High affinity oligonucleotide ligands to chorionic gonadotropin hormone and related glycoprotein hormones

• US 5,837,456 A
• Filed: 06/07/1995
• Issued: 11/17/1998
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Nieuwlandt, Dan, Davis, Ken, Gold, Larry, Jayasena, Sumedha D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/488,402
• Time in Patent Office: 1,259 Days
• Field of Search: 435/6, 435/91.2, 536/25.4, 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: 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 14/47 from mammals
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• 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: 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
• 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 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Methods are described for the identification and preparation of nucleic acid ligands to CG-related glycoprotein hormones. Included in the invention are specific RNA ligands to hCG and hTSH identified by the SELEX method.

Claims

1. A method of identifying nucleic acid ligands to a Chorionic Gonadotropin (CG)-related glycoprotein hormone, comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with a CG-related glycoprotein hormone, wherein nucleic acids having an increased affinity to the CG-related glycoprotein hormone 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 acids with relatively higher affinity and specificity for binding to the CG-related glycoprotein hormone, whereby nucleic acid ligands of CG-related glycoprotein hormone may be identified.

2. The method of claim 1 further comprising:

e) repeating steps b), c), and d).

3. The method of claim 1 further comprising contacting the increased affinity nucleic acids with one or more non-target molecules, wherein nucleic acids with affinity to the non-target molecule(s) are removed.

4. The method of claim 3 wherein the non-target molecule is a member of the CG-related glycoprotein hormone family other than the one for which increased affinity nucleic acids are being selected.

5. A method of identifying nucleic acid ligands to a human Chorionic Gonadotropin (hCG)-related glycoprotein hormone, comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with an hCG-related glycoprotein hormone, wherein nucleic acids having an increased affinity to the hCG-related glycoprotein hormone 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 acids with relatively higher affinity and specificity for binding to the hCG-related glycoprotein hormone, whereby nucleic acid ligands of hCG-related glycoprotein hormone may be identified.

6. The method of claim 5 further comprising:

e) repeating steps b), c), and d).

7. The method of claim 5 further comprising contacting the increased affinity nucleic acids with one or more non-target molecules, wherein nucleic acids with affinity to the non-target molecule(s) are removed.

8. The method of claim 7 wherein the non-target molecule is a member of the hCG-related glycoprotein hormone family other than the one for which increased affinity nucleic acids are being selected.

9. The method of claim 5 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

10. The method of claim 9 wherein said single stranded nucleic acids are ribonucleic acids.

11. The method of claim 10 wherein said nucleic acids are 2'"'"'-amino (2'"'"'-NH₂) modified ribonucleic acids.

12. The method of claim 10 wherein said nucleic acids are 2'"'"'-F modified ribonucleic acids.

13. The method of claim 5, wherein said hCG-related glycoprotein hormone is hCG.

14. The method of claim 5, wherein said hCG-related glycoprotein hormone is human thyroid-stimulating hormone (hTSH).