Aptamer-guided gene targeting
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.
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, wherein the DNA binding moiety binds to a target DNA sequence; and
donor polynucleotide sequence on the 3′ end of the bifunctional oligonucleotide that is sufficiently complementary to the target DNA sequence to anneal to the target DNA sequence under physiological conditions and wherein the donor polynucleotide sequence repairs a double-stranded DNA break produced by the DNA binding moiety at or near the target DNA sequence, wherein the DNA binding moiety is a homing endonuclease selected from the group consisting of I-SceI, LAGLIDADG (SEQ ID NO:2) homing endonucleases, HNH endonuclease, His-Cys box enzymes, HNH (I-HmuI) endonuclease, His-Cys box (I-PpoI) endonuclease and GIY-YIG (I-TevI) endonuclease.
2. The bifunctional oligonucleotide of claim 1 , wherein the homing endonuclease is I-SceI.
3. The bifunctional oligonucleotide of claim 1 , wherein the target DNA sequence is a gene or fragment thereof.
4. The bifunctional oligonucleotide of claim 1 , wherein the aptamer is a DNA aptamer.
5. The bifunctional oligonucleotide of claim 1 , wherein the donor polynucleotide sequence is 90-100% homologous to the target DNA sequence.
6. The bifunctional oligonucleotide of claim 1 , wherein the donor polynucleotide sequence is 100% complementary to the target DNA sequence.
7. A vector encoding the bifunctional oligonucleotide of claim 1 .
8. A pharmaceutical composition comprising one or more bifunctional oligonucleotides according to claim 1 .
9. The bifunctional oligonucleotide of claim 1 , wherein the aptamer comprises DNA or RNA.
10. The bifunctional oligonucleotide of claim 1 , wherein the donor polynucleotide sequence comprises DNA or RNA.