Nucleic acid ligands to integrins

• US 6,331,394 B1
• Filed: 07/29/1999
• Issued: 12/18/2001
• Est. Priority Date: 06/10/1991
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Ruckman, Judy, Janjic, Nebojsa, Gold, Larry, Stephens, Andrew
• Primary Examiner(s): Zitomer, Stephanie
• Application Number:
  US09/364,543
• Time in Patent Office: 873 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/25.4
• US Class Current: 435/6.11
• 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 2310/16 Aptamers
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3533 Halogen

Abstract

Methods are described for the isolation of nucleic acid ligands to integrins using the SELEX process. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The nucleic acid ligands of the present invention are useful as therapeutic and diagnostic agents.

Claims

1. A nucleic acid ligand to an integrin identified according to the method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with said integrin, wherein nucleic acids having an increased affinity to said integrin 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;

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 said integrin, whereby a nucleic acid ligand of said integrin may be identified.

2. The nucleic acid ligand of claim 1 wherein said integrin is a β

₃ integrin.

3. The nucleic acid ligand of claim 2 wherein said ⊖

₃ integrin is selected from the group consisting of α

_IIbβ

₃ integrin and α

_vβ

₃ integrin.

6. The nucleic acid ligand of claim 1 wherein said integrin is attached through either covalent or non-covalent bonds to a solid support, and wherein steps b)-c) take place on the surface of said solid support.

7. The nucleic acid ligand of claim 6 wherein said solid support is a bead.

8. The nucleic acid ligand of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

9. The nucleic acid ligand of claim 8 wherein said single stranded nucleic acids are ribonucleic acids.

10. The nucleic acid ligand of claim 8 wherein said single stranded nucleic acids are deoxyribonucleic acids.

11. The nucleic acid ligand of claim 9 wherein said candidate mixture of nucleic acids comprises 2′

-F(2′

-fluoro) modified ribonucleic acids.

4. A purified and isolated non-naturally occurring nucleic acid ligand to an integrin.

12. The purified and isolated non-naturally occurring nucleic acid ligand of claim 4 wherein said nucleic acid ligand is single stranded.

13. The purified and isolated non-naturally occurring nucleic acid ligand of claim 12 wherein said nucleic acid ligand is RNA.

14. The purified and isolated non-naturally occurring RNA ligand of claim 13 wherein said ligand is comprised of 2′

-fluoro (2′

-F) modified nucleotides.

5. A purified and non-naturally occurring RNA ligand to an integrin wherein said ligand is selected from the group consisting of SEQ ID NO:

5-118.

15. A method for the isolation of nucleic acid ligands to an integrin, comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with said integrin, wherein nucleic acids having an increased affinity to said integrin 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;

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 said integrin, whereby a nucleic acid ligand of said integrin may be isolated; and

e) isolating nucleic acid ligands to an integrin.

16. The method of claim 15 wherein said candidate mixture comprises single-stranded nucleic acids.

17. The method of claim 16 wherein said single-stranded nucleic acids comprise ribonucleic acids.

18. The method of claim 15 wherein said integrin is a β

₃ integrin.

19. The method of claim 18 wherein said β

₃ integrin is α

_IIbβ

₃ integrin.