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Details for Patent: 6,020,130

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Details for Patent: 6,020,130

Title: Nucleic acid ligands that bind to and inhibit DNA polymerases
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.
Inventor(s): Gold; Larry (Boulder, CO), Javasena; Sumedha (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Oct 28, 1997
Application Number:08/945,734
Claims:1. A method for inhibiting the activity of a thermostable DNA polymerase, comprising adding a nucleic acid ligand that inhibits said DNA polymerase to a DNA polymerization reaction which is being maintained at a temperature at or below which said ligand inhibits polymerization.

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

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

4. The method of claim 3 wherein said polymerase ligand is a DNA selected from one of the ligands of Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) or Table 5 (SEQ ID NO:74).

5. The method of claim 1 wherein said DNA polymerase is Tth polymerase.

6. The method of claim 5 wherein said polymerase ligand is a DNA selected from one of the ligands of Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) or Table 5 (SEQ ID NO:74).

7. A purified and isolated non-naturally occurring nucleic acid ligand to a thermostable polymerase.

8. The nucleic acid ligand of claim 7 identified according to the method 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.

9. The purified and isolated non-naturally occurring nucleic acid ligand of claim 7, wherein said polymerase is Taq polymerase and wherein said ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74).

10. The purified and isolated non-naturally occurring nucleic acid ligand of claim 7, wherein said polymerase is Tth polymerase, and wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74).

11. A purified and isolated non-naturally occurring nucleic acid ligand to a thermostable reverse transcriptase.

12. The nucleic acid ligand to the reverse transcriptase of claim 11 identified according to the method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with the reverse transcriptase, wherein nucleic acids having an increased affinity to the reverse transcriptase 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) amplifing 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 reverse transcriptase, whereby nucleic acid ligands of the reverse transcriptase may be identified.

13. The purified and isolated non-naturally occurring nucleic acid ligand of claim 11, wherein said reverse transcriptase is Tth polymerase, and wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74).

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, Taq polymerase, and a nucleic acid ligand that inhibits the polymerase at a temperature at or below which said ligand inhibits polymerization, yet allows the polymerase to be activated at elevated temperatures; wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74); 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. 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, Tth polymerase, and a nucleic acid ligand that inhibits the polymerase at a temperature at or below which said ligand inhibits polymerization, yet allows the polymerase to be activated at elevated temperatures; wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74); 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.

16. A PCR kit comprising a thermostable DNA polymerase and a nucleic acid ligand that inhibits said polymerase at a temperature below which said ligand inhibits polymerization, yet allows the polymerase to be activated at elevated temperatures, wherein said thermostable DNA polymerase is Taq polymerase and wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74).

17. A PCR kit comprising a thermostable DNA polymerase and a nucleic acid ligand that inhibits said polymerase at a temperature below which said ligand inhibits polymerization, yet allows the polymerase to be activated at elevated temperatures, wherein said thermostable DNA polymerase is Tth polymerase and wherein said nucleic acid ligand is selected from the group consisting of the sequences set forth in Table 2 (SEQ ID NOS:7-35), Table 3 (SEQ ID NOS:36-66, 76, 77), Table 4 (SEQ ID NOS:67-73) and Table 5 (SEQ ID NO:74).
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