High affinity nucleic acid ligands to lectins

• US 6,280,932 B1
• Filed: 11/21/1997
• Issued: 08/28/2001
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Gold, Larry, Hicke, Brian, Bridonneau, Philippe, Parma, David H
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/952,793
• Time in Patent Office: 1,376 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.33, 530/300
• 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: C07K 14/001 by chemical synthesis
• 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 2310/13 Decoys
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• 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: F02B 2075/027 four
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/4724 : Lectins
• CPC Class Codes: G01N 2333/575 : Hormones
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/7056 : Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
• 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 33/53 : Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

This invention discloses high-affinity oligonucleotide ligands to lectins, specifically nucleic acid ligands having the ability to bind to the lectins, wheat germ agglutinin, L-selectin and P-selectin. Also disclosed are the methods for obtaining such ligands.

Claims

1. A method for identifying modified, single-stranded ribonucleic acid ligands to a lectin comprising:

a) contacting a candidate mixture of modified, single-stranded ribonucleic acids with a lectin, said modified ribonucleic acids selected from the group consisting of 2′

-amino (2′

-NH₂) modified ribonucleic acids and 2′

-fluoro (2′

-F) modified ribonucleic acids, wherein ribonucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity ribonucleic acids from the remainder of the candidate mixture; and

c) amplifying the increased affinity ribonucleic acids to yield a mixture of ribonucleic acids enriched for ribonucleic acid sequences with relatively high affinity and specificity for binding to said lectin, whereby modified, single-stranded ribonucleic acid ligands to said lectin may be identified.

2. A method for forming a multivalent Complex of nucleic acid ligands to a lectin comprising:

a) contacting a candidate mixture of nucleic acids with a lectin, wherein nucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

c) 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 said lectin, whereby nucleic acid ligands to said lectin may be identified; and

d) forming a multivalent Complex comprising two nucleic acid ligands identified in step c).

3. A method for identifying single-stranded ribonucleic acid ligands and producing modified ribonucleic acid ligands to a lectin comprising:

a) contacting a candidate mixture of single-stranded ribonucleic acids with a lectin, wherein ribonucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity ribonucleic acids from the remainder of the candidate mixture; and

c) amplifying the increased affinity ribonucleic acids to yield a mixture of single-stranded ribonucleic acids enriched for ribonucleic acid sequences with relatively higher affinity and specificity for binding to said lectin, whereby the ribonucleic acid ligands to said lectin may be identified; and

d) substituting 2′

-O-methyl ribonucleic acids for 2′

-OH ribonucleic acids in the ribonucleic acid ligands identified in step c), whereby the modified ribonucleic acids to a ligand are produced.

4. A method for identifying nucleic acid ligands and nucleic acid ligand sequences to a lectin comprising:

a) contacting a candidate mixture of nucleic acids with a lectin, wherein nucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

c) 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 said lectin, whereby nucleic acid ligands to said lectin may be identified, wherein said lectin is a selectin.

5. The method of claim 4 wherein said selectin is selected from the group consisting of L-selectin, E-selectin, and P-selectin.

6. A purified and isolated non-naturally occurring nucleic acid ligand to a lectin.

7. The nucleic acid ligand of claim 6 which is a non-naturally occurring nucleic acid ligand having a specific binding affinity for said lectin, such lectin 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 said lectin.

8. The nucleic acid ligand of claim 6 wherein said lectin is selected from the group consisting of a mammalian lectin, a plant lectin, a microbial lectin and a viral lectin.

9. The nucleic acid ligand of claim 6 wherein said lectin is selected from the group consisting of wheat germ agglutinin, L-selectin, E-selectin and P-selectin.

10. The nucleic acid ligand of claim 6 wherein said lectin is wheat germ agglutinin.

11. The nucleic acid ligand to wheat germ agglutinin of claim 10 wherein said nucleic acid ligand is a ribonucleic acid (RNA) ligand.

12. The nucleic acid ligand of claim 11 which comprises a modified ribonucleic acid.

13. The nucleic acid ligand of claim 12 wherein said modified ribonucleic acid is a 2′

-amino (NH₂) modified ribonucleic acid.

14. The nucleic acid ligand to wheat germ agglutinin of claim 13 wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 2.

15. The nucleic acid ligand of claim 14 wherein said ligand is selected from the group consisting of SEQ ID NOS:

4-55.

16. The nucleic acid ligand of claim 11 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS:

56-63.

17. The nucleic acid ligand to wheat germ agglutinin of claim 10 wherein said ligand is substantially homologous to a ligand selected from the group consisting of the sequences set forth in Table 2.

