Nucleic acid encoding poly-zinc finger proteins with improved linkers

• US 7,153,949 B2
• Filed: 04/20/2005
• Issued: 12/26/2006
• Est. Priority Date: 03/02/1998
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Pabo, Carl O., Kim, Jin-Soo
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US11/110,594
• Publication Number:
  US 20050202498A1
• Time in Patent Office: 615 Days
• Field of Search: 536/23.1, 530/350
• US Class Current: 536/23.1
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Polynucleotides encoding chimeric proteins, and methods for their production and use are disclosed. The chimeric proteins comprise a flexible linker between two zinc finger DNA-binding domains, wherein the linker contains eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first domain and the first conserved cysteine residue of the amino-terminal zinc finger of the second domain.

Claims

1. A polynucleotide encoding 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 an engineered 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 six or more amino acids in length;

wherein the first and second domains are fused with the flexible linker.

2. The polynucleotide of claim 1, wherein the adjacent target sites are separated by one nucleotide and the flexible linker is seven, eight, or nine amino acids in length.

3. The polynucleotide of claim 2, wherein the flexible linker has the amino acid sequence RQKDGERP (SEQ ID NO:

14).

4. The polynucleotide of claim 1, wherein the adjacent target sites are separated by two nucleotides and the flexible linker is ten, eleven, or twelve amino acids in length.

5. The polynucleotide of claim 4, wherein the flexible linker has the amino acid sequence RQKDGGGSERP (SEQ ID NO:

15).

6. The polynucleotide of claim 1, wherein the adjacent target sites are separated by three nucleotides and the flexible linker is twelve or more amino acids in length.

7. The polynucleotide of claim 1, wherein the first and the second domains are zinc finger polypeptides.

8. The polynucleotide of claim 7, wherein the zinc finger polypeptides are heterologous.

9. The polynucleotide of claim 7, wherein the chimeric zinc finger protein has femtomolar affinity for the adjacent target sites.

10. The polynucleotide of claim 9, wherein the chimeric zinc finger protein has about 2–

4 femtomolar affinity for the adjacent target sites.

11. The polynucleotide of claim 1, wherein the chimeric zinc finger protein further comprises a regulatory domain polypeptide.

12. The polynucleotide of claim 1, wherein the first domain comprises a poly-finger zinc finger protein.

13. The polynucleotide of claim 12, wherein the second domain comprises a poly-finger zinc finger protein.

14. The polynucleotide of claim 11, wherein the regulatory domain is selected from the group consisting of a transcription activator domain and a transcription repressor domain.

15. The polynucleotide of claim 11, wherein the regulatory domain comprises an endonuclease.

16. The polynucleotide of claim 15, wherein the endonuclease comprises FokI.

17. A polynucleotide encoding a DNA-binding protein that binds to adjacent first and second target sites, wherein the adjacent target sites are separated by one nucleotide, the DNA-binding protein comprising first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues;

and

the first and second ZFPs are joined by an amino acid sequence of 9, 10 or 11 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP.

18. The polynucleotide of claim 17, wherein the amino acid sequence joining the first and second ZFPs comprises the amino acid sequence RQKDGERP (SEQ ID NO:

14).

19. A polynucleotide encoding a DNA-binding protein that binds to adjacent first and second target sites, wherein the adjacent target sites are separated by two nucleotides, the DNA-binding protein comprising first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues;

and

the first and second ZFPs are joined by an amino acid sequence of 12, 13 or 14 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine-residue of the amino-terminal zinc finger of the second ZFP.

20. A polynucleotide encoding a DNA-binding protein that binds to adjacent first and second target sites, the DNA-binding protein comprising

a regulatory domain;

and

first and second DNA-binding domains, wherein

the first and second DNA-binding domains are zinc finger polypeptides (ZFPs) which do not occur together in the same arrangement in nature,

the first ZFP binds to the first target site and the second ZFP binds to the second target site,

each zinc finger polypeptide comprises a plurality of zinc fingers,

each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues; and

the first and second ZFPs are joined by an amino acid sequence comprising eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP.

21. The polynucleotide of claim 20, wherein the regulatory domain is selected from the group consisting of a transcription activator domain and a transcription repressor domain.

22. The polynucleotide of claim 20, wherein the regulatory domain comprises an endonuclease.

23. The polynucleotide of claim 22, wherein the endonuclease comprises FokI.

24. A polynucleotide encoding a DNA-binding protein that binds to first and second adjacent target sites, wherein the adjacent target sites are separated by one nucleotide, the DNA-binding protein comprising:

first and second zinc finger polypeptides (ZFPs), wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by a sequence of 9, 10 or 11 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger.

25. The polynucleotide of claim 24, wherein the amino acid sequence joining the first and second ZFPs comprises the amino acid sequence RQKDGERP (SEQ ID NO:

14).

26. A polynucleotide-encoding a DNA-binding protein that binds to first and second adjacent target sites, wherein the adjacent target sites are separated by two nucleotides, the DNA-binding protein comprising:

first and second zinc finger polypeptides (ZFPs), wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by a sequence of 12, 13 or 14 amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger.

27. A polynucleotide encoding a DNA-binding protein that binds to first and second adjacent target sites, the DNA-binding protein comprising:

a regulatory domain polypeptide; and

first and second zinc finger polypeptides (ZFPs), wherein each zinc finger polypeptide comprises a plurality of zinc fingers and each zinc finger comprises two conserved cysteine residues that are amino-terminal to two conserved histidine residues, and wherein the first ZFP binds to the first target site and the second ZFP binds to the second target site;

wherein the first and second ZFPs are joined by an amino acid sequence comprising eight or more amino acids between the second conserved histidine residue of the carboxy-terminal zinc finger of the first ZFP and the first conserved cysteine residue of the amino-terminal zinc finger of the second ZFP; and

further wherein the plurality of zinc fingers within each ZFP are joined by a sequence of seven amino acids between the second conserved histidine residue of a zinc finger and the first conserved cysteine residue of an adjacent zinc finger.

28. The polynucleotide of claim 27, wherein the regulatory domain is selected from the group consisting of a transcription activator domain and a transcription repressor domain.

29. The polynucleotide of claim 27, wherein the regulatory domain comprises an endonuclease.

30. The polynucleotide of claim 29, wherein the endonuclease comprises FokI.