supports more than about 10 different oligonucleotide and/or polynucleotide compositions and preferably more than about 100 different oligonucleotide and or polynucleotide compositions, although m some embodiments more than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10^s different compositions are provided on a substrate Of course, within a region of the substrate m which a modified oligonucleotide or polynucleotide is attached, it is preferred that the modified nucleotides be substantially pure In preferred embodiments regions of the substrate contam ohgonucleotides or polynucleotides which are at least about 50%. preferably 80%, more preferably 90%, and even more preferably, 95% pure Ohgonucleotides or polynucleotides having several sequences can be intentionally provided withm a single region so as to provide an initial screening for biological activity, after which matenals withm regions exhibiting significant bmdmg are further evaluated In a preferred embodiment, each region will contam a substantially pure modified oligonucleotide or polynucleotide composition havmg a smgle sequence The substrates of the arrays of the mvention compnse at least one surface on which the pattern of associated ohgonucleotides and/or polynucleotides is present, where the surface may be smooth, substantially planar, or have irregulanties. such as depressions or elevations The surface on which the pattern of associated nucleic acids present may be modified with one or more different layers of compounds that serve to modify the properties of the surface in a desirable manner Such modification layers, when present, will generally range m thickness from a monomolecular thickness to about 1 mm, usually from a monomolecular thickness to about 0 1 mm and more usually from a monomolecular thickness to about 0 001 mm Modification la\ ers of mterest include inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like

The amount of modified oligonucleotide or polynucleotide present m each composition will be sufficient to provide for adequate hybridization and detection of nucleic acids during the assay m which the array is employed Generally, the amount of oligonucleotide or polynucleotide in each composition will be at least about 0 1 ng usually at least about 0 5 ng and more usually at least about 1 ng, where the amount may be as high as 1000 ng or higher, but will usually not exceed about 20 ng and more usually will not exceed about 10 ng The copy number of each oligonucleotide or polynucleotide m a composition will be sufficient to provide enough hybndization sites to yield a detectable signal, and will generally range from about 0 01 frnol to 50 fmol, usually from about 0 05 fmol to 20 frnol and more usually from about 0 1 frnol to 5 frnol Where the composition has an overall circular dimension, the diameter of the sample will generally range from about 10 to 5,000 μm, usually from about 20 to 2,000 μm and more usually from about 50 to 1000 μm Control composition may be present on the array mcludmg compositions compnsmg ohgonucleotides or polynucleotides correspondmg to genonuc DNA, housekeepmg genes, negative and positive control genes, and the like These latter types of compositions are not "umque" as that term is defined and used herem, I e , they are "common " In other words, they are calibrating or control genes whose function is not to tell whether a particular "key" gene of interest is expressed, but rather to provide other useful information, such as background or basal level of expression The percentage of samples which are made of unique ohgonucleotides or polynucleotide that correspond to the same type of gene is generally at least about 30%, and usually at least about 60% and more usually at least about 80% Preferably, the arrays of the present mvention will be of a specific type, where representative array types mclude human arrays, mouse arrays, cancer arrays, apoptosis arrays, human stress arrays, oncogene and tumor suppressor arrays, cell-cell mteraction arrays, cytokine and cytokine receptor arrays, rat arrays, blood anays, mouse stress arrays, neuroarrays. and the like

With respect to the oligonucleotide and or polynucleotide compositions that correspond to a particular type or land of gene, type or land can refer to a plurality of different charactenzing features, where such features mclude species specific genes, where specific species of mterest include eukaryotic species, such as mice, rats, rabbits, pigs, primates, humans, etc , function specific genes, where such genes mclude oncogenes, apoptosis genes, cytokines, receptors, protem kinases, etc , genes specific for or involved m a particular biological process, such as apoptosis, differentiation, cell cycle regulation, cancer, aging, proliferation, etc . location specific genes, where locations mclude organs, such as heart, liver, prostate, lung etc , tissue, such as nerve, muscle, connective, etc , cellular, such as axonal, lymphocytic, etc , or subcellular locations, e g , nucleus, endoplasmic reticulum, Golgi complex, endosome, lyosome, peroxisome mitochondna cytoplasm, cytoskeleton. plasma membrane, extracellular space specific genes that change expression level over time, e g . genes that are expressed at different levels during the progression of a disease condition, such as prostate genes which are induced or repressed dunng the progression of prostate cancer In a preferred embodiment, longer ohgonucleotides, preferably from 80-300 nt in length, more preferably from 100-200 nt m length, are used on the arrays These are especially useful m place of cDNAs for determining the presence of mRNA m a sample, as the modified ohgonucleotides have the advantage of rapid synthesis and punfication and analysis prior to attachments to the substrate surface In particular, ohgonucleotides with 2¹ modified sugar groups show mcreased bmdmg affimty with RNA, and these ohgonucleotides are particularly advantageous m identifying mRNA in a sample exposed to an array

