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, 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, an archaeal thermostable single-stranded DNA binding protein (SSB), a guide DNA, and a nucleic acid substrate that comprises a target sequence that is complementary to the guide DNA.
2. The composition of claim 1 , wherein the guide DNA is 16-21 nucleotides in length.
3. The composition of claim 1 , wherein the guide DNA is at least 22 nucleotides in length.
4. The composition of claim 3 , further comprising a strand-displacing polymerase.
5. The composition according to claim 1 , wherein the nucleic acid substrate is a double-stranded DNA (dsDNA) substrate.
6. The composition according to claim 1 , wherein the thermostable single-stranded DNA binding protein (SSB) is an extreme thermostable single-stranded DNA binding protein (ET SSB).
7. The composition according to claim 1 , 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.
8. A method for cleaving a nucleic acid substrate at a target sequence, comprising:
(a) combining a bacterial thermostable DNA guided Argonaute protein, an archaeal thermostable SSB, a guide DNA and the nucleic acid substrate that comprises a target sequence that is complementary to at least part of the guide DNA, to produce a reaction mix;
(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.
9. The method of claim 8 , 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.
10. The method of claim 9 , 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.
11. The method according to claim 10 , wherein the cleavage of the substrate is substantially complete within 1 hour.
12. The method according to claim 8 , wherein the nucleic acid substrate is eukaryotic genomic dsDNA.
13. The method according to claim 8 , wherein cleaving in (c) is performed under isothermal conditions.
14. A method for copying a substrate DNA, comprising:
(a) combining a bacterial thermostable DNA guided Argonaute protein, an archaeal thermostable 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; and
(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.
15. The method of claim 14 , wherein the Argonaute protein is catalytically inactive in (i) or (ii).
16. The method of claim 14 , wherein the Argonaute protein is catalytically active in (ii) or (iii).
17. The method of claim 14 , wherein (b) is an isothermal reaction.
18. The method according to claim 14 , wherein the guide DNA is at least 24 nucleotides.
19. The method of claim 14 , wherein the reaction mix further comprises a second guide DNA complementary to a second target sequence that is proximate to the opposite strand to a first target sequence, and wherein the incubation step results in extension of the second guide DNA using the opposing strand as a template, thereby resulting in copying of both strands of the nucleic acid substrate between the first and the second guide DNA.
20. A method for identifying a polymorphism, comprising:
(a) combining a bacterial thermostable DNA guided Argonaute protein, an archaeal thermostable single-stranded DNA binding protein (SSB), an allele-specific guide DNA, and a nucleic acid substrate that has a polymorphism in the region corresponding to nucleotides 12-15 of the guide DNA, to produce a reaction mix;
(b) incubating the reaction mix to cleave the nucleic acid substrate; and
(c) detecting cleavage of the substrate, wherein cleavage indicates the allele of the polymorphism.
21. The method of claim 20 , wherein the nucleic acid substrate is a double-stranded DNA (dsDNA) substrate.
22. A method according to claim 20 , wherein the guide DNA hybridizes with the nucleic acid substrate.
23. A non-naturally occurring, preparation comprising an archaeal thermostable DNA guided Argonaute protein and a single-stranded DNA binding protein (SSB), a guide DNA, and a nucleic acid substrate that comprises a target sequence that is complementary to the guide DNA, wherein the SSB is a bacterial thermostable SSB, a viral SSB, or a mammalian SSB.