Programmable dna-guided artificial restriction enzymes

Abstract

Methods and compositions for specific cleavage of DNA molecules using single-stranded guides and Argonaute (Ago) proteins are described.

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         10 . A method of site-specific modification of a target DNA molecule comprising: contacting the target DNA molecule with: (i) one or more single-stranded DNA guide molecules having complementarity to the target DNA molecule and (ii) an Ago protein. 
     
     
         11 . The method of  claim 10 , wherein the site-specific modification is cleavage of one or more double-stranded DNA molecules, wherein the method comprises:
 (a) denaturing the one or more double-stranded DNA molecules into partially denatured one or more double-stranded DNA molecules or first single-stranded DNA molecules and second single-stranded DNA molecules;   (b) contacting the partially denatured one or more double-stranded DNA molecules or the first single-stranded DNA molecules and the second single-stranded DNA molecules with (i) one or more single-stranded DNA guide molecules that can hybridize to the partially denatured one or more double-stranded DNA molecules or to the first single-stranded DNA molecules and to the second single-stranded DNA molecules and (ii) an Ago protein, wherein the partially denatured one or more double-stranded DNA molecules or the first single-stranded DNA molecules and the second single-stranded DNA molecules are cleaved; and   (c) allowing the cleaved partially denatured one or more double-stranded DNA molecules to hybridize or allowing the cleaved first single-stranded DNA molecules and the cleaved second single-stranded DNA molecules to hybridize into one or more double-stranded DNA molecules.   
     
     
         12 . The method of  claim 11 , wherein:
 (i) when the first single-stranded DNA molecules and the second single-stranded DNA molecules are cleaved, one or more target portions of the first single-stranded DNA molecules and the second single-stranded DNA molecules are removed such that when the first single-stranded DNA molecules and the second single-stranded DNA molecules hybridize, one or more target portions of the double-stranded DNA molecules are removed; or   (ii) when the partially denatured double-stranded DNA molecules are cleaved, one or more target portions of the double-stranded DNA molecules are removed such that when the partially denatured double-stranded DNA molecules hybridize, one or more target portions of the double-stranded DNA molecules are removed.   
     
     
         13 . The method of  claim 10 , wherein the site-specific modification is cleavage of one or more single-stranded DNA molecules, wherein the method comprises: (a) contacting the one or more single-stranded DNA molecules with (i) one or more single-stranded DNA guide molecules that can hybridize to the one or more single-stranded DNA molecules; and (ii) an Ago protein; wherein the one or more single-stranded DNA molecules are cleaved. 
     
     
         14 . The method of  claim 10 , wherein the site-specific modification is insertion of one or more target DNA molecules into recipient double-stranded DNA molecules, wherein the method comprises:
 (a) denaturing the recipient double-stranded DNA molecules into partially denatured recipient double-stranded DNA molecules or first recipient single-stranded DNA molecules and second recipient single-stranded DNA molecules;   (b) contacting the partially denatured recipient double-stranded DNA molecules or the first recipient single-stranded DNA molecules and the second recipient single-stranded DNA molecules with (i) one or more single-stranded DNA guides that can hybridize to the partially denatured recipient double-stranded DNA molecules or to the first recipient single-stranded DNA molecules and to the second recipient single-stranded DNA molecules (ii) an Ago protein, and (iii) one or more target DNA molecules;   wherein one or more target DNA molecules are inserted into the recipient double-stranded DNA molecules.   
     
     
         15 . A method of DNA fingerprinting comprising:
 (a) denaturing one or more double-stranded DNA molecules into partially denatured double-stranded DNA molecules or first single-stranded DNA molecules and second single-stranded DNA molecules;   (b) contacting the partially denatured double-stranded DNA molecules or the first single-stranded DNA molecules and the second single-stranded DNA molecules with (i) one or more single-stranded DNA guide molecules that can hybridize to the partially denatured double-stranded DNA molecules or to the first single-stranded DNA molecules and to the second single-stranded DNA molecules and (ii) an Ago protein, wherein the partially denatured double-stranded DNA molecules or the first single-stranded DNA molecules and the second single-stranded DNA molecules are cleaved;   (c) hybridizing the partially denatured double-stranded DNA molecules or the first single-stranded DNA molecules and the second single-stranded DNA molecules to form double-stranded DNA molecule fragments;   (d) separating the double-stranded DNA molecule fragments.   
     
     
         16 . The method of  claim 15 , wherein the double-stranded DNA molecule fragments are labeled. 
     
     
         17 . The method of  claim 15 , wherein the double-stranded DNA molecule fragments are not labeled. 
     
     
         18 . The method of  claim 15 , wherein the separated double-stranded DNA fragments are detected. 
     
     
         19 . The method of  claim 15 , wherein the one or more double-stranded DNA molecules are amplified prior to step (a). 
     
     
         20 . The method of  claim 10 , wherein the target DNA molecule, double-stranded DNA, or single-stranded DNA is methylated. 
     
     
         21 . The method of  claim 10 , wherein the target DNA molecule, double-stranded DNA, or single-stranded DNA is eukaryotic, prokaryotic, or synthetic DNA. 
     
     
         22 . The method of  claim 10 , wherein the one or more DNA guides are synthetic DNA molecules. 
     
     
         23 . The method of  claim 10 , wherein the one or more single-stranded DNA guides are about 9 to about 60 nucleotides in length. 
     
     
         24 . The method of  claim 10 , wherein the one or more single-stranded DNA guides have complementarity to the target DNA molecules, double-stranded DNA molecules, or single-stranded DNA molecules. 
     
     
         25 . The method of  claim 10 , wherein the Ago protein is a PfAgo or ttAgo protein. 
     
     
         26 . The method of  claim 10 , wherein the denaturing or partial denaturing occurs in vitro at above about 60° C. 
     
     
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         28 . A method of assembling two or more double-stranded DNA molecules into one or more larger double-stranded DNA molecules comprising:
 (a)(i) in a single reaction, denaturing or partially denaturing two or more double-stranded DNA molecules and contacting the denatured or partially denatured DNA molecules with two or more single stranded guides and an Ago protein, wherein the two or more single-stranded guides are designed to generate specific overhanging ends on the resulting two or more double-stranded DNA molecules, wherein the two or more denatured or partially denatured DNA molecules are cleaved to generate two or more double-stranded DNA molecules with specific overhanging ends; or   (a)(ii) in separate reactions, denaturing or partially denaturing two or more double-stranded DNA molecules and contacting the denatured or partially denatured DNA molecules with two or more single stranded guides and an Ago protein, wherein the two or more single-stranded guides are designed to generate specific overhanging ends on the resulting two or more double-stranded DNA molecules, and wherein the two or more denatured or partially denatured DNA molecules are cleaved to generate two or more double-stranded DNA molecules with specific overhanging ends; and combining the two or more double-stranded DNA molecules with specific overhanging ends; and   (b) allowing the two or more two or more double-stranded DNA molecules with specific overhanging ends to hybridize via the specific overhanging ends such that the two or more double-stranded DNA molecules are assembled into one or more larger double-stranded DNA molecules.   
     
     
         29 . The method of  claim 28 , wherein the two or more double-stranded DNA molecules are assembled into one or more larger double-stranded DNA molecules with ligation dependent or ligation-independent processes.