The length of the modified ohgonucleotides allows the compositions to bind with the same affinity as a much longer unmodified nucleic acid, e g an unmodified cDNA In the case where additional complementanty is needed to certain domains or regions found in cDNA, multiple ohgonucleotides may be used Multiple ohgonucleotides directed at a particular gene or RNA molecule may be mterspersed in a single region or the different ohgonucleotides may each be m a discrete region, e g to determine presence or absence of related molecules m a sample

As mentioned above, the arrays of the present mvention typically compnse one or more additional associated oligonucleotide composition which does not correspond to the array type, I e , the type or land of gene represented on the arra In other words, the array may comprise one or more compositions that are made of non "umque" ohgonucleotides, e g , ohgonucleotides correspondmg to commonly expressed genes For example compositions compnsmg ohgonucleotides that bmd to plasmid and bactenophage ohgonucleotides. ohgonucleotides which bind to genes from the same or another species which are not expressed and do not cross-hybndize with the test nucleic acid, and the like, may be present and serve as negative controls In addition, compositions compnsmg housekeepmg genes and other control genes from the same or another species may be present, e g , to serve in the normalization of mRNA abundance and standardization of hybndization signal intensity m the sample assayed with the anay

Patents and patent applications descnbmg arrays of ohgonucleotides and methods for their fabncation mclude 5,242,974, 5,384,261. 5,405,783, 5,412,087, 5,424,186. 5,429,807, 5,436,327, 5,445,934, 5,472,672, 5,527,681, 5,529.756, 5,545,531, 5,554,501, 5,556,752, 5,561,071, 5,599,895, 5,624,711, 5,639,603, 5,658.734, 5,700,637, 5,744,305, 5,837,832, 5,843,655, 5,861,242, 5,874.974, 5,885,837, WO 93/17126, WO 95/11995, WO 95/35505, EP 742 287, and EP 799 897 Patents and patent applications descnbmg methods of using arrays m vanous applications mclude 5,143,854, 5,288,644, 5,324,633 5,432,049, 5,470,710, 5,492,806. 5.503,980, 5,510,270, 5,525,464, 5.547,839, 5,580,732. 5.661,028. 5 848.659. 5.874,219, WO 95/21265, WO 96/31622, WO 97/10365, WO 97/27317, EP 373 203, and EP 785 280 References that disclose the synthesis of arrays and reagents for use with arrays include Matteucci M D and Caruthers M H , J Am Chem Soc (1981) 103 3185-3191, Beaucage S L and Caruthers M H , Tetrahedron Letters, (1981) 22(20) 1859-1862, Adams S P et al . JAm Chem Soc (1983) 105 661-663, Sproat D S and Brown D M , Nucleic Acids Research, (1985) 13(8) 2979-2987, Crea R and Horn T , Nucleic Acids Research. (1980) 8(10) 2331-48, Andrus A et al . Tetrahedron Letters, (1988) 29(8) 861-4, Applied Biosystems User Bulletin, Issue No 43 Oct 1, 1987, "Methyl phosphonamidite reagents and the synthesis and punfication of methyl phosphonate analogs of DNA", Miller P S et al , Nucleic Acids Research, (1983) 11 6225-6242 Each of these is mcorporated herein by reference as exemplary methods of construction and use of anays of the present invention The methods of these publications can be readily modified to produce the arrays of the invention with the modified ohgonucleotides of the invention on their surface

In a preferred embodiment, the modified ohgonucleotides for use with the present invention are synthesized pnor to attachment onto the substrate This affords the advantages that (1) ohgonucleotides of known composition and sequence can be produced, (2) ohgonucleotides can be analyzed and punfied prior to attachment, which eliminates "shortmers," I e , ohgonucleotides with insufficient length and/or incorrect sequence, (3) the methods used to produce ohgonucleotides are less prone to error than current methods for production of cDNA, e g PCR with Taq polymerase, and (4) attachment to the substrate may be momtored or assayed without destroymg the array

