Methods for synthetic unrandomization of oligomer fragments

• US 5,698,391 A
• Filed: 12/16/1994
• Issued: 12/16/1997
• Est. Priority Date: 08/23/1991
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Brown-Driver, Vickie, Hanecak, Ronnie, Freier, Susan M., Davis, Peter, Vickers, Timothy, Cook, Phillip Dan, Ecker, David J., Bruice, Thomas W., Anderson, Kevin, Wyatt, Jacqueline, Sanghvi, Yogesh S.
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/357,396
• Time in Patent Office: 1,096 Days
• Field of Search: 435/5, 435/6
• US Class Current: 506/1
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Methods useful for the determination of oligomers which have specific activity for a target molecule from a pool of primarily randomly assembled oligomers are provided. The disclosed methods involve repeated syntheses of increasingly simplified sets of oligomers coupled with selection procedures for determining oligomers having the highest activity. Freedom from the use of enzymes allows the application of these methods to any molecules which can be oligomerized in a controlled fashion.

Claims

1. A method for determining an oligonucleotide having an assayable activity for a target molecule comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotide, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), and having in an additional defined common position a different nucleotide, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(e) assaying each of the sets of said further group for said assayable activity;

(f) selecting the set of said further group having the highest assayable activity; and

(g) repeating steps (d) through (f) until an oligonucleotide having said assayable activity for said target molecule is determined.

2. The method of claim 1 further comprising the steps of:

(h) selecting an oligonucleotide of step (g) with the highest assayable activity;

(i) preparing additional oligonucleotides, each additional oligonucleotide differing from the oligonucleotide of step (h) by at least one nucleotide which has been added, deleted, or substituted by a different nucleotide;

(j) assaying said additional oligonucleotides for said assayable activity; and

(k) selecting the oligonucleotide of said additional oligonucleotides having the highest assayable activity.

4. The method of claim 1 wherein said oligonucleotide is a cyclic oligonucleotide.

5. The method of claim 1 wherein said oligonucleotide is from 3 to 20 nucleotides in length.

6. The method of claim 1 wherein said oligonucleotide is labeled with a detectable label.

7. The method of claim 1 wherein said assaying is by gel shift assay, streptavidin capture of biotinylated target, filter binding assay or affinity chromatography.

8. The method of claim 1 wherein the oligonucleotide is sterically constrained.

9. The method of claim 1 wherein said assayable activity is binding activity for a target molecule.

10. The method of claim 9 wherein the target molecule is a nucleic acid.

11. The method of claim 9 wherein said target molecule is a carbohydrate.

12. The method of claim 9 wherein said target molecule is a protein.

13. The method of claim 9 wherein said target molecule is a glycoprotein.

14. The method of claim 9 wherein the target molecule is an immunoglobulin, receptor, receptor binding ligand, antigen, enzyme, or transcription factor.

15. The method of claim 9 wherein the target molecule is a phospholipase, tumor necrosis factor, endotoxin, interleukin, leukotriene, plasminogen activator, protein kinase, cell adhesion molecule, lipoxygenase, hydrolase, transacylase, or transcription factor.

16. The method of claim 9 wherein the target molecule is derived from Candida, papilloma virus, Epstein-Barr virus, rhinovirus, hepatitis virus, human immunodeficiency virus, herpes simplex virus, influenza virus or cytomegalovirus.

17. The method of claim 9 wherein the target molecule is an RNA molecule or fragment thereof.

18. The method of claim 16 wherein the target molecule is the TAR element of human immunodeficiency virus.

19. The method of claim 16 wherein the target molecule is the gag-pol stem loop of human immunodeficiency virus.

20. The method of claim 16 wherein the target molecule is the 47-base pair stem loop of ras.

21. The method of claim 15 wherein the target molecule is endothelin-1.

22. The method of claim 15 wherein the target molecule is leukotriene B-4.

23. The method of claim 16 wherein the target molecule is human immunodeficiency virus gp120.

24. The method of claim 22 wherein the target molecule is human immunodeficiency virus tat protein.

25. The method of claim 1 wherein the assayable activity is catalytic activity.

26. The method of claim 1 wherein the assayable activity is induction of cellular production of interferon.

3. The method of claim 3 wherein steps (i) through (k) are performed iteratively until no additional oligonucleotides having an increase in said assayable activity is identified.

