METHODS OF USING OLIGONUCLEOTIDE-GUIDED ARGONAUTE PROTEINS

20160289734

15/089243

April 01, 2016

The invention relates to the use of Argonaute polypeptide:guide molecule complexes as fast and specific nucleic acid probes, as specific, nucleic acid-guided restriction enzymes for DNA and RNA substrates, and as a means to detect RNA-protein interactions, RNA detection, DNA detection, and RNA depletion. Using such Argonaute polypeptide:guide molecule complexes enables fast and specific detection, purification, and enzymatic activity.

1. A method of cleaving an RNA or DNA molecule, comprising binding to a target RNA or DNA sequence a complex comprising an Argonaute polypeptide and a heterologous, single-stranded oligonucleotide guide molecule that comprises a recruiting domain comprising at least 8 nucleotides at the 5′ end of the guide molecule (g1-g8) and a stabilization domain adjacent and 3′ to the recruiting domain and comprising at least 4 nucleotides (g9-g12) in a sample, wherein the stabilization domain of the guide molecule has sufficient complementarity to its target RNA or DNA sequence such that the Argonaute polypeptide:guide molecule complex binds stably to the target RNA or DNA sequence, and allowing the Argonaute polypeptide:guide molecule to cleave the RNA or DNA molecule.

2. The method of claim 1, wherein the stabilization domain consists of 4 to 8 nucleotides.

3. The method of claim 1, wherein the recruiting domain consists of 8 nucleotides, and the stabilization domain consists of 4 to 8 nucleotides.

4. The method of claim 1, wherein the oligonucleotide guide molecule is a DNA guide molecule.

5. The method of claim 1, wherein the target RNA or DNA is single-stranded or double-stranded.

6.-7. (canceled)

8. The method of claim 1, wherein the guide molecule comprises one or more mismatches 3′ of g5.

9. (canceled)

10. The method of claim 8, wherein the guide molecule comprises two mismatches 3′ of g5 and 5′ of g9.

11. The method of claim 8, wherein the guide molecule comprises two mismatches 3′ of g8 to the 3′ end of the molecule.

12.-16. (canceled)

17. The method of claim 1, wherein the sample comprises a solution comprising a salt.

18.-22. (canceled)

23. The method of claim 17, wherein the solution further comprises a buffer.

24. The method of claim 23, wherein the buffer is selected from the group consisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes], N-tris-(hyrdroxymethyl)-methyl-2-aminoethanesulfonic acid (TES), 3-[N-tris (hydroxymethyl) methylamino]-2-hydroxypropanesulfonic acid (TAPSO), and 3-[N-tris-(hydroxymethyl-mettlylamino]-propanesulfonic acid (TAPS); and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

25.-28. (canceled)

29. The method of claim 24, wherein the pH of the buffer is from about 7 to about 8.8.

30. (canceled)

31. The method of claim 17, wherein the solution further comprises a reducing agent.

32. The method of claim 31, wherein the reducing agent comprises dithiothreitol (DTT) or 2-mercaptoethanol (β-mercaptoethanol).

33.-34. (canceled)

35. The method of claim 17, wherein the solution further comprises a detergent.

36. The method of claim 35, wherein the detergent is a nonionic, non-denaturing detergent or a zwitterionic nondenaturing detergent.

37.-40. (canceled)

41. The method of claim 17, wherein the solution further comprises glycerol or a sugar.

42.-43. (canceled)

44. The method of claim 17, wherein the solution further comprises a divalent metal cation.

45.-47. (canceled)

48. The method of claim 17, wherein the solution comprises (1) 18 mM HEPES-KOH, pH 7.4; 50 mM NaCl, 3 mM MnCl2, 0.01% octylphenoxy poly(ethyleneoxy)ethanol, 5 mM DTT, and 10% glycerol or (2) 18 mM HEPES-KOH, pH 7.4; 75 mM C5H8NNaO4, 3 mM MnCl2, 0.01% octylphenoxy poly(ethyleneoxy)ethanol, 5 mM DTT, and 10% glycerol.

49.-52. (canceled)

53. A method of subcloning a desired double stranded nucleic acid fragment from a donor double stranded nucleic acid molecule (donor fragment) to an acceptor double stranded nucleic acid molecule (acceptor molecule), comprising the steps of: (a) cleaving the desired donor fragment according to the method of claim 1, wherein (i) a first Argonaute:guide molecule complex targets a first region of a first strand of the donor fragment; (ii) a second Argonaute guide molecule complex that targets the first region of a second strand of the donor fragment, such that the targeted region of the first strand and the targeted region of the second strand partially overlap such that cleavage by the first and second Argonaute:guide molecule complex creates a first sticky end; (iii) a third Argonaute:guide molecule complex that targets a second region of the first strand of the donor fragment; (iv) a fourth Argonaute:guide molecule complex targets the second region of the second strand of the donor fragment as that of the third Argonaute:guide molecule complex, such that the targeted region of the first strand of the target nucleic acid and the targeted region of the second strand of target nucleic acid partially overlap such that cleavage by the third and fourth Argonaute:guide molecule complex creates a second sticky end; (v) isolating the cleaved desired fragment; (b) cleaving the acceptor molecule according to the method of claim 1, wherein (i) steps (a)(i)-(a)(iv) are repeated for the acceptor molecule, thus creating third and fourth sticky ends that are complementary to the first and second sticky ends; (ii) isolating the cleaved acceptor molecule; and (c) combining the molecules from steps (a) and (b) to create a mixture and incubating the mixture under appropriate conditions to form a new molecule comprising the desired donor fragment subcloned into the acceptor molecule.