Numerous methods can be used for attachment of the ohgonucleotides of the mvention to the substrate For example, modified ohgonucleotides can be attached usmg the techmques of, for example U S Patent No 5,807,522, which is mcorporated herein by reference for teaching methods of polymer attachment Other similar methods may be used, as will be apparent to one skilled m the art upon reading the present technology

USE OF ARRAYS OF THE INVENTION

Oligonucleotide and/or polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotides m a sample A vanety of different array formats have been developed and are known to those of skill m the art The arrays of the subject mvention find use m a vanety of applications, mcludmg gene expression analysis, drug screening, mutation analysis and the like.

Arrays can be used, for example, to examine differential expression of genes and can be used to determine gene function For example, anays can be used to detect differential expression of a polynucleotide between a test cell and control cell (e g , cancer cells and normal cells) For example, high expression of a particular message in a cancer cell, which is not observed in a conesponding normal cell, can indicate a cancer specific gene product. Exemplary uses of arrays are further described in, for example. Pappalarado et al , Sem. Radiation Oncol. 8:217 (1998), and Ramsay, Nature Biotechnol. 16:40 (1998). Methods for analyzing the data collected from hybridization to arrays are well known in the art. For example, where detection of hybridization involves a fluorescent label, data analysis can include the steps of determining fluorescent intensity as a function of substrate position from the data collected, removing outliers, i.e., data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the test nucleic acids from the remaining data. The resulting data can be displayed as an image with the intensity in each region varying according to the binding affinity between associated ohgonucleotides and or polynucleotides and the test nucleic acids.

Ohgonucleotides having a sequence unique to that gene are preferably used in the present invention. Different methods may be employed to choose the specific region of the gene to be targeted. A rational design approach may also be employed to choose the optimal oligonucleotide sequence for the hybridization array. Preferably, the region of the gene that is selected is chosen based on the following criteria. First, the sequence that is chosen should yield an oligonucleotide composition that preferably does not cross-hybridize with any other oligonucleotide composition present on the array. Second, the sequence should be chosen such that the oligonucleotide composition has a low probability of cross- hybridizing with an oligonucleotide having a nucleotide sequence found in any other gene, whether or not the gene is to be represented on the anay from the same species of origin, e.g. , for a human array, the sequence will not be present in any other human genes. As such, sequences that are avoided include those found in: highly expressed gene products, structural RNAs, repeated sequences found in the sample to be tested with the array and sequences found in vectors. A further consideration is to select ohgonucleotides with sequences that provide for minimal or no secondary structure, structure which allows for optimal hybridization but low non-specific binding, equal or similar thermal stabilities, and optimal hybridization characteristics.

EXEMPLARY ARRAY TYPES OF THE INVENTION

A variety of specific array types are also provided by the subject invention. As discussed above, array type refers to the nature of the oligonucleotide and/or polynucleotide compositions present on the array and the types of genes to which the associated compositions correspond. These array types include, but are not limited to: human array; mouse anay; developmental array; cancer array; apoptosis array; oncogene and tumor suppressor array; cell cycle gene array; cytokine and cytokine receptor array; growth factor and growth factor receptor array; neuroarrays; and the like. In certain embodiments of the human ana\ human genes that mav be represented by the composition on the arrays include those for (a) oncogenes and tumor suppressors, (b) cell cycle regulators, (c) stress response proteins, (d) ion channel and transport protems (e) intracellular signal transduction modulators and effectors, (f) apoptosis-related proteins (g) DNA synthesis, repair and recombination proteins, (h) transcription factors and general DNA binding proteins, (1) growth factor and chemokme receptors, (j) interleulαn and interferon receptors, (k) hormone receptors, (1) neurotransmitter receptors, (m) cell surface antigens and cell adhesion protems, (n) growth factors, cytokines and chemokmes, (o) mterleukins and mterferons, (p) hormones, (q) extracellular matrix protems, (r) cytoskeleton and motihty protems (s) RNA processmg and turnover protems, (t) post- translational modification, trafficking and targeting protems, (u) protem turnover, and (v) metabolic pathway protems

