Enzymatic DNA molecules

• US 6,326,174 B1
• Filed: 08/25/1997
• Issued: 12/04/2001
• Est. Priority Date: 12/02/1994
• Status: Expired due to Term

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Breaker, Ronald R., Joyce, Gerald F.
• Primary Examiner(s): Schwartzman, Robert A.
• Application Number:
  US08/849,567
• Time in Patent Office: 1,562 Days
• Field of Search: 435/91.1, 435/91.2, 435/91.31, 536/23.1, 536/24.5
• US Class Current: 435/91.31
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12N 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

The present invention discloses deoxyribonucleic acid enzymes—catalytic or enzymatic DNA molecules—capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

Claims

1. A method of selecting a catalytic DNA molecule that cleaves a substrate nucleic acid sequence at a specific site, comprising the following steps:

a. obtaining a population of single-stranded DNA molecules;

b. admixing nucleotide-containing substrate molecules with said population of single-stranded DNA molecules to form an admixture;

c. maintaining said admixture for a sufficient period of time and under predetermined reaction conditions to allow single-stranded DNA molecules in said population to cause cleavage of said substrate sequences, thereby producing substrate cleavage products;

d. separating said population of single-stranded DNA molecules from said substrate sequences and substrate cleavage products; and

e. isolating single-stranded DNA molecules that cleave nucleotide-containing substrate at a specific site from said population.

2. The method of claim 1, wherein said substrate comprises RNA.

3. The method of claim 1, wherein said DNA molecules that cleave said substrate at a specific site are tagged with an immobilizing agent.

4. The method of claim 3, wherein said agent comprises biotin.

5. The method of claim 4, wherein said isolating step further comprises exposing said tagged DNA molecules to a solid surface having avidin linked thereto, whereby said tagged DNA molecules become attached to said solid surface.

6. A method of in vitro selection of catalytic DNA molecules that cleave phosphoester bonds in a nucleic acid substrate, comprising the following steps:

a. obtaining a population of single-stranded DNA molecules;

b. introducing genetic variation into said population to produce a variant population;

c. selecting individuals from said variant population that meet predetermined selection criteria;

d. separating said selected individuals from the remainder of said variant population; and

e. amplifying said selected individuals, thereby obtaining in vitro selected catalytic DNA molecules that cleave phosphoester bonds in a nucleic acid substrate.

7. A non-naturally-occurring catalytic DNA molecule comprising a nucleotide sequence defining a conserved core flanked by one or more recognition domains, variable regions, and spacer regions.

8. The catalytic DNA molecule of claim 7, wherein said nucleotide sequence defines a first variable region contiguous or adjacent to the 5′

-terminus of the molecule, a first recognition domain located 3′

-terminal to the first variable region, a first spacer region located 3′

-terminal to the first recognition domain, a first conserved region located 3′

-terminal to the first spacer region, a second spacer region located 3′

-terminal to the first conserved region, a second conserved region located 3′

-terminal to the second spacer region, a second recognition domain located 3′

-terminal to the second conserved region, and a second variable region located 3′

-terminal to the second recognition domain.

9. The catalytic DNA molecule of claim 7, wherein said nucleotide sequence defines a first variable region contiguous or adjacent to the 5′

-terminus of the molecule, a first recognition domain located 3′

-terminal to the first variable region, a first spacer region located 3′

-terminal to the first recognition domain, a first conserved region located 3′

-terminal to the first spacer region, a second spacer region located 3′

-terminal to the first conserved region, a second conserved region located 3′

-terminal to the second spacer region, a second recognition domain located 3′

-terminal to the second conserved region, a second variable region located 3′

-terminal to the second recognition domain, and a third recognition domain located 3′

-terminal to the second variable region.

10. A catalytic DNA molecule having site-specific endonuclease activity, wherein said molecule includes one or more hairpin loop structures.

11. The catalytic DNA molecule of claim 10, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

12. The catalytic DNA molecule of claim 10, wherein said molecule is single-stranded.

13. The catalytic DNA molecule of claim 11, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

14. The catalytic DNA molecule of claim 11, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

15. The catalytic DNA molecule of claim 11, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

16. A catalytic DNA molecule having site-specific endonuclease activity, wherein said substrate nucleic acid sequence is attached to said catalytic DNA molecule.

17. The catalytic DNA molecule of claim 16, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

18. The catalytic DNA molecule of claim 16, wherein said molecule is single-stranded.

19. The catalytic DNA molecule of claim 17, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

20. The catalytic DNA molecule of claim 17, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

21. The catalytic DNA molecule of claim 17, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

22. A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule comprises a nucleotide sequence selected from the group consisting of:

SEQ ID NOS 52 through 101.

23. The catalytic DNA molecule of claim 22, wherein said endonuclease activity is enhanced by the presence of Mg²⁺.

24. A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule has a substrate binding affinity of about 1 μ

M or less.