27. A method for determining an oligonucleotide having an assayable activity for a target molecule comprising the steps of:

(a) binding a group of target molecules to a solid support;

(b) incubating the solid support with a group of oligonucleotides nucleotides labeled with a detectable label having all positions randomized, under binding conditions;

(c) detecting the presence or absence of binding wherein binding of an oligonucleotide indicates activity of the oligonucleotide for one or more target molecules, thereby identifying selected target molecules;

(d) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(e) assaying each of the sets for activity against said selected target molecules;

(f) selecting the set having the highest activity;

(g) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (f), and having in an additional defined common position a different nucleotide, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(h) assaying each of the sets of said further group for said assayable activity for said selected target molecules;

(i) selecting the set of said further group having the highest activity; and

(j) repeating steps (g) through (i) until an oligonucleotide having an assayable activity for a target molecule is determined.

28. The method of claim 27 wherein said oligonucleotide is a cyclic oligonucleotide.

29. A method for determining an oligonucleotide cassette having an assayable activity for a target molecule comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), each set of said further group of sets having a different nucleotide in an additional defined common position, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(e) assaying each of the sets of said further group for specific activity for the target molecule;

(f) selecting the set of said further group having the highest activity; and

(g) repeating steps (d) through (f) until an oligonucleotide cassette having said assayable activity for said target molecule is determined.

30. The method of claim 29 wherein said oligonucleotide is a cyclic oligonucleotide.

31. A method for determining an oligonucleotide having an assayable activity for a target molecule comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least one predefined oligonucleotide cassette and at least one flanking region by;

(i) defining a common position in said flanking region of the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides of said flanking region which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), each set of said further group of sets having a different nucleotide in an additional defined common position, the nucleotides in the positions of the oligonucleotides which are not in a defined position being randomized;

(e) assaying each of the sets of said further group for said assayable activity;

(f) selecting the set of said further group having the highest activity;

(g) repeating steps (d) through (f) until an oligonucleotide having said assayable activity for said target molecule is determined.

32. The method of claim 31 wherein said oligonucleotide is a cyclic oligonucleotide.

33. The method of claim 31 wherein said cassette is prepared by a method comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), each set of said further group of sets having a different nucleotide in an additional defined common position, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(e) assaying each of the sets of said further group for specific activity for the target molecule;

(f) selecting the set of said further group having the highest activity; and

(g) repeating steps (d) through (f) until an oligonucleotide cassette having said assayable activity for said target molecule is determined.

34. A method of determining an optimal set of monomers for oligomers having an assayable activity for a target molecule comprising:

(a) preparing a group of sets of oligomers of substantially the same length, each oligomer comprising at least three monomer units, by;

(i) selecting a group of monomer units, and

(ii) synthesizing sets of randomized oligomers, each set of randomized oligomers using all but at least one defined monomer unit from said group of monomer units, said excluded monomer unit being different for each set of oligomers;

(b) assaying each of the sets for activity against the target molecule;

(c) identifying each set of oligomers which expresses little or no activity against the target molecule;

(d) selecting monomers for inclusion in said optimal set wherein each defined monomer unit excluded from the sets identified in step (c) are selected.