The arrays of the invention can be used m among other applications differential gene expression assays Thus, anays are useful m the differential expression analysis of (a) diseased and normal tissue, e g , neoplastic and normal tissue (b) different tissue or tissue types, (c) developmental stage, (d) response to external or internal stimulus, (e) response to treatment and the like The arrays are also useful m broad scale expression screening for drug discovery and research, such as the effect of a particular active agent on the expression pattern of genes m a particular cell, where such information can be used to reveal drug toxicity, carcinogenicity, etc , environmental momtonng, disease research and

HYBRIDIZATION AND DETECTION Following preparation of the test nucleic acids from the tissue or cell of mterest, the test sample is contacted with the array under hybndization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity m view of the particular assay being performed In analyzmg the differences m the population of labeled test bmdmg agents generated from two or more physiological sources usmg the anays descnbed above, each population of labeled test samples are separately contacted to identical arrays or together to the same array under conditions of hybridization, preferably under stnngent hybndization conditions (for example, at 50 °C or higher and 0 1X SSC (15 mM sodium chlonde/01 5 πiM sodium citrate)), such that test nucleic acids hybndize to complementary ohgonucleotides and/or polynucleotides on the substrate surface

Where all of the test nucleic acids have the same label, different arrays can be employed for each physiological source Preferably, the same array can be employed sequentially for each physiological source, with test samples removed from the array as described below Alternatively, where the labels of the test nucleic acids are different and distinguishable for each of the different physiological sources being assayed, the opportunity anses to use the same array at the same time for each of the different test populations Examples of distinguishable labels are well known in the art and include two or more different emission wavelength fluorescent dyes, like C\ 3 and Cy5, two or more isotopes with different energies of emission, like ³²P and ³³P, labels which generate signals under different treatment conditions, like temperature pH treatment by additional chemical agents, etc , or generate signals at different time points after treatment Using one or more enzymes for signal generation allows for the use of an even greater \ anet} of distinguishable labels, based on different substrate specificity of enzymes (e g , alkaline phosphatase/peroxidase)

Following hybridization, non-hybndized labeled nucleic acid is removed from the support surface, convemently by washing, generating a pattern of hybndized oligonucleotide and/or polynucleotide on the substrate surface A vanety of wash solutions are known to those of skill m the art and may be used The resultant hybndization patterns of labeled, hybridized ohgonucleotides and/or polynucleotides may be visualized or detected m a vanety of ways, with the particular manner of detection being chosen based on the particular label of the test nucleic acid, where representative detection means include scintillation countmg autoradiography, fluorescence measurement, colonmetnc measurement, light emission measurement and the like

Following detection or visualization, the hybndization patterns may be compared to identify differences between the patterns Where arrays in which each of the different ohgonucleotides and/or polynucleotides corresponds to a known gene are employed, any discrepancies can be related to a differential expression of a particular gene m the physiological sources bemg compared

CLEARING OF TEST NUCLEIC ACIDS FROM ARRAY Following bmdmg and visualization of a test sample on an array, the array may be treated to remove the bound test nucleic acids The associated nucleic acid compositions remam mtact following treatment, allowing reuse of the treated arra> The array of the mvention substantially retams its bmdmg capabilities, and any differences m bmdmg ability may be determmed usmg control sequences associated on the array Preferably, the array of the mvention retams at least 75% of its bmdmg capabilities, more preferably the array retams at least 85% of its bmdmg capabilities, and even more preferably the array of the mvention retams at least 95% of its bmdmg capabilities

Arrays with associated modified oligonucleotide and/or polynucleotide compositions can be exposed to a low pH environment, e g . pH from 0 5 -4 5, which results m the degradation of non- modified nucleic acids Following the treatment, the arrays of the mvention are rinsed to remove any unwanted test nucleic acid fragments, residual label and the like, and the arrays are prepared for reuse After detection of the test sample is complete, the array may be regenerated by removal and/or degradation of the test sample For example, a two hour incubation of the sample-bound array m an acid solution at pH 1 5, 39 °C, results m complete loss of a full-length unmodified 14-mer oligonucleotide Under these conditions the bound arra\ ohgonucleotides of the mvention maintain full length structural integrity Following the acid incubation a variety of wash conditions may be used to clear the test sample from the probe array For example, mcreased temperature incubation of a low salt wash solution would result in the dissociation of short test fragments from the array Alternatively, a chemical denaturant (e g , urea) could be used as a wash to remove the test sample Additional steps, such as an alkaline solution rinse may also be added to the protocol to speed up the cycle time for regeneration

