Zinc finger domain libraries
OwnerJin-Soo Kim, Kwang-Hee Bae +4inventors
InventorsJin-Soo Kim, Kwang-Hee Bae, Kyung-Soon Park, Young Do Kwon, Eun-Hyun Ryu, Moon-Sun Hwang
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Abstract
Disclosed are libraries of chimeric zinc finger domains. The libraries can include two or more zinc finger domains from naturally occurring proteins, e.g., mammalian proteins and particularly human proteins. Useful chimeric zinc finger domains can be identified from the library. Also disclosed are the amino acid sequences of zinc finger domains that recognize particular sites.
Claims52
A library comprising a plurality of polypeptides, each polypeptide comprising a first and a second zinc finger domain,
- wherein (1) the first and second zinc finger domains of each polypeptide are each identical to a zinc finger domain from a naturally occurring protein and either (i) do not occur in the same naturally occurring protein or (ii) occur in the same naturally occurring protein in a different configuration than in the polypeptide, (2) the first zinc finger domain varies among polypeptides of the plurality, and (3) the second zinc finger domain varies among polypeptides of the plurality.
A library comprising a plurality of polypeptides, each polypeptide comprising a first and a second zinc finger domain,
- wherein (1) the first and second zinc finger domains of each polypeptide are each identical to a zinc finger domain from a naturally occurring protein and do not occur in the same naturally occurring protein, (2) the first zinc finger domain varies among polypeptides of the plurality, and (3) the second zinc finger domain varies among polypeptides of the plurality.
A library comprising a plurality of nucleic acids, each nucleic acid of the plurality encoding a polypeptide comprising a first and a second zinc finger domain,
- wherein (1) the first and second zinc finger domains of the polypeptide encoded by each nucleic acid of the plurality are each identical to zinc finger domains from naturally occurring proteins and either (i) do not occur in the same naturally occurring protein or (ii) occur in the same naturally occurring protein in a different configuration than in the polypeptide, (2) the first zinc finger domain varies among nucleic acids of the plurality, and (3) the second zinc finger domain varies among nucleic acids of the plurality.
A method of producing a plurality of chimeric nucleic acids, the method comprising:
- (a) providing a set of nucleic acids, each nucleic acid of the set comprising a sequence encoding a zinc finger domain from a naturally occurring protein;
- and
- (b) joining each nucleic acid of the set to one or more other nucleic acids of the set to form a plurality of chimeric nucleic acids.
A method of identifying a nucleic acid encoding a polypeptide that recognizes a target DNA site, the method comprising:
- providing a plurality of nucleic acids, each nucleic acid of the plurality encoding a polypeptide comprising a first and a second zinc finger domain, wherein the first and second zinc finger domains of the polypeptide encoded by each nucleic acid of the plurality are identical to zinc finger domains from different naturally occurring mammalian proteins, the first zinc finger domain varies among nucleic acids of the plurality, and the second zinc finger domain varies among nucleic acids of the plurality;
- providing cells containing a reporter gene operably linked to a target DNA site;
- expressing the plurality of nucleic acids in the cells;
- identifying a cell having altered expression of the reporter gene relative to the level of expression in the absence of a polypeptide that recognizes the target DNA site level;
- and
- identifying a nucleic acid expressed in the cell, the nucleic acid being a nucleic acid of the plurality, thereby identifying a nucleic acid encoding a polypeptide that recognizes the target DNA site.
A polypeptide comprising a first and a second zinc finger domain, wherein the first and second zinc finger domains are each from naturally occurring proteins and are selected from the zinc finger domains of Tables 5, 6, and 7.
A purified polypeptide comprising an amino acid sequence selected from the group consisting of:
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-His-X-Ser-Ser-Xb-X-Arg-His-X3-5-His (SEQ ID NO:
- 167),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Ile-X-Ser-Asn-Xb-X-Arg-His-X3-5-His (SEQ ID NO:
- 168),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Lys-X-Ser-Asn-Xb-X-Arg-His-X3-5-His (SEQ ID NO:169),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Gln-X-Ser-Asn-Xb-X-Lys-His-X3-5-His (SEQ ID NO:170),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Gln-X-Ser-His-Xb-X-Thr-His-X3-5-His (SEQ ID NO:171),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Val-X-Ser-Asn-Xb-X-Val-His-X3-5-His (SEQ ID NO:
- 172),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Asp-X-Ser-Cys-Xb-X-Arg-His-X3-5-His (SEQ ID NO:193),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Ile-X-Ser-Asn-Xb-X-Val-His-X3-5-His (SEQ ID NO:
- 194),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Trp-X-Ser-Asn-Xb-X-Arg-His-X3-5-His (SEQ ID NO:195), and
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Asp-X-Ser-Ala-Xb-X-Arg-His-X3-5-His (SEQ ID NO:196),
- wherein Xa is phenylalanine or tyrosine, and Xb is a hydrophobic residue.
A purified polypeptide comprising between two and ten segments, each segment having an amino acid sequence selected from the group consisting of:
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-His-X-Ser-Ser-Xb-X-Arg-His-X3-5-His (SEQ ID NO:167),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Ile-X-Ser-Asn-Xb-X-Arg-His-X3-5-His (SEQ ID NO:
- 168),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Lys-X-Ser-Asn-Xb-X-Arg-His-X3-5-His (SEQ ID NO:169),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Gln-X-Ser-Asn-Xb-X-Lys-His-X3-5-His (SEQ ID NO:170),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Gln-X-Ser-His-Xb-X-Thr-His-X3-5-His (SEQ ID NO:171),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Val-X-Ser-Asn-Xb-X-Val-His-X3-5-His (SEQ ID NO:
- 172),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Asp-X-Ser-Cys-Xb-X-Arg-His-X3-5-His (SEQ ID NO:
- 193),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Ile-X-Ser-Asn-Xb-X-Val-His-X3-5-His (SEQ ID NO:194),
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Trp-X-Ser-Asn-Xb-X-Arg-His-X3-5-His (SEQ ID NO:195), and
- Xa-X-Cys-X2-5-Cys-X3-Xa-X-Asp-X-Ser-Ala-Xb-X-Arg-His-X3-5-His (SEQ ID NO:
- 196),
- wherein Xa is phenylalanine or tyrosine, and Xb is a hydrophobic residue.
Dependencies are inferred from the claim text.