35. The method of claim 34 wherein said oligomer has an ethylene glycol phosphate (egp) backbone.

36. The method of claim 34 wherein said oligomer has a hydroxy pyrrolidine phosphate (hpp) backbone.

37. The method of claim 34 wherein said oligomer is an oligonucleotide.

38. The method of claim 34 wherein said oligomer is a cyclic oligomer.

39. The method of claim 38 wherein said cyclic oligomer is a cyclic oligonucleotide.

40. The method of claim 38 wherein said cyclic oligomer is a cyclic amine.

41. A method for determining an oligonucleotide having an assayable activity for a non-antibody target molecule comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), and having in an additional defined common position a different nucleotide, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(e) assaying each of the sets of said further group for said assayable activity;

(f) selecting the set of said further group having the highest assayable activity; and

(g) repeating steps (d) through (f) until an oligonucleotide having said assayable activity for said target molecule is determined.

42. The method of claim 41 further comprising the steps of:

(h) selecting an oligonucleotide of step (g) with the highest assayable activity;

(i) preparing additional oligonucleotides, each additional oligonucleotide differing from the oligonucleotide of step (h) by at least one oligonucleotide which has been added, deleted, or substituted with a different nucleotide;

(j) assaying said additional oligonucleotides for said assayable activity; and

(k) selecting the oligonucleotide of said additional oligonucleotides having the highest assayable activity.

43. The method of claim 42 wherein steps (i) through (k) are performed iteratively until no additional oligonucleotide having an increase in said assayable activity is identified.

44. The method of claim 42 wherein said oligonucleotide is a cyclic oligonucleotide.

45. The method of claim 41 wherein said oligonucleotide is from 3 to 20 nucleotides in length.

46. The method of claim 41 wherein said oligonucleotide is labeled with a detectable label.

47. The method of claim 41 wherein said assaying is by gel shift assay, streptavidin capture of biotinylated target, filter binding assay or affinity chromatography.

48. The method of claim 41 wherein the oligonucleotide is sterically constrained.

49. The method of claim 41 wherein said assayable activity is binding activity for a target molecule.

50. The method of claim 49 wherein the target molecule is a nucleic acid.

51. The method of claim 49 wherein said target molecule is a carbohydrate.

52. The method of claim 49 wherein said target molecule is a protein.

53. The method of claim 49 wherein said target molecule is a glycoprotein.

54. The method of claim 49 wherein the target molecule is a receptor, receptor binding ligand, antigen, enzyme, or transcription factor.

55. The method of claim 49 wherein the target molecule is a phospholipase, tumor necrosis factor, endotoxin, interleukin, leukotriene, plasminogen activator, protein kinase, cell adhesion molecule, lipoxygenase, hydrolase, transacylase, or transcription factor.

56. The method of claim 49 wherein the target molecule is derived from Candida, papilloma virus, Epstein-Barr virus, rhinovirus, hepatitis virus, human immunodeficiency virus, herpes simplex virus, influenza virus or cytomegalovirus.

57. The method of claim 49 wherein the target molecule is an RNA molecule or fragment thereof.

58. The method of claim 56 wherein the target molecule is the TAR element of human immunodeficiency virus.

59. The method of claim 56 wherein the target molecule is the gag-pol stem loop of human immunodeficiency virus.

60. The method of claim 56 wherein the target molecule is the 47-base pair stem loop of ras.

61. The method of claim 55 wherein the target molecule is endothelin-1.

62. The method of claim 55 wherein the target molecule is leukotriene B-4.

63. The method of claim 56 wherein the target molecule is human immunodeficiency virus gp120.

64. The method of claim 56 wherein the target molecule is human immunodeficiency virus tat protein.

65. The method of claim 41 wherein the assayable activity is catalytic activity.

66. The method of claim 41 wherein the assayable activity is induction of cellular production of interferon.

67. A method for determining an oligonucleotide having an assayable activity for a non-antibody target molecule comprising the steps of:

(a) binding a group of target molecules to a solid support;

(b) incubating the solid support with a group of oligonucleotides labeled with a detectable label having all positions randomized, under binding conditions;

(c) detecting the presence or absence of binding wherein binding of an oligonucleotide indicates activity of the oligonucleotide for one or more target molecules, thereby identifying selected target molecules;

(d) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(e) assaying each of the sets for activity against said target molecules;

(f) selecting the set having the highest activity;

(g) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (f), and having in an additional defined common position a different nucleotide, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(h) assaying each of the sets of said further group for said assayable activity for said selected target molecules;

(i) selecting the set of said further group having the highest activity; and

(j) repeating steps (g) through (i) until an oligonucleotide having said assayable activity for a target molecule is determined.