25. The catalytic DNA molecule of claim 24, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

26. The catalytic DNA molecule of claim 24, wherein said molecule is single-stranded.

27. The catalytic DNA molecule of claim 25, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

28. The catalytic DNA molecule of claim 25, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

29. The catalytic DNA molecule of claim 25, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

30. A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule binds substrate with a K_D of less than about 0.1 μ

M.

31. The catalytic DNA molecule of claim 30, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

32. The catalytic DNA molecule of claim 30, wherein said molecule is single-stranded.

33. The catalytic DNA molecule of claim 31, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

34. The catalytic DNA molecule of claim 31, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

35. The catalytic DNA molecule of claim 31, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

36. A catalytic DNA molecule having site-specific endonuclease activity, wherein said endonuclease activity is enhanced by the presence of a divalent cation.

37. The catalytic DNA molecule of claim 36, wherein said divalent cation is selected from the group consisting of Pb²⁺, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺.

38. The catalytic DNA molecule of claim 36, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

39. The catalytic DNA molecule of claim 36, wherein said molecule is single-stranded.

40. The catalytic DNA molecule of claim 38, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

41. The catalytic DNA molecule of claim 38, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

42. The catalytic DNA molecule of claim 38, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

43. A catalytic DNA molecule having site-specific endonuclease activity, wherein said endonuclease activity is enhanced by the presence of a monovalent cation.

44. The catalytic DNA molecule of claim 43, wherein said monovalent cation is selected from the group consisting of Na⁺ and K⁺.

45. The catalytic DNA molecule of claim 43, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

46. The catalytic DNA molecule of claim 43, wherein said molecule is single-stranded.

47. The catalytic DNA molecule of claim 45, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

48. The catalytic DNA molecule of claim 45, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

49. The catalytic DNA molecule of claim 45, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

50. A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule comprises a conserved core flanked by first and second substrate binding regions and wherein one or more spacer nucleotides are present between said conserved core and said substrate binding region.

51. The catalytic DNA molecule of claim 50, wherein said conserved core comprises one or more conserved regions.

52. The catalytic DNA molecule of claim 50, wherein said first substrate binding region includes a nucleotide sequence selected from the group consisting of:

53. The catalytic DNA molecule of claim 50, wherein said second substrate binding region includes a nucleotide sequence selected from the group consisting of:

54. The catalytic DNA molecule of claim 50, further comprising a third substrate binding region, wherein said third region includes a nucleotide sequence selected from the group consisting of:

55. The catalytic DNA molecule of claim 51, wherein said one or more conserved regions includes a nucleotide sequence selected from the group consisting of:

56. The catalytic DNA molecule of claim 51, further comprising one or more variable or spacer nucleotides between said conserved regions in said conserved core.

57. The catalytic DNA molecule of claim 54, further comprising one or more spacer regions between said substrate binding regions.

58. The catalytic DNA molecule of claim 54, wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence.

59. The catalytic DNA molecule of claim 54, wherein said molecule is single-stranded.

60. The catalytic DNA molecule of claim 54, wherein said single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

61. The catalytic DNA molecule of claim 58, wherein said substrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, or composites thereof.

62. The catalytic DNA molecule of claim 58, wherein said endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at said cleavage site.

63. A composition comprising two or more populations of catalytic DNA molecules having site-specific endonuclease activity, wherein each population of catalytic DNA molecules cleaves a different nucleotide sequence in a substrate.

64. A composition comprising two or more populations of catalytic DNA molecules having site-specific endonuclease activity, wherein each population of catalytic DNA molecules recognizes a different substrate.

65. A method of cleaving a phosphoester bond, comprising:

a. admixing a catalytic DNA molecule capable of cleaving a substrate nucleic acid sequence at a defined cleavage site with a phosphoester bond-containing nucleic acid substrate, to form a reaction admixture;

b. maintaining said admixture under predetermined reaction conditions to allow said catalytic DNA molecule to cleave said phosphoester bond, thereby producing a population of nucleic acid substrate products;

c. separating said products from said catalytic DNA molecule; and

d. adding additional substrate to said catalytic DNA molecule to form a new reaction admixture.

66. The method of claim 65, wherein said substrate comprises RNA.

67. A method of cleaving a phosphoester bond, comprising:

a. admixing a catalytic DNA molecule capable of cleaving a substrate nucleic acid sequence at a defined cleavage site with a phosphoester bond-containing nucleic acid substrate, to form a reaction admixture; and

b. maintaining said admixture under predetermined reaction conditions to allow said catalytic DNA molecule to cleave said phosphoester bond, thereby producing a population of nucleic acid substrate products, wherein said predetermined reaction conditions include the presence of a monovalent cation, a divalent cation, or both.

68. The method of claim 67, wherein said substrate comprises RNA.