Combinatorial selection of oligonucleotide aptamers

• US 7,179,894 B2
• Filed: 04/11/2002
• Issued: 02/20/2007
• Est. Priority Date: 10/26/1998
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Gorenstein, David G., Brasier, Allan R., King, David J., Ventura, Daniel A.
• Primary Examiner(s): Shibuya; Mark L.
• Assistant Examiner(s): Steele; Amber D.
• Application Number:
  US10/120,815
• Publication Number:
  US 20030027184A1
• Time in Patent Office: 1,776 Days
• Field of Search: 536/23.1, 536/24.5, 435/6
• US Class Current: 536/23.1
• 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: 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 15/1048 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 2310/313 Phosphorodithioates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

A random combinatorial selection method is disclosed for the construction of oligonucleotide aptamers in which nuclease resistance is conferred by the inclusion of modified nucleotides. The modified nucleotides are incorporated during PCR amplification to form achiral modified oligonucleotides. Thio-substituted aptamers are provided that bind tightly to the nuclear factor for human IL6 (NF-IL6).

Claims

1. A NF-IL6 protein binding aptamer comprising a mix of unmodified and thiophosphate-modified nucleotides that form a partially thiophosphate-modified oligonucleotide identified by SEQ ID NO:

7, wherein at least one but no more than four of any one single nucleotide base is thiophosphate-modified, wherein the aptamer binds NF-IL6.

2. The aptamer of claim 1, wherein the modified nucleotide base comprises a phosphorothioate.

3. The aptamer of claim 1, wherein the modified nucleotide base comprises a phosphorodithioate.

4. The aptamer of claim 1, wherein the modified nucleotide base is selected from the group consisting of dATP(α

S), dTTP(α

S), dCTP(α

S) and dGTP(α

S).

5. The aptamer of claim 1, wherein no more than three adjacent phosphate sites are replaced with phosphorothioate groups.

6. The aptamer of claim 1, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorothioate groups.

7. The aptamer of claim 1, wherein no more than three adjacent phosphate sites are replaced with phosphorodithioate groups.

8. The aptamer of claim 1, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorodithioate groups.