In vitro cleavage of DNA using argonaute
Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, extreme thermostable single-stranded DNA binding protein (ET SSB), and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute/guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and/or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.
1 . A composition comprising:
a bacterial thermostable DNA guided Argonaute protein and
an archaeal thermostable single-stranded DNA binding protein (SSB),
wherein the composition excludes helicase and excludes recombinase.
2 . A composition according to claim 1 further comprising a guide DNA.
3 . A composition according to claim 2 further comprising a nucleic acid substrate that comprises a target sequence that is complementary to the guide DNA.
4 . A composition according to claim 3 , wherein the nucleic acid substrate is a double-stranded DNA (dsDNA) substrate.
5 . A composition according to claim 2 , wherein the guide DNA is 16-21 nucleotides in length.
6 . A composition according to claim 2 , wherein the guide DNA is at least 22 nucleotides in length.
7 . A composition according to claim 6 further comprising a strand-displacing polymerase.
8 . A composition according to claim 1 , wherein the bacterial DNA guided Argonaute protein is an Aquifex aeolicus , a, an Exiguobacterium , an Anoxybacillus flavithermus , a Thermus thermophilus , a Synechococcus , or a Thermosynechococcus elongatus Argonaute.
9 . A cell-free composition comprising:
a bacterial DNA guided Argonaute protein, and
a viral single-stranded DNA binding protein (SSB),
wherein the composition excludes helicase and excludes recombinase.
10 . A composition according to claim 9 , wherein the bacterial DNA guided Argonaute protein is a bacterial thermostable DNA guided Argonaute protein.
11 . A composition according to claim 9 further comprising a guide DNA.
12 . A composition according to claim 11 further comprising a nucleic acid substrate that comprises a target sequence that is complementary to the guide DNA.
13 . A composition according to claim 9 further comprising a guide DNA, wherein the guide DNA is 16-21 nucleotides in length.
14 . A composition according to claim 9 further comprising a guide DNA, wherein the guide DNA is at least 22 nucleotides in length.
15 . A composition according to claim 14 further comprising a strand-displacing polymerase.
16 . A composition according to claim 9 further comprising a double-stranded DNA (dsDNA) substrate.
17 . A composition according to claim 9 , wherein the bacterial DNA guided Argonaute protein is an Aquifex aeolicus , a Microsystis aeruginosa , a Clostridium bartlettii , an Exiguobacterium , an Anoxybacillus flavithermus , an Aromatoleum aromaticum , a Thermus thermophilus , a Synechococcus , a Synechococcus elongatus , or a Thermosynechococcus elongatus Argonaute.
18 . A cell-free kit comprising a bacterial thermostable DNA guided Argonaute protein, an archaeal thermostable single-stranded binding protein (SSB) and a non-naturally occurring buffering agent.
19 . A cell-free kit according to claim 18 further comprising at least one of a guide DNA, a polymerase, and dNTPs.
20 . A cell-free kit comprising
a bacterial DNA guided Argonaute protein,
a eukaryotic or viral single-stranded DNA binding protein (SSB), and
a non-naturally occurring buffering agent.
21 . A kit according to claim 20 further comprising at least one of a guide DNA, a polymerase, and dNTPs.
22 . A cell-free in vitro reagent for targeted cleavage of DNA comprising:
a bacterial DNA guided Argonaute protein, and
a eukaryotic or viral single-stranded DNA binding protein (SSB).
23 . A cell-free composition comprising:
a bacterial DNA guided Argonaute protein, and
a eukaryotic single-stranded DNA binding protein (SSB),
wherein the composition excludes helicase and excludes recombinase.
24 . A method for cleaving a nucleic acid substrate at a target sequence, comprising:
(a) combining
a bacterial DNA guided Argonaute protein,
a eukaryotic or viral SSB,
a guide DNA and
a nucleic acid substrate that comprises a target sequence that is complementary to at least part of the guide DNA,
to produce a reaction mix, wherein the reaction mix excludes helicase and excludes recombinase;
(b) incubating the reaction mix under conditions suitable for cleaving the nucleic acid substrate at the target sequence; and
(c) cleaving the nucleic acid substrate.
25 . A method according to claim 24 , wherein the nucleic acid substrate is a double-stranded DNA (dsDNA) substrate and step (b) results in a nick in the dsDNA substrate after cleavage of one strand.
26 . A method according to claim 24 , wherein the nucleic acid substrate is eukaryotic genomic dsDNA.
27 . A method according to claim 24 , wherein the method further comprises nicking the second strand of the dsDNA substrate at a site on a strand that is complementary to or proximate to the target sequence but not the guide DNA.
28 . A method for copying a substrate DNA, comprising:
(a) combining
a bacterial DNA guided Argonaute protein,
a eukaryotic or viral single-stranded DNA binding protein (SSB),
a double-stranded DNA (dsDNA) substrate that comprises a target sequence,
a strand-displacing polymerase, and
a guide DNA that is complementary to the target sequence,
to produce a reaction mix, wherein the reaction mix excludes helicase and excludes recombinase;
(b) incubating the reaction mix, so that a strand of the DNA substrate provides a template for the polymerase, and extending (i) the 3′ end of the guide DNA (ii) a separate primer that hybridizes to a site that is proximal to the target sequence or (iii) the 3′ end formed by the Argonaute induced nick; and
(c) copying the nucleic acid substrate.
29 . A method according to claim 28 , wherein the Argonaute protein is catalytically inactive in (i) or (ii).
30 . A method according to claim 28 , wherein the Argonaute protein is catalytically active in (ii) or (iii).
31 . A method according to claim 28 , wherein (b) is an isothermal reaction.
32 . A method according to claim 28 , wherein the guide DNA is at least 24 nucleotides.