The above-described washes and rinses can be avoided if the acid incubation is mcreased resulting in almost complete degradation of the test sample under conditions where the array probe mamtams its mtegnty Actual incubation tunes required will vary somewhat from array type to array type, and may be shorter than those given below As a consequence of the degradation of the test sample the array probe/test sample hybrids become unstable under expenmental conditions and may be removed usmg rmses of the hybridization or strmgent wash buffer

Exemplary clearmg conditions for use with the arrays of the invention are (1) Incubation of the bound array with pH 1-2 acid solution, 8 hours at 39 °C Follow with three rmses at 39 °C with strmgent wash buffer. 0 1 X SSC pH 7 0, and two rmses with hybndization buffer, pH approximately 7 0 These two solutions are for removal of degraded sample and the regeneration of the substrate array and hence do not require a low pH Array may then be reused

(2) Incubation of the bound array with pH 1-2 acid solution, 4 hours at 39 °C Follow with three 15 mmute rmses at 39 °C with 8 0 molar urea Rinse once with strmgent wash buffer, and twice with hybridization buffer Array can be reused at this pomt

(3) Incubation of the bound array with pH 1-2 acid solution, 4 hours at 39 °C Rinse twice at 39 °C with strmgent wash buffer Incubate 20 mmutes m 60 °C strmgent wash buffer, and raise twice more with 60 ° C strmgent wash buffer Rinse twice with hybndization buffer Array can be reused at

(4) Incubation of the bound array with pH 1-2 acid solution, 4 hours at 39°C Rinse twice with strmgent wash buffer Wash twice with 39 ° C alkaline solution for 15 mmutes followed by two washes with strmgent wash buffer Incubate 20 minutes m 60 ° C strmgent wash buffer Rinse twice more with 60 °C strmgent wash buffer, and twice with hybndization buffer Array can be reused at this pomt

(5) Incubation of the bound anay with nuclease (actual conditions vary with nuclease type) at 37°C for 1 hour Wash twice with protein denaturing solution for 20 minutes Rinse twice with strmgent wash buffer Incubate 20 minutes m 60°C strmgent wash buffer Rinse twice with 60°C stringent wash buffer Rinse twice with hybridization buffer Array can be reused at this pomt (6) Incubation of the bound arra\ with pH 10-13 base solution (e g , NaOH) at room temperature for 1-30 mmutes followed by additional washes with pH 10-13 base solutions, water, or acidic solution washes followed by a buffer wash

Following treatment, the associated acid stable ohgonucleotides of the array remain 1) associated to the substrate surface, 2) structuralh mtact, and 3) capable of bmding with another test bmdmg partner

In addition, as an alternative way.

s with associated ohgonucleotides charactenzed as nuclease resistant may be treated with a nuclease to remove bound test nucleic acids and label The nuclease used can be chosen depending on the nature of the binding between the associated oligonucleotide and/or polynucleotide and the molecules of the test sample and the attachment of the oligonucleotide and/or polynucleotide to the arra} For example, if the associated ohgonucleotides are end-blocked ohgonucleotides. and the test sample is compnsed of mRNA molecules, then the appropnate nuclease would be one that recognizes RNA-DNA hybrids, e g , Ribonuclease H In another example, if the associated ohgonucleotides are end-blocked ohgonucleotides, and the test sample is compnsed of cDNA molecules, then the appropnate nuclease would be one that recognizes double stranded DNA complexes, e g , Deoxyπbonuclease I or II, and Exodeoxynbonuclease III or V In yet another example, if the associated ohgonucleotides are end-blocked cRNA and the test sample is compnsed of mRNA, the appropnate nuclease is one that recognizes RNA-RNA hybnds, such as micrococcal nuclease Similarly, nucleases that are 5' or 3' specific may be chosen dependmg on the attachment site of the oligonucleotide and/or polynucleotide to the array Since the ohgonucleotides of this embodiment of the invention are nuclease-resistant, the test samples will be specifically targeted and degraded by the nuclease

Actual choice of regeneration conditions should take into consideration the type of substrate, the type of attachment of probe to substrate, test sample type, and whether there are clearing time constraints In cases where the substrate is acid sensitive it would be more advantageous to use nuclease digestion to remove the test sample from the array Such modifications would be well within the skill of one m the art upon reading the present disclosure and descnption of the subject anays

