Methods of using oligonucleotide-guided argonaute proteins

Patent metadata

Field	Value
Publication number	US-2016289734-A1
Application number	US-201615089243-A
Country	US
Kind code	A1
Filing date	Apr 1, 2016
Priority date	Apr 3, 2015
Publication date	Oct 6, 2016
Grant date	—

Abstract

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.

First claim

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 MnCl 2 , 0.01% octylphenoxy poly(ethyleneoxy)ethanol, 5 mM DTT, and 10% glycerol or (2) 18 mM HEPES-KOH, pH 7.4; 75 mM C 5 H 8 NNaO 4 , 3 mM MnCl 2 , 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 , wherei

Assignees

• Univ Massachusetts

Inventors

• Zamore Phillip David
• Moore Melissa Jeanne
• Jolly Samson Michael
• Salomon William Edward
• Serebrov Victor
• Zhang Han

Classifications

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12N15/64

  General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title