High affinity nucleic acid ligands containing modified nucleotides

• US 5,958,691 A
• Filed: 08/25/1997
• Issued: 09/28/1999
• 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, Gold, Larry, Pieken, Wolfgang, Kirschenheuter, Gary P., Tasset, Diane
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/918,304
• Time in Patent Office: 764 Days
• Field of Search: 435/6, 435/91.2, 536/24.5, 536/22.1, 536/23.1, 536/24.31, 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: 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 for identifying nucleic acid ligands comprising a modified nucleotide to a target molecule comprising:

a) preparing a candidate mixture of single-stranded nucleic acids wherein each nucleic acid of said candidate mixture comprises a modified nucleotide;

b) contacting the candidate mixture with said target molecule;

c) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands comprising modified nucleotides of the target molecule are identified.

2. The method of claim 1 further comprising e) repeating steps c) and d).

3. The method of claim 1 wherein said single-stranded nucleic acids are selected from the group consisting of single-stranded DNA and single-stranded RNA.

4. The method of claim 1 wherein said modified nucleotides have been chemically modified at the ribose and/or phosphate and/or base positions.

5. A nucleic acid ligand comprising a modified nucleotide to a target molecule, wherein said nucleic acid is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for a target molecule, said target molecule being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, obtained by the process of identifying a nucleic acid ligand of a target from a candidate mixture of nucleic acids wherein each nucleic acid of said candidate mixture comprises a modified nucleotide, said process comprising:

(a) contacting the candidate mixture with said target molecule;

(b) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture; and

(c) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands comprising modified nucleotides of the target molecule are identified.

6. The nucleic acid ligand of claim 5 wherein said nucleic acid is selected from the group consisting of single-stranded RNA, double-stranded RNA, single-stranded DNA and double-stranded DNA.

7. The nucleic acid ligand of claim 5 wherein said nucleic acid comprises one or more or a combination of nucleotides which have been chemically modified at the ribose and/or phosphate and/or base positions.

8. The nucleic acid ligand of claim 7 wherein said nucleotide is modified at the 2'"'"'- or 5- position.

9. The nucleic acid ligand of claim 8 wherein said 2'"'"'-position modification to said nucleotide is a 2'"'"'-amino modification.

10. The nucleic acid ligand of claim 8 wherein said 2'"'"'-position modification to said nucleotide is a 2'"'"'-fluoro modification.

11. The nucleic acid ligand of claim 8 wherein said 5-position modified nucleotide is selected from the group consisting of 5-(3-aminoallyl)uridine triphosphate (5-AA-UTP), 5-fluorescein-12-uridine triphosphate (5-F-12-UTP), 5-digoxygenin-11-uridine triphosphate (5-Dig-11-UTP), and 5-bromouridine triphosphate (5-Br-UTP).

12. The nucleic acid ligand of claim 8 wherein said modified nucleotide is selected from the group consisting of a 2'"'"'-position modified pyrimidine and a 5-position modified pyrimidine.

13. The nucleic acid ligand of claim 9 wherein said modified nucleotide is selected from the group consisting of a 2'"'"'-amino pyrimidine and a 2'"'"'-amino purine.

14. The nucleic acid ligand of claim 9 wherein said modified nucleotide is selected from the group consisting of a 2'"'"'-fluoro pyrimidine and a 2'"'"'-fluoro purine.

15. A nucleic acid ligand comprising a modified nucleotide to a target molecule, wherein said nucleic acid is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.

16. The nucleic acid ligand of claim 15 wherein said nucleic acid is selected from the group consisting of single-stranded RNA, double-stranded RNA, single-stranded DNA and double-stranded DNA.

17. The nucleic acid ligand of claim 15 wherein said nucleic acid comprises one or more or a combination of nucleotides which have been chemically modified at the ribose and/or phosphate and/or base positions.

18. The nucleic acid ligand of claim 17 wherein said nucleotide is modified at the 2'"'"'- or 5- position.

19. The nucleic acid ligand of claim 18 wherein said 2'"'"'-position modification to said nucleotide is a 2'"'"'-amino modification.

20. The nucleic acid ligand of claim 18 wherein said 2'"'"'-position modification to said nucleotide is a 2'"'"'-fluoro modification.

21. The nucleic acid ligand of claim 18 wherein said 5-position modified nucleotide is selected from the group consisting of 5-(3-aminoallyl)uridine triphosphate (5-AA-UTP), 5-fluorescein-12-uridine triphosphate (5-F-12-UTP), 5-digoxygenin-11-uridine triphosphate (5-Dig-11-UTP), and 5-bromouridine triphosphate (5-Br-UTP).

22. The nucleic acid ligand of claim 18 wherein said modified nucleotide is selected from the group consisting of a 2'"'"'-position modified pyrimidine and a 5-position modified pyrimidine.

23. The nucleic acid ligand of claim 19 wherein said modified nucleotide is selected from the group consisting of a 2'"'"'-amino pyrimidine and a 2'"'"'-amino purine.

24. The nucleic acid ligand of claim 19 wherein said modified nucleotide is selected from the group consisting of a 2'"'"'-fluoro pyrimidine and a 2'"'"'-fluoro purine.