Claims
- 1. An isolated, non-canonical zinc finger binding protein comprising one or more non-canonical zinc finger components that bind to a target sequence.
- 2. The isolated zinc finger binding protein of claim 1, wherein the target sequence is a nucleic acid sequence.
- 3. The isolated zinc finger binding protein of claim 1, wherein the target sequence is an amino acid sequence.
- 4. The isolated zinc finger binding protein of claim 2, wherein the target sequence is DNA.
- 5. The isolated zinc finger binding protein of claim 2, wherein the target sequence is RNA.
- 6. The isolated zinc finger binding protein of claim 1, wherein the amino acid sequence of one or more of the zinc finger components is selected from the group consisting of: X3-B-X2-4-Cys-X12-His-X1-7-His-X4; X3-Cys-X2-4-B-X12-His-X1-7-His-X4;X3-Cys-X2-4-Cys-X12-Z-X-1-7-His-X4; X3-Cys-X2-4-Cys-X12-His-X1-7-Z-X4; X3-B-X2-4-B-X12His-X1-7-His-X4; X3-B-X2-4-Cys-X12-Z-X-1-7-His-X4; X3-B-X2-4-Cys-X12-His-X1-7-Z-X4; X3-Cys-X2-4-B-X12-Z-X1-7-His-X4; X3-Cys-X2-4-B-X12-His-X-1-7-Z-X4; X3-Cys-X2-4-Cys-X12-Z-X1-7-Z-X4; X3-Cys-X2-4-B-X12-Z-X-1-7-Z-X4; X3-B-X2-4-Cys-X12-Z-X-1-7-Z-X4; X3-B-X2-4-B-X12-His-X1-7-Z-X4; X3-B-X2-4-B-X12-Z-X1-7-His-X4; and X3-B-X2-4-B-X12-Z-X1-7Z-X4,wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 7. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-Cys-X12-His-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 8. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-B-X12-His-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 9. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-Cys-X12-Z-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 10. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-Cys-X12-His-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 11. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-B-X12-His-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 12. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-Cys-X12-Z-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 13. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-Cys-X12-His-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 14. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-B-X12-Z-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 15. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-B-X12-His-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 16. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-Cys-X12-Z-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 17. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-Cys-X2-4-B-X12-Z-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 18. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-CYS-X12-Z-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 19. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-B-X12-His-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 20. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-B-X12-Z-X1-7-His-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 21. The isolated zinc finger binding protein of claim 6, wherein the zinc finger component comprises the sequence X3-B-X2-4-B-X12-Z-X1-7-Z-X4, wherein X is any amino acid, B is any amino acid except cysteine and Z is any amino acid except histidine.
- 22. The isolated zinc finger binding protein of claim 1, wherein the target sequence is in a plant cell.
- 23. The isolated zinc finger binding protein of claim 1, wherein the target sequence is in an animal cell.
- 24. The isolated zinc finger binding protein of claim 23, wherein the target sequence is in a human cell.
- 25. The isolated zinc finger binding protein of claim 1, wherein the target sequence is a promoter sequence.
- 26. The isolated zinc finger binding protein of claim 1, comprising three zinc finger components.
- 27. The isolated zinc finger binding protein of claim 1, wherein the target sequence comprises about 9 to about 14 contiguous base pairs.
- 28. The isolated zinc finger binding protein of claim 26, wherein the third finger component comprises a non-canonical zinc finger component
- 29. The isolated zinc finger binding protein of claim 1, comprising a modified plant ZFP backbone.
- 30. An isolated polynucleotide encoding a zinc-finger binding protein according to claim 1.
- 31. An expression vector comprising the polynucleotide of claim 30.
- 32. A host cell comprising the polynucleotide of claim 30.
- 33. A fusion polypeptide comprising: (a) an isolated zinc finger binding protein according to claim 1 and (b) at least one functional domain.
- 34. The fusion polypeptide of claim 33, wherein the functional domain is a repressive domain.
- 35. The fusion polypeptide of claim 34, wherein the repressive domain is selected from the group consisting of KRAB, MBD-2B, v-ErbA, MBD3, TR and members of the DNMT family.
- 36. The fusion polypeptide of claim 35, wherein the functional domain is an activation domain.
- 37. The fusion polypeptide of claim 36, wherein the activation domain is selected from the group consisting of VP16, p65 subunit of NF-kappa B, and VP64.
- 38. The fusion polypeptide of claim 37, wherein the functional domain is selected from the group consisting of an insulator domain, a chromatin-remodeling protein or a methyl-binding domain.
- 39. An isolated polynucleotide encoding the fusion polypeptide of claim 33.
- 40. An expression vector comprising the polynucleotide of claim 39.
- 41. A host cell comprising the polynucleotide of claim 39.
- 42. A method of modulating expression of a gene, the method comprising the step of contacting a region of DNA with a fusion molecule according to claim 33.
- 43. The method of claim 42, wherein the zinc finger binding protein of the fusion molecule binds to a target site in a gene encoding a product selected from the group consisting of vascular endothelial growth factor, erythropoietin, androgen receptor, PPAR-γ2, p16, p53, pRb, dystrophin and e-cadherin.
- 44. The method of claim 42, wherein the functional domain comprises a repressive domain.
- 45. The method of claim 44, wherein the repressive domain is selected from the group consisting of KRAB, MBD-2B, v-ErbA, MBD3, TR and members of the DNMT family.
- 46. The method of claim 42, wherein the functional domain comprises an activation domain.
- 47. The method of claim 46, wherein the activation domain is selected from the group consisting of VP16, p65 subunit of NF-kappa B, and VP64.
- 48. The method of claim 42, wherein the functional domain is selected from the group consisting of an insulator domain, a chromatin-remodeling protein or a methyl-binding domain.
- 49. The method of claim 42, wherein the gene is in a plant cell.
- 50. The method of claim 42, wherein the gene is in an animal cell.
- 51. The method of claim 50, wherein the gene is in a human cell.
- 52. A pharmaceutical composition comprising a non-canonical zinc finger protein according to claim 1 and a pharmaceutically acceptable excipient.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional patent application Serial No. 60/263,445 filed Jan. 22, 2001 and also claims the benefit of U.S. provisional patent application Serial No. 60/290,716 filed May 11, 2001; both of which disclosures are hereby incorporated by reference in their entireties.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60263445 |
Jan 2001 |
US |
|
60290716 |
May 2001 |
US |