Poly zinc finger proteins with improved linkers
AssigneeMassachusetts Institute of Technology
InventorsJin-Soo Kim, Carl O. Pabo
Life of this patent
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Abstract
The present invention provides methods for making chimeric zinc finger proteins with improved linkers, providing zinc finger proteins with enhanced affinity and specificity.
Claims58
A method of making a chimeric zinc finger protein that binds to adjacent target sites, the method comprising the steps of:
- (i) selecting a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent;
- (ii) using structure-based design to determine the physical separation between the first and second domains when they are individually bound to the first and second target sites;
- (iii) selecting a flexible linker that is at least 1-2 Å longer than the physical separation between the first and second domains;
- and
- (iv) fusing the first and second domains with the flexible linker, thereby making a chimeric zinc finger protein that binds to adjacent target sites.
A method of making a chimeric zinc finger protein that binds to adjacent target sites, the method comprising the steps of:
- (i) selecting a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent;
- (ii) selecting a flexible linker that is five or more amino acids in length;
- and
- (iv) fusing the first and second domains with the flexible linker, thereby making a chimeric zinc finger protein that binds to adjacent target sites.
A chimeric zinc finger protein that binds to adjacent target sites, the chimeric zinc finger protein comprising:
- (i) a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent;
- and
- (ii) a flexible linker that is at least 1-2 Å longer than the physical separation between the first and second domains when they are individually bound to the first and second target sites, as determined by structure-based modeling;
- wherein the first and second domains are fused with the flexible linker.
A chimeric zinc finger protein that binds to adjacent target sites, the chimeric zinc finger protein comprising:
- (i) a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent;
- and
- (ii) a flexible linker that is five or more amino acids in length;
- wherein the first and second domains are fused with the flexible linker.
Dependencies are inferred from the claim text.