KITS HAVING ARRAYS OF PRESENT INVENTION Also covered are kits for performing analyte bmdmg assays usmg the arrays of the present mvention Such kits accordmg to the subject mvention will at least compnse the arrays of the mvention having associated modified ohgonucleotides and/or polynucleotides Kits also preferably compnse an agent for removal of test bmdmg agents, e g , a solution with low pH and or with nuclease activity The kits may further compnse one or more additional reagents employed m the vanous methods, such as 1) primers for generatmg test nucleic acids. 2) dNTPs and/or rNTPs (either premixed or separate), optionally with one or more uniquely labeled dNTPs and/or rNTPs (e g , biotinylated or Cy3 or Cy5 tagged dNTPs) 3) post synthesis labeling reagents, such as chemically actπe derivatives of fluorescent dyes, 4) enzymes, such as reverse transcnptases DNA polymerases, and the like, 5) various buffer mediums, e g hybridization and washmg buffers, 6) labeled probe purification reagents and components like spin columns etc , and 7) signal generation and detection reagents, e g streptavidm- alkahne phosphatase conjugate chemifluorescent or chemiluminescent substrate, and the like

EXAMPLES The present invention and its particular embodiments are illustrated m the following examples The examples are not intended to limit the scope of this invention but are presented to illustrate and support the claims of this present invention

EXAMPLE 1 Synthesis and Purification of Modified Nucleic Acids

Ohgonucleotides were synthesized using commercial phosphoramidites on commercially purchased DNA synthesizers from <1 uM to >lmM scales usmg standard phosphoramidite chemistry and methods that are well known m the art, such as, for example, those disclosed in Stec et al , J Am Chem Soc 106 6077-6089 (1984), Stec et al J Org Chem 50(20) 3908-3913 (1985), Stec et al , J Chromatog 326 263-280 (1985), LaPlanche et al , Nuc Acid Res 14(22) 9081-9093 (1986), and Fasman, Practical Handbook of Biochemistry and Molecular Biology, 1989. CRC Press, Boca Raton, FL, herem incorporated by reference

Ohgonucleotides were deprotected following phosphoramidite manufacturer's protocols Unpunfied ohgonucleotides were either dned down under vacuum or precipitated and then dned Sodium salts of ohgonucleotides were prepared usmg the commercially available DNA-Mate (Barkosigan Inc ) reagents or conventional techmques such as commercially available exchange resm, e g , Dowex, or by addition of sodium salts followed by precipitation, diafiltration, or gel filtration, etc A vanety of standard methods were used to punfy and produce the presently descnbed ohgonucleotides In bnef, ohgonucleotides were purified by chromatography on commercially available reverse phase (for example, see the RAININ Instrument Co , Inc instruction manual for the DYNAMAX®-300A, Pure-DNA reverse-phase columns, 1989, or current updates thereof, herem incorporated by reference) or ion exchange media such as Waters' Protem Pak or Pharmacia's Source Q (see generally Warren and Vella, 1994, "Analysis and Punfication of Synthetic Nucleic Acids by High- Performance Liquid Chromatography". mMethods in Molecular Biology, vol 26, Protocols for Nucleic Acid Conjugates, S Agrawal, Ed Humana Press, Inc , Totowa, NJ, Aharon et al , 1993, J Chrom 698 293-301, and Milhpore Technical Bulletin, 1992, Antisense DNA Synthesis, Purification, and Analysis) Peak fractions were combmed and the samples were concentrated and desalted via alcohol (ethanol, butanol, isopropanol. and isomers and mixtures thereof, etc ) precipitation, reverse phase chromatography, diafiltration, or gel filtration or size-exclusion chromatography

Lyophilized or dried-down preparations of ohgonucleotides were dissolved in pyrogen-free, stenle, physiological salme (1 e , 0 85% salme). sterile Sigma water, and filtered through a 0 45 micron Gelman filter

EXAMPLE 2 Stability of Modified Oligonucleotide Duplexes

The stability of duplexes havmg 2'-substιtuted nucleotides versus duplexes without such modification was tested by exammmg the T_m of these complexes 4 μM each of 20-mer oligonucleotide (5' - ggt ggt tec tec tea gtc gg -3', SEQ ID NO 1) and its complement (5'- ccg act gag aag gaa cca cc - 3') were bound in a solution of 50 mM NaCl, 10 mM P04 buffer, pH 7 4 Each of the nucleotides of the oligonucleotide had the same 2' group Following bmdmg, the meltmg temperature was determmed as descnbed (See L L Cummins et al . Nucleic Acids Research 23 2019-2024 (1995) Results were as follows SEQ ID NO 1 SEQ ID NO 2 T_m