54. The method of claim 53, wherein ligase is added to the mixture of step (c).

55. The method of claim 53, wherein the sticky ends are from about 18 to 24 nucleotides long, and ligase is not added to the mixture of step (c).

56. The method of claim 53, wherein the sticky ends are not complementary, and further comprising in step (c) combining a first single-stranded oligonucleotide that is complementary to a sticky end of the desired fragment and to a sticky end of the acceptor molecule such that the oligonucleotide bridges the sticky ends, and a second single-stranded oligonucleotide that is complementary to the other sticky ends of the desired fragment and of the acceptor molecule, such that the oligonucleotide bridges the sticky ends; and treating the mixture with polymerase and ligase.

57. A kit, comprising an Argonaute polypeptide and a single-stranded oligonucleotide guide molecule that comprises a recruiting domain comprising 8 nucleotides at the 5′ end of the guide molecule (g1-g8) and a stabilization domain adjacent and 3′ to the recruiting domain and comprising at least 4 nucleotides (g9-g12) and having a sequence sufficiently complementary to a target RNA or DNA molecule nucleic acid sequence such that the Argonaute polypeptide:guide molecule complex binds stably to the target RNA or DNA sequence.

58. The kit of claim 57, wherein the oligonucleotide guide molecule is a DNA guide molecule.

59. The kit of claim 57, further comprising a buffer.

60. (canceled)

61. The kit of claim 59, wherein the buffer further comprises at least one selected from the group consisting of: a salt, a detergent, a reducing agent, a divalent metal cation, glycerol and sugar.

62. The kit of claim 61, wherein the buffer is selected from the group consisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes], N-tris-(hyrdroxymethyl)-methyl-2-aminoethanesulfonic acid (TES), 3-[N-tris (hydroxymethyl) methylamino]-2-hydroxypropanesulfonic acid (TAPSO), and 3-[N-tris-(hydroxymethyl-mettlylamino]-propanesulfonic acid (TAPS); and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

63. The kit of claim 61, wherein the salt is NaCl, KCl, or C5H8NNaO4.

64. The kit of claim 61, wherein the detergent is a nonionic non-denaturing detergent or a nondenaturing zwitterionic detergent.

65. (canceled)

66. The kit of claim 59, wherein the buffer comprises (1) 18 mM HEPES-KOH, pH 7.4; 50 mM NaCl, 3 mM MnCl2, 0.01% octylphenoxy poly(ethyleneoxy)ethanol, 5 mM DTT, and 10% glycerol, or (2) 18 mM HEPES-KOH, pH 7.4; 75 mM C5H8NNaO4, 3 mM MnCl2, 0.01% octylphenoxy poly(ethyleneoxy)ethanol, 5 mM DTT, and 10% glycerol.

67. (canceled)

68. The kit of claim 57, wherein the guide molecule binds a disease marker sequence, a disorder marker sequence, or an infectious agent sequence.

69. The kit of claim 57, wherein the guide molecule further comprises a detectable label.

70.-71. (canceled)

72. The kit of claim 57, further comprising a probe to detect the target RNA or DNA sequence.

73. The kit of claim 69, wherein the detectable label is at least one fluorophore, the fluorophore localized to the recruiting or stabilization domain forming a first arm, wherein the guide molecule comprises additional sequence at the 3′ end that is complementary to the domain comprising the at least one fluorophore, the sequence labeled with at least one quencher and forming a second arm; the first arm separated from the second arm by not more than about 60 nucleotides; the guide molecule forming with the target RNA or DNA sequence under detection conditions a double-stranded hybrid having a first strength; the first and second arm sequences having sufficient complementarity to one another to form under detection conditions a double-stranded stem hybrid having a second strength less than the first strength, whereby in the absence of the target RNA or DNA sequence fluorescence of the at least one fluorophore is quenched; and the first and second hybrid strengths being selected such that the guide molecule fluoresces when the at least one fluorophore is stimulated under detection conditions in the presence of the target RNA or DNA sequence.

74.-81. (canceled)

82. A method of recruiting an Argonaute polypeptide to a heterologous target RNA or DNA sequence comprising combining the Argonaute polypeptide with a heterologous, single-stranded oligonucleotide guide molecule that comprises a recruiting domain of at least 8 nucleotides at its 5′ end of the guide molecule (g1-g8) and a stabilization domain adjacent and 3′ to the recruiting domain and comprising at least 4 nucleotides (g9-g12), wherein the stabilization domain of the guide molecule has sufficient complementarity to the target RNA or DNA sequence such that the Argonaute polypeptide:guide molecule complex stably binds to the target RNA or DNA sequence.

