High affinity nucleic acid ligands containing modified nucleotides

• US 6,184,364 B1
• Filed: 09/27/1999
• Issued: 02/06/2001
• 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: Janjic, Nebojsa, Pieken, Wolfgang, Kirschenheuter, Gary P, Gold, Larry, Tasset, Diane
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/407,234
• Time in Patent Office: 498 Days
• Field of Search: 435/6, 536/22.1, 536/4.1, 536/28.1, 536/28.2, 536/28.3, 536/28.4
• US Class Current: 536/4.1
• 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: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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: 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: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• 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 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• 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 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Nucleic acid ligands containing modified nucleotides are described as are methods for producing such oligonucleotides. Such ligands enrich the chemical diversity of the candidate mixture for the SELEX process. Specific examples are provided of nucleic acids containing nucleotides modified at the 2′- and 5-position. Specific 2-OH and 2′-NH₂ modified RNA ligands to thrombin are described.

Claims

1. A method of preparing 5-iodo,2′

-amino deoxyuridine from uridine comprising;

a) dehydrating uridine to 2,2′

-anhydrouridine;

b) converting 2,2′

-anhydrouridine to an unprotected 2′

-azido,2′

-deoxyuridine;

c) iodinating the unprotected 2′

-azido,2′

-deoxyuridine to 5-iodo, 2′

-azido,2′

-deoxyuridine; and

d) reducing the unprotected 5-iodo,2′

-azido,2′

-deoxyuridine to 5-iodo,2′

-amino,2′

-deoxyuridine.

2. A method of preparing 5-iodo,2′

-amino,2′

-deoxy uridine 5′

-triphosphate from 2′

-amino,2′

-deoxyuridine, comprising;

a) trifluoroacetylating 2′

-amino,2′

-deoxyuridine to form 2′

-trifluoroacetylamino,2′

-deoxyuridine;

b) phosphorylating 2′

-trifluoroacetylamino,2′

-deoxyuridine to 2′

-amino,2′

-deoxyuridine 5′

-triphosphate; and

c) iodinating 2′

-trifluoroacetylamino,2′

-deoxyuridine by mercuration to form 5′

-iodo,2′

-amino,2′

-deoxyuridine 5′

-triphosphate.