Aptamer-guided gene targeting

Abstract

Compositions and methods for modifying genetic material are provided. One embodiment provides aptamers capable of binding to a site-specific DNA binding moiety to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting or AGT). One embodiment provides an oligonucleotide containing a aptamer, preferably a DNA aptamer at the 5′ end. The oligonucleotide also contains a region of homology, also referred to as donor DNA, to a desired nucleic acid, locus, or gene. The DNA binding moiety can be a nucleic acid, a protein, or a complex of proteins. In a preferred embodiment the DNA binding moiety is a homing endonuclease that cuts DNA to facilitate the modification of the DNA by the donor DNA.

We claim: 
     
         1 . A bifunctional oligonucleotide having 5′ and 3′ ends comprising:
 an aptamer on the 5′ end of the bifunctional oligonucleotide that specifically binds to a DNA binding moiety; and 
 donor DNA on the 3′ end of the bifunctional oligonucleotide that is sufficiently complementary to a target nucleic acid sequence to hybridize to the target sequence under physiological conditions. 
 
     
     
         2 . The bifunctional oligonucleotide of  claim 1 , wherein the DNA binding moiety is a homing endonuclease. 
     
     
         3 . The bifunctional oligonucleotide of  claim 2 , wherein the homing endonuclease is I-SceI. 
     
     
         4 . The bifunctional oligonucleotide of  claim 1 , wherein the target nucleic acid is a gene or fragment thereof. 
     
     
         5 . The bifunctional oligonucleotide of  claim 1 , wherein the aptamer is a DNA aptamer. 
     
     
         6 . The bifunctional oligonucleotide of  claim 1 , wherein the DNA binding moiety is a DNA binding protein. 
     
     
         7 . The bifunctional oligonucleotide of  claim 1 , wherein the donor DNA is 90-100% homologous to the target nucleic acid sequence. 
     
     
         8 . The bifunctional oligonucleotide of  claim 1 , wherein the donor DNA is 100% complementary to the target nucleic acid sequence. 
     
     
         9 . The bifunctional oligonucleotide of  claim 1 , wherein the DNA binding moiety is selected from the group consisting of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the Cas9 nuclease of the clustered regularly interspaced short palindromic repeat (CRISPR) system. 
     
     
         10 . A vector encoding the bifunctional oligonucleotide of  claim 1 . 
     
     
         11 . A pharmaceutical composition comprising one or more bifunctional oligonucleotides according to  claim 1 . 
     
     
         12 . A method of modifying a target nucleic acid in a subject comprising:
 administering to the subject, the pharmaceutical composition of  claim 11 , wherein the donor DNA is sufficiently complementary to the target nucleic acid to hybridize to the target nucleic acid under physiological conditions.   
     
     
         13 . The method of  claim 12 , wherein the aptamer is specific for a homing endonuclease expressed by the subject. 
     
     
         14 . The method of  claim 12 , wherein the DNA binding moiety is administered before, concurrently, or after the pharmaceutical composition of  claim 11 .