Nucleic acid ligands

• US 6,331,398 B1
• Filed: 02/10/2000
• Issued: 12/18/2001
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Gold, Larry, Tuerk, Craig
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/502,344
• Time in Patent Office: 677 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/254, 514/44
• US Class Current: 435/6.11
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 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
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2541/101 : Selex
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 2333/16 : HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin G01N2333/665, corticotropin G01N2333/695
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/9726 : Tissue plasminogen activator
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 2333/976 : Trypsin; Chymotrypsin
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

This invention comprises nucleic acid ligand for use as a diagnostic reagent for detecting the presence or absence of a target molecule in a sample, and a diagnostic reagent to measure the amount of a target molecule in a sample. In a preferred embodiment the nucleic acid ligands are identified by the method of the invention referred to as the Systematic Evolution of Ligands by EXponential enrichment (SELEX), wherein a candidate mixture of nucleic acids are iteratively enriched in high affinity nucleic acids and amplified by further partitioning.

Claims

1. A method of identifying a function-modifying nucleic acid ligand that modifies a function of a target molecule comprising:

(a) identifying a nucleic acid ligand to the target molecule;

(b) contacting said identified nucleic acid ligand with the target molecule; and

(c) evaluating whether said identified nucleic acid ligand modifies a function of the target molecule, whereby said function-modifying nucleic acid ligand may be identified.

2. The method of claim 1 wherein said nucleic acid ligand is identified from a candidate mixture of nucleic acids by a method comprising:

(a) contacting the candidate mixture with said target molecule, wherein nucleic acids having an increased affinity to said target molecule relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

(b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

(c) amplifying the increased affinity nucleic acid to yield a ligand-enriched mixture of nucleic acids, whereby said nucleic acid ligand may be identified.

3. The method of claim 1 wherein the target molecule is selected from the group consisting of a protein, a nucleoside, a nucleotide and a coenzyme nucleotide.

4. A function-modifying nucleic acid ligand that modifies a function of a target molecule identified by the method of claim 1.

5. A method of modifying a function of a target molecule comprising:

contacting the target molecule with the function-modifying nucleic acid ligand identified by the method of claim 2 whereby the function of the target molecule is modified.

6. The method of claim 5 wherein the function of the target molecule is a biological function and wherein said method further comprises inhibiting the biological function of the target molecule.

7. The method of claim 6 wherein the biological function of the target molecule is catalysis.

8. The method of claim 7 wherein the function-modifying nucleic acid ligand comprises a nucleic acid ligand to a transition state analogue.

9. The method of claim 7 wherein the function-modifying nucleic acid ligand binds irreversibly to the target molecule.

10. The method of claim 7 wherein the function-modifying nucleic acid ligand comprises a nucleic acid ligand that modifies substrate binding of the target molecule.

11. The method of claim 5 wherein the function of the target molecule is binding a receptor and wherein said function-modifying nucleic acid ligand comprises a nucleic acid ligand that inhibits binding of the target molecule to the receptor or modifies the specificity of binding of the target molecule to the receptor.

12. The method of claim 5 wherein the target molecule is a protein and said method comprises affecting formation of the protein quaternary structure from the protein subunits.

13. The method of claim 12 wherein said affecting formation comprises disrupting quaternary structure of protein subunits.

14. The method of claim 5 wherein said method further comprises modifying transport properties of a protein.

15. The method of claim 14 wherein modifying the transport properties comprises disrupting transport of small molecules or ions.