Nucleic acid ligand inhibitors to DNA polymerases

• US 6,183,967 B1
• Filed: 03/01/1999
• Issued: 02/06/2001
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Jayasena, Sumedha
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US09/258,797
• Time in Patent Office: 708 Days
• Field of Search: 435/6, 435/91.219, 435/194, 536/25.4, 536/23.1
• US Class Current: 435/5
• 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: 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 2310/16 Aptamers
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2549/101 Hot start
• CPC Class Codes: G01N 2333/96433 Serine endopeptidases (3.4.21)

Abstract

This invention discloses high-affinity oligonucleotide ligands to the thermostable Taq polymerase, Tth polymerase and TZ05 polymerase. Specifically, this invention discloses DNA ligands having the ability to bind to the Taq, Tth and TZ05 polymerases and the methods for obtaining such ligands. The ligands are capable of inhibiting polymerases at any predetermined temperature.

Claims

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

a) preparing a candidate mixture of nucleic acids;

b) heating the candidate mixture of nucleic acids with said polymerase to a predetermined temperature, wherein nucleic acids having an increased affinity to the polymerase relative to the candidate mixture at that temperature may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture at said predetermined temperature; 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 selected from a DNA polymerase and a reverse transcriptase.

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

5. The method of claim 3 wherein said DNA polymerase is selected from Thermus aquaticus polymerase (Taq polymerase), Thermus thermophilus polymerase (Tth polymerase) and TZ05 polymerase.

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

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

11. A purified and isolated non-naturally occurring nucleic acid ligand identified according to the method of claim 3.

12. The purified and isolated non-naturally occurring nucleic acid ligand of claim 11, wherein said ligand is selected from the group consisting of the sequences set forth in Table 4 (SEQ ID NOS:

78-84), Table 5 (SEQ ID NOS;

85-88), Table 6 (SEQ ID NOS;

89-106), FIG. 33 (SEQ ID NOS;

107-115), and FIG. 38 (SEQ ID NOS;

116-118).

13. The purified and isolated non-naturally occurring nucleic acid ligand of claim 11, wherein said ligand is substantially homologous to and has substantially the same ability to bind the polymerase as a ligand selected from the group consisting of the sequences set forth in Table 4 (SEQ ID NOS:

78-84), Table 5 (SEQ ID NOS;

85-88), Table 6 (SEQ ID NOS;

89-106), FIG. 33 (SEQ ID NOS;

107-115), and FIG. 38 (SEQ ID NOS;

116-118).

18. A method for inhibiting the activity of a thermostable DNA polymerase, comprising adding a nucleic acid ligand that inhibits said DNA polymerase, identified according to the method of claim 1, to a DNA polymerization reaction which is being maintained at a temperature at or below which said ligand inhibits polymerization.

19. The method of claim 18 wherein said DNA polymerase is selected from Taq polymerase and TZ05 polymerase.

20. The method of claim 18 wherein said polymerase ligand is a DNA selected from the ligands of Table 4 (SEQ ID NOS:

78-84), Table 5 (SEQ ID NOS;

85-88), Table 6 (SEQ ID NOS;

89-106), FIG. 33 (SEQ ID NOS;

107-115), and FIG. 38 (SEQ ID NOS;

1 16-118).

8. A method for inhibiting a DNA polymerase, comprising adding a nucleic acid ligand that inhibits said DNA polymerase DNA at a predetermined temperature to a DNA polymerization reaction.

9. The method of claim 8 wherein said DNA polymerase is selected from Taq polymerase, Tth polymerase and TZ05 polymerase.

10. The method of claim 9 wherein said polymerase ligand is a DNA selected from the ligands of Table 4 (SEQ ID NOS:

78-84), Table 5 (SEQ ID NOS;

85-88), Table 6 (SEQ ID NOS;

89-106), FIG. 33 (SEQ ID NOS;

107-115), and FIG. 38 (SEQ ID NOS;

116-118).

14. A method for performing the Polymerase Chain Reaction (PCR) comprising:

a) mixing a sample containing a nucleic acid sequence that is to be amplified with primers that are complementary to the sequences that flank the sequence to be amplified, a thermostable polymerase, and a nucleic acid ligand that is capable of inhibiting the polymerase at a predetermined temperature, yet allows the polymerase to be activated at temperatures above said predetermined temperature; and

b) performing the standard PCR steps of melting the target nucleic acid, annealing the primers to the target nucleic acid, and synthesizing the target nucleic acid, by thermal cycling of the mixture.

15. The method of claim 14 wherein said thermostable polymerase is selected from Taq polymerase and TZ05 polymerase.

16. The method of claim 14 wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 4 (SEQ ID NOS:

78-84), Table 5 (SEQ ID NOS;

85-88), Table 6 (SEQ ID NOS;

89-106), FIG. 33 (SEQ ID NOS;

107-115), and FIG. 38 (SEQ ID NOS;

116-118).

17. The method of claim 14 wherein said acid ligand substantially homologous to and has substantially the same ability to bind the thermostable polymerase as a ligand selected from the group consisting of the sequences set forth in Table 4 (SEQ ID NOS:

78-84), Table 5 (SEQ ID NOS;

85-88), Table 6 (SEQ ID NOS;

89-106), FIG. 33 (SEQ ID NOS;

107-115), and FIG. 38 (SEQ ID NOS;

116-118).

21. A method of identifying a nucleic acid ligand switch for a polymerase, comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with a 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

d) further partitioning the increased affinity nucleic acids of step c) based on their lack of affinity to the polymerase upon the variation of an environmental parameter; and

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