Methods of producing nucleic acid ligands

• US 7,368,236 B2
• Filed: 04/06/2004
• Issued: 05/06/2008
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Tuerk, Craig, Gold, Larry
• Primary Examiner(s): VIVLEMORE, TRACY ANN
• Application Number:
  US10/818,954
• Publication Number:
  US 20040241731A1
• Time in Patent Office: 1,491 Days
• Field of Search: None
• US Class Current: 435/5
• 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: 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 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: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C07K 7/22 Tachykinins, e.g. Eledoisins , Substance P; Related peptides
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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 2740/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 : incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/301 : Hairpin oligonucleotides
• CPC Class Codes: C12Q 2541/101 : Selex
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 2333/16 : 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
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/918 : Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• 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 33/532 : 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 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

The present invention includes methods for the identification and production of improved nucleic acid ligands based on the SELEX process. Also included are nucleic acid ligands to the HIV-RT protein identified according to the methods described therein.

Claims

1. A method for modifying a nucleic acid ligand for a given target so that the modified ligand has at least one of the following improved properties:

decreased size;

improved stability;

improved binding to the target;

modification of biological activity of the target;

resistance to clearance;

wherein said modifying comprises adding, removing or substituting nucleotide residues, wherein said residues may be chemically modified, and/or chemically modifying said nucleic acid ligand based on;

(a) determining which nucleotides are crucial to the interaction with the target molecule;

(b) chemical modification experiments;

or

(c) determining the structural conformation of said nucleic acid ligand; and

(d) modifying said ligand based on said determinations made in steps a) through c).

2. A method according to claim 1 wherein (a), (b) or (c) comprises performing covariance analysis.

3. A method according to claim 1 wherein (a), (b) or (c) comprises determining which nucleotide residues are necessary for maintaining the three dimensional structure of the ligand.

4. A method according to claim 1 wherein (a), (b) or (c) comprises determining the positions of the ligand which are crucial to the bound structure of the ligand with the target.

5. A method according to claim 1 wherein (a), (b) or (c) comprises determining which nucleotide residues interact with the target.

6. A method according to claim 5 wherein (a), (b) or (c) comprises determining which nucleotide residues interact with the target to facilitate the formation of ligand-target binding pairs.

7. A method according to claim 5 wherein (a), (b) or (c) comprises determining which nucleotide residues are involved in proximal contacts with the target.

8. A method according to claim 1 wherein (a), (b) or (c) comprises determining the binding affinity of a modified ligand relative to the ligand before modification, where the modification is provided by nucleotide substitution in the ligand.

9. A method according to claim 1 wherein (a), (b) or (c) comprises determining the binding affinity of a modified ligand relative to the ligand before modification, where the modification is provided by the absence of one or more nucleotides of the ligand.

10. A method according to claim 1 wherein (a), (b) or (c) comprises determining the binding affinity of a chemically modified form of the ligand relative to the ligand before chemical modification.

11. A method according to claim 1 wherein (a), (b) or (c) comprises chemically modifying the ligand in the presence of the target, and determining which nucleotides of the ligand are not chemically modified.

12. A method according to claim 1 wherein (a), (b) or (c) comprises denaturing the ligand and chemically modifying both the denatured and non-denatured forms of the ligand, and determining which nucleic acid residues are modified in the denatured ligand that are not modified in the non-denatured ligand.

13. A method according to claim 1 wherein said modifying comprises truncation of the ligand.

14. A method according to claim 1 wherein said modifying comprises incorporation of a cross-linking agent to covalently link the ligand to the target.

15. A method according to claim 1 wherein said modifying produces a small molecule that mimics the structure of the initial ligand framework.

16. A method according to claim 1 wherein said modifying has the effect of increasing the in vivo stability of the ligand.

17. A method according to claim 1 wherein said modifying has the effect of enhancing or mediating the delivery of the ligand.

18. A method according to claim 1 wherein said modifying provides resistance to enzymatic or chemical degradation.

19. A method according to claim 1 wherein said modifying reduces the rate of clearance of the modified ligand from the body during therapy.

20. A method according to claim 1 wherein said modifying comprises chemical modification or derivatization of the ligand at the ribose and/or phosphate and/or base positions.

21. A method according to claim 1 wherein the chemical modification experiments (b) are performed using a chemical selected from:

ethylnitrosourea;

dimethyl sulfate;

carbodiimide;

diethylpyrocarbonate; and

kethoxal.

22. A method according to claim 1 wherein the target is selected from:

HIV-RT protein;

HIV-1 Rev protein;

Thrombin.

23. The method of claim 1 wherein said nucleic acid ligand has been obtained from a candidate mixture of nucleic acids by:

(1) contacting the candidate mixture with the target molecule, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

(2) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

(3) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; and

(4) repeating steps (1)-(3) as necessary to identify a nucleic acid ligand, wherein the ligand-enriched mixture of nucleic acids produced in step (3) is used as the candidate mixture in step (1).

