Ligands of thrombin

• US 5,476,766 A
• Filed: 11/06/1992
• Issued: 12/19/1995
• 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, Tasset, Diane
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/973,333
• Time in Patent Office: 1,138 Days
• Field of Search: 435/6, 435/91, 435/91.2, 536/22.1, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 14/001 by chemical synthesis
• 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 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• 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 2525/301 Hairpin oligonucleotides
• 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 2333/16 HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 basic FGF [bFGF]
• CPC Class Codes: G01N 2333/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin G01N2333/665, corticotropin G01N2333/695
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/9726 : Tissue plasminogen activator
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 2333/976 : Trypsin; Chymotrypsin
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Methods are described for the identification of nuclei acid ligand solutions to thrombin. The present invention utilizes the SELEX (Systematic Evolution of Ligands for EXponential enrichment) method for identifying and preparing RNA ligands to thrombin. Further included in the present invention are modified nucleotide sequences based on the sequences of the RNA ligands identified. The modified RNA ligands to thrombin exhibit increased in vivo stability.

Claims

1. A method for identifying nucleic acid ligands to thrombin comprising:

a) preparing a candidate mixture of RNA nucleic acids;

b) contacting the candidate mixture with thrombin, wherein RNA nucleic acids having an increased affinity to thrombin relative to the candidate mixture RNA nucleic acids may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity RNA nucleic acids from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of RNA nucleic acids, whereby RNA nucleic acid ligands to thrombin 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 candidate mixture of RNA nucleic acids is comprised of single stranded nucleic acids.

4. A nucleic acid ligand to thrombin identified according to the method of claim 1.

5. The nucleic acid ligand of claim 4 being a single stranded nucleic acid.

9. The RNA ligand of claim 6 identified by the method of claim 1.

6. A purified and isolated non-naturally occurring RNA ligand to thrombin wherein the nucleotide sequence of said RNA ligand is selected from the group consisting of the nucleotide sequences set forth in FIG. 1.

7. The RNA ligand of claim 6 consisting of the nucleotide sequence (SEQ ID NO:

2);

space="preserve" listing-type="equation">5'"'"'-GGAUCGAAG(N).sub.2 AGUAGGC-3'"'"'

8. The RNA ligand of claim 6 consisting of having the nucleotide sequence (SEQ ID NO:

3);

space="preserve" listing-type="equation">5'"'"'-GCGGCUUUGGGCGCCGUGCUU-3'"'"'