83. The method of claim 82, wherein the stabilization domain consists of 4 to 8 nucleotides.

84. The method of claim 82, wherein the recruiting domain consists of 8 nucleotides, and the stabilization domain consists of 4 to 8 nucleotides.

85. The method of claim 82, wherein the oligonucleotide guide molecule is a DNA guide molecule.

86. The method of claim 82, wherein the target RNA or DNA is single-stranded or double-stranded.

87.-88. (canceled)

89. The method of claim 82, wherein the guide molecule comprises one or more mismatches 3′ of g5.

90. (canceled)

91. The method of claim 89, wherein the guide molecule comprises two mismatches 3′ of g5 and 5′ of g9.

92. The method of claim 89, wherein the guide molecule comprises two mismatches 3′ of g8 to the 3′ end of the molecule.

93.-99. (canceled)

100. The method of claim 82, wherein the heterologous target RNA or DNA is a eukaryotic, prokaryotic, or viral mRNA or gene.

101. The method of claim 82, wherein the Argonaute polypeptide:guide molecule complex is attached to a solid support.

102. The method of claim 101, wherein contacting the Argonaute polypeptide:guide molecule complex to a sample depletes the sample of the target RNA or DNA molecule.

103. The method of claim 102, wherein the depleted sequence is an rRNA sequence.

104. (canceled)

105. The method of claim 82, wherein the target comprises an RNA molecule, and the guide molecule targets a splice site on the mRNA molecule.

106.-109. (canceled)

110. The method of claim 82, further comprising detecting the Argonaute polypeptide:guide molecule complex.

111. The method of claim 110, wherein the guide molecule further comprises a detectable label.

112.-113. (canceled)

114. The method of claim 111, wherein the detecting comprises detecting the target RNA or DNA nucleotide sequence.

115. The method of claim 114, wherein detecting the target RNA or DNA nucleotide sequence comprising using a probe to detect the target RNA or DNA nucleotide sequence.

116. The method of claim 111, wherein the detectable label is at least one fluorophore, the fluorophore localized to the recruiting or stabilization domain forming a first arm, wherein the guide molecule comprises additional sequence at the 3′ end that is complementary to the domain comprising at least one fluorophore, the sequence labeled with at least one quencher and forming a second arm; the first arm separated from the second arm by not more than about 60 nucleotides; the guide molecule forming with the target RNA or DNA sequence under detection conditions a double-stranded hybrid having a first strength; the first and second arm sequences having sufficient complementarity to one another to form under detection conditions a double-stranded stem hybrid having a second strength less than the first strength, whereby in the absence of the target RNA or DNA sequence fluorescence of the at least one fluorophore is quenched; and the first and second hybrid strengths being selected such that the guide molecule fluoresces when the at least one fluorophore is stimulated under detection conditions in the presence of the target RNA or DNA sequence.

117. The method of claim 110, wherein the guide molecule further comprises an additional sequence added to the 3′ end of the guide molecule.

118. The method of claim 117, wherein the detecting is detecting the additional sequence of the guide molecule.

119.-123. (canceled)

124. The method of claim 110, wherein the target RNA or DNA molecule encodes a disease marker sequence, a disorder marker sequence, or an infectious agent sequence.

125. A method of identifying an RNA binding polypeptide comprising binding to a target RNA sequence in an RNA molecule a complex comprising an Argonaute polypeptide and a heterologous, single-stranded oligonucleotide guide molecule that comprises a recruiting domain comprising at least 8 nucleotides at its 5′ end of the guide molecule (g1-g8) and a stabilization domain adjacent and 3′ to the recruiting domain and comprising at least 4 nucleotides (g9-g12) in a sample, wherein the stabilization domain of the guide molecule has sufficient complementarity to the target RNA sequence such that the Argonaute polypeptide:guide molecule complex binds stably to the target RNA sequence, isolating the Argonaute polypeptide:guide molecule complex bound to the target RNA sequence, and detecting polypeptides bound to the RNA molecule comprising the target RNA sequence.

126. The method of claim 125, wherein the stabilization domain consists of 4 to 8 nucleotides.

127. The method of claim 125, wherein the recruiting domain consists of 8 nucleotides, and the stabilization domain consists of 4 to 8 nucleotides.

128. The method of claim 125, wherein the oligonucleotide guide molecule is a DNA guide molecule.

129. The method of claim 125, wherein the target RNA is single-stranded or double-stranded.

130.-131. (canceled)

132. The method of claim 125, wherein the guide molecule comprises one or more mismatches 3′ of g5.

133. (canceled)

134. The method of claim 132, wherein the guide molecule comprises two mismatches 3′ of g5 and 5′ of g9.

135. The method of claim 132, wherein the guide molecule comprises two mismatches 3′ of g8 to the 3′ end of the molecule.

136.-146. (canceled)

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