18. The nucleic acid ligand to wheat germ agglutinin of claim 10 wherein said ligand has substantially the same structure as a ligand selected from the group consisting of the sequences set forth in Table 2.

19. The nucleic acid ligand of claim 6 wherein said lectin is a selectin.

20. The nucleic acid ligand of claim 19 wherein said selectin is selected from the group consisting of L-selectin, E-selectin and P-selectin.

21. The nucleic acid ligand of claim 20 wherein said selectin is L-selectin.

22. The nucleic acid ligand to L-selectin of claim 21 wherein said nucleic acid ligand is ribonucleic acid (RNA) ligand.

23. The nucleic acid ligand of claim 22 which comprises a modified ribonucleic acid.

24. The nucleic acid ligand of claim 23 wherein said modified ribonucleic acid is selected from the group consisting of a 2′

-amino (2′

-NH₂) modified ribonucleic acid and a 2′

-fluoro (2′

-F) modified ribonucleic acid.

25. The nucleic acid ligand to L-selectin of claim 24 wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Tables 8 and 16.

26. The nucleic acid ligand of claim 25 wherein said ligand is selected from the group consisting of SEQ ID NOS:

67-117 and 293-388.

27. The nucleic acid ligand of claim 22 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS:

118-125.

28. The nucleic acid ligand to L-selectin of claim 21 wherein said ligand is substantially homologous to a ligand selected from the group consisting of the sequences set forth in Tables 8, 12 and 16.

29. The nucleic acid ligand to L-selectin of claim 21 wherein said ligand has substantially the same structure as a ligand selected from the group consisting of the sequences set forth in Tables 8, 12 and 16.

30. The nucleic acid ligand to L-selectin of claim 21 wherein said nucleic acid ligand is deoxyribonucleic acid (DNA).

31. The nucleic acid ligand to L-selectin of claim 30 wherein said ligand is an DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 12.

32. The nucleic acid ligand of claim 31 wherein said ligand is selected from the group consisting of SEQ ID NOS:

129-180 and 185-196.

33. The nucleic acid ligand of claim 30 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS:

181-184.

34. The nucleic acid ligand of claim 20 wherein said selectin is P-selectin.

35. The nucleic acid ligand to P-selectin of claim 34 wherein said nucleic acid ligand is ribonucleic acid (RNA) ligand.

36. The nucleic acid ligand of claim 35 which comprises a modified ribonucleic acid.

37. The nucleic acid ligand of claim 36 wherein said modified ribonucleic acid is selected from the group consisting of a 2′

-amino (2′

-NH₂) modified ribonucleic acid, a 2′

-fluoro (2′

-F) modified ribonucleic acid, and a 2′

-O-Methyl (2′

-O-Me) modified ribonucleic acid.

38. The nucleic acid ligand to P-selectin of claim 37 wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Tables 19, 21 and 25.

39. The nucleic acid ligand of claim 38 wherein said ligand is selected from the group consisting of SEQ ID NOS:

199-219 and 236-290.

40. The nucleic acid ligand of claim 35 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NO:

291.

41. A nucleic acid ligand to P-selectin of claim 34 wherein said ligand is substantially homologous to a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and 25.

42. The nucleic acid ligand to P-selectin of claim 34 wherein said ligand has substantially the same structure as a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and 25.

43. The nucleic acid ligand to P-selectin of claim 37 wherein said nucleic acid ligand is deoxyribonucleic acid (DNA).

44. The nucleic acid ligand of claim 6 wherein said ligand is a ribonucleic acid ligand.

45. The nucleic acid ligand of claim 44 which comprises a modified ribonucleic acid.

46. The nucleic acid ligand of claim 45 wherein said modified ribonucleic acid is selected from the group consisting of 2′

-amino (2′

-NH₂) modified ribonucleic acids, 2′

-fluoro (2′

-F) modified ribonucleic acids and 2′

-O-Methyl (2′

-O-Me) modified ribonucleic acids.

47. The nucleic acid ligand of claim 6 wherein said ligand is a deoxyribonucleic acid.

48. The nucleic acid ligand of claim 6 wherein said ligand has been chemically modified at a sugar and/or phosphate and/or base.

49. A multivalent Complex comprising a plurality of ligands of claim 6.

50. A nucleic acid ligand to a lectin identified according to the method comprising:

a) contacting a candidate mixture of nucleic acids with a lectin, wherein nucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

c) 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 said lectin, whereby nucleic acid ligand of said lectin may be identified.