24. A method according to claim 1 wherein the modified ligand is a single stranded nucleic acid.

25. A method according to claim 1 wherein the modified ligand is an RNA or DNA.

26. A method according to claim 1 wherein the modified ligand is an RNA and said ligand modification includes a substitution on ribose of 2′

-methoxy for 2′

-hydroxyl.

27. A method according to claim 1 wherein the modified ligand is an RNA and said ligand modification includes modification of certain riboses to contain 2′

-NH₂.

56. The method according to claim 1 wherein the modified ligand is an RNA and said modifying comprises a 2′

substitution on ribose.

28. A method for modifying a nucleic acid ligand for a given target, such target 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, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target so that the modified ligand has at least one of the following improved properties:

decreased size;

improved stability;

improved binding to the target;

modification of biological activity of the target;

capability to cross tissue or cell membrane barriers;

resistance to clearance;

wherein said modifying comprises adding, removing or substituting nucleotide residues, wherein said residues may be chemically modified, and/or chemically modifying said nucleic acid ligand based on;

(a) determining which nucleotides are crucial to the interaction with the target molecule;

(b) chemical modification experiments;

or

(c) determining the structural conformation of said nucleic acid ligand; and

(d) modifying said ligand based on said determinations made in steps a) through c).

29. The method according to claim 28 wherein (a), (b) or (c) comprises performing covariance analysis.

30. The method according to claim 28 wherein (a), (b) or (c) comprises determining which nucleotide residues are necessary for maintaining the three dimensional structure of the ligand.

31. The method according to claim 28 wherein (a), (b) or (c) comprises determining the positions of the ligand which are crucial to the bound structure of the ligand with the target.

32. The method according to claim 28 wherein (a), (b)or (c) comprises determining which nucleotide residues interact with the target.

33. The method according to claim 28 wherein (a), (b) or (c) comprises determining which nucleotide residues interact with the target to facilitate the formation of ligand-target binding pairs.

34. The method according to claim 33 wherein (a), (b) or (c) comprises determining which nucleotide residues are involved in proximal contacts with the target.

35. The method according to claim 28 wherein (a), (b) or (c) comprises determining the binding affinity of a modified ligand relative to the ligand before modification, where the modification is provided by nucleotide substitution in the ligand.

36. The method according to claim 28 wherein (a), (b) or (c) comprises determining the binding affinity of a modified ligand relative to the ligand before modification, where the modification is provided by the absence of one or more nucleotides of the ligand.

37. The method according to claim 28 wherein (a), (b) or (c) comprises determining the binding affinity of a chemically modified form of the ligand relative to the ligand before chemical modification.

38. The method according to claim 28 wherein (a), (b) or (c) comprises chemically modifying the ligand in the presence of the target, and determining which nucleotides of the ligand are not chemically modified.

39. The method according to claim 28 wherein (a), (b) or (c) comprises denaturing the ligand and chemically modifying both the denatured and non-denatured forms of the ligand, and determining which nucleic acid residues are modified in the denatured ligand that are not modified in the non-denatured ligand.

40. A method according to claim 28 wherein said modifying comprises truncation of the ligand.

41. The method according to claim 28 wherein said ligand modifying comprises incorporation of a cross-linking agent to covalently link the ligand to the target.

42. The method according to claim 28 wherein said modifying produces a small molecule that mimics the structure of the initial ligand framework.

43. The method according to claim 28 wherein said modifying has the effect of enhancing or mediating the delivery of the ligand.

44. The method according to claim 28 wherein said ligand modification provides resistance to enzymatic or chemical degradation.

46. The method according to claim 28 wherein said modifying has the effect of increasing the in vivo stability of the ligand.

47. A method according to claim 28 wherein said modifying reduces the rate of clearance of the modified ligand from the body during therapy.

48. The method according to claim 28 wherein the chemical modification experiments (b) are performed using a chemical selected from:

ethylnitrosourea;

dimethyl sulfate;

carbodiimide;

diethylpyrocarbonate; and

kethoxal.

49. The method according to claim 28 wherein the target is selected from:

HIV-RT protein;

HIV-1 Rev protein;

or

Thrombin.

50. The method of claim 28 wherein said nucleic acid ligand has been obtained from a candidate mixture of nucleic acids by:

(1) contacting the candidate mixture with the target molecule, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

(2) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

(3) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; and

(4) repeating steps (1)-(3) as necessary to identify a nucleic acid ligand, wherein the ligand-enriched mixture of nucleic acids produced in step (3) is used as the candidate mixture in step (1).

51. The method according to claim 28 wherein the modified ligand is a single stranded nucleic acid.

52. A method according to claim 28 wherein the modified ligand is an RNA or DNA.

53. The method according to claim 28 wherein the modified ligand is an RNA and said ligand modification includes a substitution on ribose of 2′

-methoxy for 2′

-hydroxyl.

54. The method according to claim 28 wherein the modified ligand is an RNA and said modifying comprises a 2′

substitution on ribose.

55. The method according to claim 28 wherein the modified ligand is an RNA and said ligand modification includes modification of certain riboses to contain 2′

-NH₂.

45. The method according to 28 wherein said ligand modifying comprises chemical modification or derivatization of the ligand at the ribose and/or phosphate and/or base positions.