Nucleic acid ligand inhibitors to DNA polymerases

• US 5,763,173 A
• Filed: 06/07/1995
• Issued: 06/09/1998
• 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: Gold, Larry, Jayasena, Sumedha D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/487,426
• Time in Patent Office: 1,098 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/16 Aptamers
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2740/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• 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 2333/9125 : with a definite EC number (2.7.7.-)
• CPC Class Codes: G01N 33/5308 : for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/573 : for enzymes or isoenzymes

Abstract

This invention discloses high-affinity oligonucleotide ligands to the thermostable Taq polymerase and Tth polymerase. Specifically, this invention discloses DNA ligands having the ability to bind to the Taq and Tth polymerases and the methods for obtaining such ligands. The ligands are capable of inhibiting polymerases at ambient temperatures.

Claims

1. A method of identifying nucleic acid ligands to a polymerase, comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with said polymerase, wherein nucleic acids having an increased affinity to the polymerase 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; and

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

2. The method of claim 1 further comprising:

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

3. The method of claim 1 wherein said polymerase is a DNA polymerase.

4. The method of claim 1 wherein said polymerase is a reverse transcriptase.

5. The method of claim 1 wherein said polymerase is thermostable.

6. The method of claim 3 wherein said DNA polymerase is isolated from Thermus aquaticus (Taq polymerase).

7. The method of claims 3 or 4 wherein said polymerase is isolated from Thermus thermophilus (Tth polymerase).

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

9. The method of claim 1 wherein said single stranded nucleic acids are deoxyribonucleic acids.

11. The method of claim 10 wherein said DNA polymerase nucleic acid ligand is identified according to the method of claim 1.

10. A method for inhibiting the activity of a DNA polymerase, comprising adding an effective amount of a high affinity DNA polymerase nucleic acid ligand to a DNA polymerization reaction.

12. The method of claim 10 wherein said DNA polymerase is Taq polymerase.

13. The method of claim 12 wherein said polymerase ligand is selected from one of the ligands of Table 2 (SEQ ID NOS:

7-35).

14. The method of claim 10 wherein said DNA polymerase is Tth polymerase.

15. The method of claim 14 wherein said polymerase ligand is selected from one of the ligands of Table 3 (SEQ ID NOS:

36-73).