Regular RNA and Regular DNA 66 °C

Regular RNA and 2'-0-methyl 79 °C

Regular DNA and p-ethoxy DNA 55 °C

Regular RNA and p-ethoxy RNA 56 °C Regular RNA and p-ethoxy 2'-0-methyl 71 °C

The duplexes with the 2'-0-methyl substitutions display a significantly mcreased T_m compared to RNA or DNA with a 2' H or 2' OH, respectively RNA or DNA with propyl or fluoro substitutions at the 2' position display an even higher T_m than does the 2'-0-methyl

EXAMPLE 3 Acid Stability of the Ohgonucleotides of the Invention

Homopolymers of 2'-0-methyl A. C, G, and U twelve bases long, were synthesized with 3' and 5' inverted T-blocked ends They were punfied. desalted, lyophilized, and dissolved at 300 A₂₆₀ per ml in stenle water Samples were removed and diluted 1 to 4 with either 0 1 N HCI or 1 0 N HCI to give final pHs of approximately 1 and 0, respectively, and placed m a heat block at 39 °C Aliquots were taken at 0, 2, 4 and 24 hours, diluted 1 20 mto a solution of 0 025 M NaOH and 0 03 M NaCl, stored at -20 °C until bemg run on an analytical HPLC under strongly denaturing conditions on an anion exchange column % Full LenjTth

Homopolvmer EH O hr 2 hr 4 hr 24 hr

A 1 99 99 99 99

C 1 99 99 99 96 G 1 96 98 98 98

U 1 97 97 97

A 0 99 99 99 99

C 0 99 99 98 97 G 0 96 97 97 89

U 0 97 97 96

It was evident that there is essentially no degradation at pH 1 and 39 °C and only slight degradation over 24 hours at pH 0 and 39°C

EXAMPLE 4 Acid Stability of the Ohgonucleotides of the Invention

A 14 mer heteropolymer was synthesized as a regular phosphodiester DNA (O), a phosphorothioate DNA (S), an unblocked 2 -O-methyl RNA (2'om), a 2 -O-methyl RNA with 3' and 5' butanol blocked ends (B2'om), and a phosphorothioate chimera havmg four 2'-0-methyl phosphorothioate bases on either side of 6 mtenor phosphorothioate DNA bases (SD) They were punfied, desalted, lyophilized, and dissolved at 300 A₂₆₀ per ml m sterile water Samples were removed and diluted 1 to 4 with 0 1 N HCI to give a final pH of approximately 1 5, and placed m a heat block at 39 °C Aliquots were taken at the times mdicated and diluted 1 20 mto a solution of 0 025 M NaOH and 0 03 M NaCl, and were run on an analytical HPLC under strongly denatunng conditions on an anion exchange column Imtially all but the end-blocked 2'-0-methyl RNA solutions became cloudy upon addition of the HCI Upon heatmg. both the phosphodiester DNA and the unblocked 2'-0-methyl RNA became clear The two ohgonucleotides with phosphorothioate linkages appeared cloudy until about 2 hours when they slowly began to clear as they decomposed

% Full Length

Oligo O hr 0.5 hr 1.0 hr 2 hr 4 hr 6 hr 1 d 2 d 3 d 5 d 10 d 20 d

0 99 38 10 0 0 0 0 - — — -

S 95 65 29 1 0 0 0 - - -

SD 97 83 70 49 0 0 0 ~ ~ ~ -

2'om 99 99 99 99 98 98 98 96 94 94 87 80

B2'om 100 100 100 100 99 99 98 97 97 95 90 81

The 2'-0-methyl ohgonucleotides, both unblocked and blocked, are far more stable than the correspondmg phosphodiester, phosphorothioate. or a mixed 2'-0-methyl phosphorothioate structure that Agrawal et al recommended to mcrease bioavailabihty

While the present invention has been descnbed with reference to the specific embodiments thereof, it should be understood by those skilled m the art that various changes may be made and eqmvalents may be substituted without departmg from the true spmt and scope of the mvention In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spint and scope of the present mvention All such modifications are mtended to be within the scope of the claims appended hereto

