Nucleic acid ligand inhibitors to DNA polymerases

• US 5,693,502 A
• Filed: 06/07/1995
• Issued: 12/02/1997
• 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, Ph.D., Amy
• Application Number:
  US08/484,557
• Time in Patent Office: 909 Days
• Field of Search: 435/91.2, 435/6, 435/810
• US Class Current: 435/91.2
• 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 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• 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 1/686 Polymerase chain reaction [PCR]
• 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 2527/101 : Temperature
• CPC Class Codes: C12Q 2527/127 : the enzyme inhibitor or activator used
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: Y10S 435/81 : Packaged device or kit

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 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 single-stranded nucleic acid ligand to the polymerase that is capable of inhibiting said polymerase, wherein the inhibition of the polymerase by said nucleic acid is temperature dependent;

b) heating said mixture to a temperature at which said nucleic, acid ligand no longer inhibits said polymerase; and

c) 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.

3. The method of claim 1 wherein said thermostable polymerase is selected from Thermus aquaticus (Taq polymerase) or Thermus thermophilus (Tth polymerase).

2. A PCR kit comprising a thermostable DNA polymerase and a single-stranded nucleic acid ligand to said thermostable DNA polymerase that inhibits the activity of said polymerase, wherein the inhibition of said polymerase by said nucleic ligand is temperature dependent.