WO2002083837B1 - Methods for identifying small molecules that bind specific rna structural motifs - Google Patents

Methods for identifying small molecules that bind specific rna structural motifs

Info

Publication number
WO2002083837B1
WO2002083837B1 PCT/US2002/011758 US0211758W WO02083837B1 WO 2002083837 B1 WO2002083837 B1 WO 2002083837B1 US 0211758 W US0211758 W US 0211758W WO 02083837 B1 WO02083837 B1 WO 02083837B1
Authority
WO
WIPO (PCT)
Prior art keywords
test compound
libraries
rna
target rna
test compounds
Prior art date
Application number
PCT/US2002/011758
Other languages
French (fr)
Other versions
WO2002083837A1 (en
Inventor
Neil G Almstead
Original Assignee
Ptc Therapeutics Inc
Neil G Almstead
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ptc Therapeutics Inc, Neil G Almstead filed Critical Ptc Therapeutics Inc
Priority to US10/475,026 priority Critical patent/US20050142545A1/en
Publication of WO2002083837A1 publication Critical patent/WO2002083837A1/en
Publication of WO2002083837B1 publication Critical patent/WO2002083837B1/en
Priority to US11/359,721 priority patent/US20060194234A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX

Abstract

The present invention relates to a method for screening and identifying test compounds that bind to a preselected target ribonucleic acid ('RNA'). Direct, non-competitive binding assays are advantageously used to screen bead-based libraries of compounds for those that selectively bind to a preselected target RNA. Binding of target RNA molecules to a particular test compound is detected using any physical method that measures the altered physical property of the target RNA bound to a test compound. The structure of the test compound attached to the labeled RNA is also determined. The methods used will depend, in part, on the nature of the library screened. The methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads.

Claims

AMENDED CLAIMS
[received by the International Bureau on 15 November 2002 (15.11.02); original claim 1 amended; new claims 2-18 added (3 pages)]
L A method for identifying a test compound that binds to a target RN A molecule, comprising the steps of:
(a) contacting a detectably labeled target RNA molecule with a library of solid support-attached test compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of solid support-attached test compounds so that a detectably labeled target RNA:support- attached test compound complex is formed;
(b) separating the detectably labeled target RNA:support-attached test compound complex formed in step (a) from uncornplexed target RNA molecules and test compounds; and
(c) determining a structure of the test compound of the RNA:support-attached test compound complex.
2. The method of claim 1 in which tlie target RNA molecule contains an HIV TAR element, internal ribosome entry site, "slippery site", instability element, or adenylate uridylate-rich element.
3. The method of claim 1 in which the RNA molecule is an element derived from the mRNA for tumor necrosis factor alpha ("TNF-α1"), granulocyte- macrophage colony stimulating factor ("GM-CSF"), interleukin 2 ("IL-2"), interleukin 6 ("IL-6"), vascular endothelial growth factor ("VEGF"), human immunodeficiency virus J ("HTV-r), hepatitis C virus ("HCV" - genotypes la & lb), ribonuclease P RNA ("RNaseF"), X-liπked inhibitor of apoptosis protein ("XIAP"), or survivin,
4. The method of claim 1 in which the detectably labeled RNA is labeled with a fluorescent dye, phosphorescent dye, ultraviolet dye, infrared dye, visible dye, radiolabel, enzyme, spectroscopic colorimetric label, affinity tag, or nanoparticlc.
5. The method of claim 1 in which the test compound is selected from a combinatorial library of solid support-attached test compounds comprising peptoids; random bio-oligomers; diversomers such as hydantoins, benzodiazepines and dipeptides;
86 vinylogous polypeptides; πonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and small organic molecule libraries.
6. The method of claim 5 in which the small organic molecule libraries are libraries of benzodiazepines, isoprenoids, thiarølidiriones, metathiazanones, pyrrølidines, morpholino compounds, or diazepindioncs.
7. The method of claim 1 in which screening a library of solid support- attached test compounds comprises contacting the test compound with the target nucleic acid in the presence of an aqueous solution wherein the aqueous solution comprises a buffer and a combination of salts.
8. The method of claim 7 wherein the aqueous solution approximates or mimics physiologic conditions.
9. The method of claim 7 in which the aqueous solution optionally further comprises non-specific nucleic acids comprising DNA, yeast IRNA, salmon spenn DNA, homoribopolymers, and nonspecific RNAs.
10. The method of claim 7 in which the aqueous solution further comprises a buffer, a combination of salts, and optionally, a detergent or a surfactant.
11. The method of claim 10 in which the aqueous solution further comprises a combination of salts, from about 0 mM to about 100 mM KC1, from about 0 mM to about 1 M NaCl, and from about 0 mM to about 200 mM MgCI,.
12, The method of claim 11 wherein the combination of salts is about
100 M KC1, 500 mM NaCl, and 10 mM MgCl2.
13. The method of claim 10 wherein the solution optionally comprises from about 0.01% to about 0.5% (w/v) of a detergent or a surfactant.
87
14. The method of claim 1 in which separating the detectably labeled target RNArsupport-attached test compound complex formed in step (a) from uncomplcxcd target RNA and test compounds is by flow cytometry, aff ity chromatography, manual batch mode separation, suspension of beads in electric fields, or microwave.
15. The method of claim 1 in which the library of solid support-attached test compounds are small organic molecule libraries.
16. The method of claim J 5 in which the structure of the test compound is determined by mass spectroscopy, NMR, or vibration spectroscopy,
17. The method of claim 1 in which the library of solid support-attached test compounds are peptide or peptide-based libraries.
18. The method of claim 17 in which the structure of the test compound is determined by Edman degradation.
88
PCT/US2002/011758 2001-04-11 2002-04-11 Methods for identifying small molecules that bind specific rna structural motifs WO2002083837A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/475,026 US20050142545A1 (en) 2001-04-11 2002-04-11 Methods for identifying small molecules that bind specific rna structural motifs
US11/359,721 US20060194234A1 (en) 2001-04-11 2006-02-21 Methods for identifying small molecules that bind specific RNA structural motifs