Claims

CLAIMS That which is claimed is

1 An array comprising a

of modified oligonucleotide compositions stably associated with the surface of a support wherem each oligonucleotide composition is characterized by an oligonucleotide backbone structure modified from that of a naturally occurring nucleotide polymer, wherein the ohgonucleotides of the composition is characterized by a pH stability of at least one hour at 37° C at a pH in a range of about 0 5 to about 6 0

2 The anay of claim 1, further compnsmg a blocking chemical modification at or near at least one end of said oligonucleotide, wherem the oligonucleotide is further characterized by havmg a nuclease resistance of at least twice that of a naturally occurring oligonucleotide having the same sequence and number of bases

3 The array of claim 1, wherem the number of oligonucleotide compositions on said array ranges from about 2 to about 10⁹

4 The array of claim 1, wherem the modified oligonucleotide is further charactenzed by modification of at least 25% of the internucleoside linkages of the oligonucleotide

5 An array compnsmg a plurality of oligonucleotide compositions stably associated with the surface of a support, wherem each oligonucleotide composition is charactenzed by an oligonucleotide backbone structure modified from that of a naturally occurring nucleotide polymer, and a blockmg chemical modification at or near at least one end of the oligonucleotide, wherem the oligonucleotide is charactenzed by a nuclease resistance of at least twice that of a naturally occurnng polymer having the same number of nucleotides 6 An array of modified ohgonucleotides. the array compnsmg a planar, non-porous solid support a surface, a plurality of different modified ohgonucleotides attached to the surface of the solid support at a density exceeding 400 different modified ohgonucleotides/cm², wherein each of the different modified ohgonucleotides is attached to the surface of the solid support m a different predefined region, has a different determmable sequence, and is at least 80 nucleotides m length, and further wherem the modified ohgonucleotides are characterized by a charactenstic selected from the group consisting of (a) a binding affinity of at least about 1 25 times that of a correspondmg. non- modified oligonucleotide, (b) a pH stability of at least one hour at 37° C at a pH m a range of about 0 5 to 6 0, and (c) a nuclease resistance of at least twice that of a naturally occurnng oligonucleotide having the same sequence and number of bases

7 A method of analyzing compnsmg the steps of (a) contacting a first sample of naturally occurnng nucleic acid sequences with an array compnsed of a solid support sequence havmg bound to its surface a plurality of modified nucleic acid sequences,

(b) allowing sequences of the sample to hybndize to the modified sequence of the array,

(c) analyzmg results of the hybndizmg, (d) removing sequences hybndized to sequences of the array usmg a removmg agent selected from the group consistmg of a solution having a pH of less than 6 0 and a nuclease which enzymatically destroys natural nucleic acid sequences, and

(e) repeating (a), (b), (c) and (d) with a second sample of naturally occurnng nucleic acid sequences.

8 The method of claim 7. wherem (a), (b), (c) and (d) are repeated a plurality of times with different samples of naturally occurnng nucleic acid sequences

9 A method for detecting nucleic acid sequences in two or more collections of nucleic acid molecules, the method compπsmg

(a) providing an array of modified polynucleotides bound to a solid surface each said modified polynucleotide comprising a determinable nucleic acid, (b) contacting the array of modified polynucleotides with

(1) a first collection of labeled nucleic acid compnsmg a sequence substantially complementary to a nucleic acid of said array, and (n) at least a second collection of labeled nucleic acid compnsmg a sequence substantially complementary to a modified polynucleotide of said array, wherem the first and second labels are distinguishable from each other, and

(c) detecting hybridization of the first and second labeled complementary nucleic acids to nucleic acids of said arra\ s wherem the modified ohgonucleotides are characterized by a charactenstic selected from the group consistmg of (a) a bmdmg affinity of at least about 1 25 times that of a correspondmg, non- modified oligonucleotide, (b) a pH stability of at least one hour at 37° C at a pH m a range of about 0 5 to 6, and (c) a nuclease resistance of at least twice that of a naturally occurnng oligonucleotide havmg the same sequence and number of bases

10 A method of using a label to detect hybndization with modified polynucleotide probes of known sequence, said method compnsmg

(a) contactmg under hybndization conditions a labeled polynucleotide sequence with a collection of modified polynucleotide probes of known sequences wherem said probes are attached to a substrate at known locations, and

(b) determining the sequences of the probes which hybndize with the labeled polynucleotide, said collection compnsmg at least 100 different probes per square centimeter of substrate