High-affinity nucleic acid ligands that discriminate between theophylline and caffeine

• US 5,580,737 A
• Filed: 05/18/1995
• Issued: 12/03/1996
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Polisky, Barry, Gold, Larry, Jenison, Robert D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/443,957
• Time in Patent Office: 565 Days
• Field of Search: 435/6, 435/91.2, 536/22.1
• 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: 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• 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: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• 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 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Methods are described for the identification and preparation of nucleic acid ligand ligands to theophylline and caffeine. Included in the invention is a method for identifying nucleic acid ligands with high affinity and selectivity for a target molecule, termed counter-SELEX. One embodiment of counter-SELEX is comprised of the steps of a) preparing a candidate mixture of nucleic acids; b) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned form the remainder of the candidate mixture; partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; d) contacting the increased affinity nucleic acids with one or more non-target molecules such that nucleic acid ligands to the non-target molecule(s) are removed; and e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity and specificity for binding to the target molecule. Included in the invention are specific nucleic acid ligands to theophylline identified by the method of counter-SELEX.

Claims

1. A method for identifying nucleic acid ligands to a target molecule comprising:

a) preparing a candidate mixture of nucleic acids;

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

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

d) contacting the increased affinity nucleic acids with one or more non-target molecules, wherein nucleic acids with affinity to the non-target molecule(s) are removed; and

e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to the target molecule, whereby nucleic acid ligands of the target molecule may be identified.

2. The method of claim 1 further comprising f) repeating steps b), c), d) and e).

3. The method of claim 1 wherein said target molecule is immobilized on a column.

4. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

6. A purified and isolated non-naturally occurring RNA ligand to caffeine, identified by the method of claim 1, wherein the nucleotide sequence of said RNA ligand is selected from the group consisting of the nucleotide sequences set fourth in FIG. 8 (SEQ ID NOS:

16-27).

11. The method of claim 1 wherein said target molecule is theophylline.

12. A nucleic acid ligand to theophylline, identified according to the method of claim 11.

13. The method of claim 1 wherein said target molecule is caffeine.

14. A nucleic acid ligand to caffeine identified according to the method of claim 13.

5. A purified and isolated non-naturally occurring RNA ligand to theophylline, wherein the nucleotide sequence of said RNA ligand is selected from the group consisting of the nucleotide sequences set forth in FIGS. 2A and 2B (SEQ ID NOS:

3-15).

7. Nucleic acid ligands to theophylline identified according to a method for identifying nucleic acid ligands from a candidate mixture comprised of nucleic acids, said method comprising:

a) preparing a candidate mixture of nucleic acids;

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

c) partitioning the increased affinity nucleic acid ligands from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acid, whereby nucleic acid ligands of theophylline may be identified.

8. The method of claim 7 further comprising

e) repeating steps b), c) and d).

9. Nucleic acid ligands to caffeine identified according to a method for identifying nucleic acid ligands from a candidate mixture comprised of nucleic acids, said method comprising:

a) preparing a candidate mixture of nucleic acids;

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

c) partitioning the increased affinity nucleic acid ligands from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acid, whereby nucleic acid ligands of caffeine may be identified.

10. The method of claim 9 further comprising

e) repeating steps b), c) and d).

15. A non-naturally occurring nucleic acid ligand, having a specific binding affinity for a target molecule, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, and wherein said target molecule is selected from the group consisting of theophylline and caffeine.

16. A method for identifying nucleic acid ligands to a target molecule comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with one or more non-target molecules, wherein nucleic acids with affinity to the non-target molecule(s) are removed;

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

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

(e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to the target molecule, whereby nucleic acid ligands of the target molecule may be identified.