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28296601P 2001-04-11 2001-04-11
US60/282,966 2001-04-11

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/359,721 Continuation US20060194234A1 (en) 2001-04-11 2006-02-21 Methods for identifying small molecules that bind specific RNA structural motifs

Publications (2)

Publication Number Publication Date
WO2002083837A1 WO2002083837A1 (en) 2002-10-24
WO2002083837B1 true WO2002083837B1 (en) 2003-01-30

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/011758 WO2002083837A1 (en) 2001-04-11 2002-04-11 Methods for identifying small molecules that bind specific rna structural motifs

Country Status (2)

Country Link
US (2) US20050142545A1 (en)
WO (1) WO2002083837A1 (en)

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US7888005B2 (en) * 2003-02-12 2011-02-15 The Curators Of The University Of Missouri Inhibitors of macromolecular activity
CA2520664A1 (en) * 2003-03-27 2004-10-14 Ptc Therapeutics, Inc. Targeting enzymes of the trna splicing pathway for identification of anti-fungal and/or anti-proliferative molecules
WO2004087069A2 (en) * 2003-03-27 2004-10-14 Ptc Therapeutics, Inc. METHODS OF IDENTIFYING COMPOUNDS THAT TARGET tRNA SPLICING ENDONUCLEASE AND USES OF SAID COMPOUNDS AS ANTI-PROLIFERATIVE AGENTS
WO2004087070A2 (en) * 2003-03-27 2004-10-14 Ptc Therapeutics, Inc. METHODS OF IDENTIFYING COMPOUNDS THAT TARGET tRNA SPLICING ENDONUCLEASE AND USES OF SAID COMPOUNDS AS ANTI-FUNGAL AGENTS
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CA2651815A1 (en) * 2006-05-10 2007-11-22 Dxterity Diagnostics Detection of nucleic acid targets using chemically reactive oligonucleotide probes
US7598040B2 (en) * 2006-11-22 2009-10-06 Trana Discovery, Inc. Compositions and methods for the identification of inhibitors of protein synthesis
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AU2008298712A1 (en) * 2007-09-14 2009-03-19 Trana Discovery Compositions and methods for the identification of inhibitors of retroviral infection
WO2009129236A2 (en) * 2008-04-14 2009-10-22 Integrated Nano-Technologies Llc Method for manipulating samples with magnetic nucleation nanoparticles
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WO2010114599A1 (en) * 2009-04-01 2010-10-07 Dx Terity Diagnostics Inc. Chemical ligation dependent probe amplification (clpa)
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EP3688187A4 (en) * 2017-09-25 2021-09-29 Skyhawk Therapeutics, Inc. Methods and compositions for screening and identification of splicing modulators

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Publication number Publication date
US20050142545A1 (en) 2005-06-30
WO2002083837A1 (en) 2002-10-24
US20060194234A1 (en) 2006-08-31

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