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

• US 5,849,890 A
• Filed: 06/07/1995
• Issued: 12/15/1998
• 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: Gold, Larry, Nieuwlandt, Dan, Davis, Ken, Jayasena, Sumedha D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/484,552
• Time in Patent Office: 1,287 Days
• Field of Search: 536/23.1, 536/22.1, 435/91.2, 435/6, 436/501, 436/87
• US Class Current: 536/23.1
• 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: 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: 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: 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 2333/8125 Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/966 Elastase
• 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 purified and isolated non-naturally occurring nucleic acid ligand to a chorionic gonadotropin (CG)-related glycoprotein hormone.

2. The nucleic acid ligand to a CG-related glycoprotein hormone of claim 1 identified according to the method 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 acid sequences with relatively higher affinity and specificity for binding to the CG-related glycoprotein hormone, whereby nucleic acid ligands of the CG-related glycoprotein hormone may be identified.

3. The nucleic acid ligand to a CG-related glycoprotein hormone of claim 2 wherein the method further comprises 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, and 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.

4. A purified and isolated non-naturally occurring nucleic acid ligand to an hCG-related glycoprotein hormone.

5. The nucleic acid ligand to an hCG-related glycoprotein hormone of claim 4 identified according to the method 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 acid sequences with relatively higher affinity and specificity for binding to the hCG-related glycoprotein hormone, whereby nucleic acid ligands of the hCG-related glycoprotein hormone may be identified.

6. The nucleic acid ligand to an hCG-related glycoprotein hormone of claim 5 wherein the method further comprises 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, and 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.

7. The purified and isolated non-naturally occurring nucleic acid ligand of claim 4, wherein said hCG-related glycoprotein hormone is hCG, and wherein said ligand is an RNA selected from the group consisting of the sequences set forth in FIGS. 3 and 4 and Tables 2, 5, 11 and 13 (SEQ ID NOS:

4-30, 34-35, 41, 80-169).

8. The purified and isolated non-naturally occurring nucleic acid ligand of claim 4, wherein said hCG-related glycoprotein hormone is hTSH, and wherein said ligand is an RNA selected from the group consisting of the sequences set forth in Table 7 (SEQ ID NOS:

42-63).

9. The purified and isolated non-naturally occurring nucleic acid ligand of claim 5, wherein said hCG-related glycoprotein hormone is hCG.

10. The purified and isolated non-naturally occurring nucleic acid ligand of claim 5, wherein said hCG-related glycoprotein hormone is hTSH.