68. The method of claim 67 wherein said oligonucleotide is a cyclic oligonucleotide.

69. A method for determining an oligonucleotide cassette having an assayable activity for a non-antibody target molecule comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), each set of said further group of sets having a different nucleotide in an additional defined common position, the nucleotides in the positions of the oligonucleotides which are not in a defined common position being randomized;

(e) assaying each of the sets of said further group for assayable activity for the target molecule;

(f) selecting the set of said further group having the highest activity; and

(g) repeating steps (d) through (f) until an oligonucleotide cassette having said assayable activity for said target molecule is determined.

70. The method of claim 69 wherein said oligonucleotide is a cyclic oligonucleotide.

71. A method for determining an oligonucleotide having an assayable activity for a non-antibody target molecule comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least one predefined oligonucleotide cassette and at least one flanking region by;

(i) defining a common position in said flanking region of the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides of said flanking region which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of the sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), each set of said further group of sets having a different nucleotide in an additional defined common position, the nucleotides in the positions of the oligonucleotides which are not in a defined position being randomized;

(e) assaying each of the sets of said further group for said assayable activity;

(f) selecting the set of said further group having the highest activity; and

(g) repeating steps (d) through (f) until an oligonucleotide having said assayable activity for said target molecule is determined.

72. The method of claim 71 wherein said oligonucleotide is a cyclic oligonucleotide.

73. The method of claim 71 wherein said cassette is prepared by a method comprising the steps of:

(a) preparing a group of sets of oligonucleotides of substantially the same length, each oligonucleotide comprising at least three nucleotides, by;

(i) defining a common position in the oligonucleotides of the sets, and

(ii) synthesizing said sets of oligonucleotides such that each set has a different nucleotide in said common position, the nucleotides which are not in said common position being randomized;

(b) assaying each of the sets for activity against the target molecule;

(c) selecting the set having the highest activity;

(d) preparing a further group of sets of oligonucleotides of said substantially same length, each of sets of said further group having in the previously defined common position the nucleotide appearing in that position in the set selected in step (c), each set of said further group of sets having a different nucleotide in an additional defined common position, the nucleotides in the positions of the oligonucleotides which are not in a defined, common position being randomized;

(e) assaying each of the sets of said further group for specific activity for the target molecule;

(f) selecting the set of said further group having the highest activity; and

(g) repeating steps (d) through (f) until an oligonucleotide cassette having said assayable activity for said target molecule is determined.

74. A method of determining an optimal set of monomers for oligomers having an assayable activity for a non-antibody target molecule comprising:

(a) preparing a group of sets of oligomers of substantially the same length, each oligomer comprising at least three monomer units, by;

(i) selecting a group of monomer units, and

(ii) synthesizing sets of randomized oligomers, each set of randomized oligomers using all but at least one defined monomer unit from said group of monomer units, said excluded monomer unit being different for each set of oligomers;

(b) assaying each of the sets for activity against the target molecule;

(c) identifying each set of oligomers which expresses little or no activity against the target molecule;

(d) selecting monomers for inclusion in said optimal set wherein each defined monomer unit excluded from the sets identified in step (c) are selected.

75. The method of claim 74 wherein said oligomer has an ethylene glycol phosphate (egp) backbone.

76. The method of claim 74 wherein said oligomer has a hydroxy pyrrolidine phosphate (hpp) backbone.

77. The method of claim 74 wherein said oligomer is an oligonucleotide.

78. The method of claim 74 wherein said oligomer is a cyclic oligomer.

79. The method of claim 78 wherein said cyclic oligomer is a cyclic oligonucleotide.

80. The method of claim 78 wherein said cyclic oligomer is a cyclic amine.