Nucleic acid ligand inhibitors to DNA polymerases

• US 5,874,557 A
• Filed: 06/07/1995
• Issued: 02/23/1999
• 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.
• Application Number:
  US08/487,720
• Time in Patent Office: 1,357 Days
• Field of Search: 536/22.1, 536/23.1, 435/5, 435/91.2
• US Class Current: 536/23.1
• 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: C07K 7/22 Tachykinins, e.g. Eledoisins , Substance P; Related peptides
• 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 2310/16 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 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 33/573 : for enzymes or isoenzymes

Abstract

High-affinity oligonucleotide ligands to the thermostable Taq polymerase and Tth polymerase are disclosed. Specifically disclosed are 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 purified and isolated non-naturally occurring nucleic acid ligand to a DNA polymerase.

5. The purified and isolated non-naturally occurring nucleic acid ligand of claim 1, wherein said DNA polymerase is Thermus aguaticus (Taq) polymerase and wherein said ligand is selected from the group set forth in Table 3 (SEQ ID NOS:

36-74).

6. The purified and isolated non-naturally occurring nucleic acid ligand of claim 1, wherein said DNA polymerase is Thermus thermophilus (Tth) polymerase, and wherein said DNA ligand is selected from the group set forth in Table (SEQ ID NOS:

7-35).

2. A nucleic acid ligand to a DNA polymerase identified according to the method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with the DNA polymerase, wherein nucleic acids having an increased affinity to the DNA 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 DNA polymerase, whereby a nucleic acid ligand of the DNA polymerase may be identified.

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

4. A nucleic acid ligand to a reverse transcriptase 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) 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 reverse transcriptase, whereby a nucleic acid ligand of the reverse transcriptase may be identified.