Bibliographic Details
| Title: |
ENGINEERING OF ZINC FINGER ARRAYS BY CONTEXT-DEPENDENT ASSEMBLY |
| Document Number: |
20120178647 |
| Publication Date: |
July 12, 2012 |
| Appl. No: |
13/386995 |
| Application Filed: |
August 03, 2010 |
| Abstract: |
A method of designing a multi-zinc-finger polypeptide predicted to bind to a sequence of interest that has at least three subsites includes the steps of: a) providing a nucleotide sequence of interest having first, second, and third consecutive subsites, wherein each of the first and third subsites are adjacent to the second subsite; b) identifying first and second adjacent zinc finger polypeptide sequences previously shown to bind to the first and second subsites in the context of a multi-zinc finger polypeptide; c) identifying a third zinc finger polypeptide previously shown to bind to a third subsite adjacent to the second subsite when present in the context of a multi-zinc finger polypeptide adjacent to the second zinc finger polypeptide; and d) combining the first, second, and third zinc finger polypeptide sequences in linear order, thereby designing a multi-zinc finger polypeptide predicted to bind to the sequence of interest. |
| Inventors: |
Joung, J. Keith (Winchester, MA, US); Sander, Jeffry D. (Woburn, MA, US) |
| Assignees: |
THE GENERAL HOSPITAL CORPORATION (Boston, MA, US) |
| Claim: |
1. A method of designing a multi-zinc-finger polypeptide sequence predicted to bind to a nucleic acid sequence of interest comprising at least three subsites, the method comprising: a) providing a nucleotide sequence of interest comprising first, second, and third consecutive subsites, wherein each of the first and third subsites are adjacent to the second subsite; b) identifying first and second adjacent zinc finger polypeptide sequences previously shown to bind to the first and second subsites in the context of a multi-zinc finger polypeptide; c) identifying a third zinc finger polypeptide sequence shown to bind to a third subsite adjacent to the second subsite when present in the context of a multi-zinc finger polypeptide adjacent to the second zinc finger polypeptide sequence; and d) combining the first, second, and third zinc finger polypeptide sequences in linear order, thereby designing a multi-zinc finger polypeptide sequence predicted to bind to the sequence of interest. |
| Claim: |
2. The method of claim 1, further comprising producing a polynucleotide comprising a sequence that encodes a polypeptide comprising the multi-zinc-finger polypeptide. |
| Claim: |
3. The method of claim 1, further comprising producing a polypeptide comprising the multi-zinc-finger polypeptide sequence. |
| Claim: |
4. The method of claim 1, wherein the first subsite is located 5′ to the second subsite. |
| Claim: |
5. The method of claim 1, wherein the first subsite is located 3′ to the second subsite. |
| Claim: |
6. The method of claim 1, wherein the second zinc finger sequence comprises a sequence selected from SEQ ID NOs: 1-18. |
| Claim: |
7. The method of claim 6, wherein the first zinc finger sequence comprises a sequence selected from SEQ ID NOs: 19-337. |
| Claim: |
8. The method of claim 6, wherein the third zinc finger sequence comprises a sequence selected from SEQ ID NOs: 338-681. |
| Claim: |
9. A polypeptide produced by the method of claim 3. |
| Claim: |
10. The polypeptide of claim 9, wherein the polypeptide comprises one or more functional domains. |
| Claim: |
11. The polypeptide of claim 10, wherein the functional domain is selected from the group comprising transcriptional activation domain, transcriptional repressor domain, transcriptional silencing domain, acetylase domain, de-acetylase domain, methylation domain, de-methylation domain, kinase domain, phosphatase domain, dimerization domain, multimerization domain, nuclear localization domain, nuclease domain, endonuclease domain, resolvase domain and integrase domain. |
| Claim: |
12. The polypeptide of claim 9, wherein the functional domain is an endonuclease domain. |
| Claim: |
13. A method of regulating the expression of a gene comprising contacting a polypeptide according to claim 10 with a sequence of interest in the gene to form a binding complex, such that expression of the gene is regulated. |
| Claim: |
14. A method of altering the structure of a gene comprising contacting a zinc finger polypeptide according to claim 10 with a sequence of interest within the gene to form a binding complex, such that the structure of the gene is altered. |
| Claim: |
15. A method of cleaving a sequence of interest comprising contacting a zinc finger polypeptide according to claim 10 with the sequence of interest to form a binding complex, such that the sequence of interest is cleaved. |
| Claim: |
16. A set of multi-zinc finger array sequences, wherein each array comprises at least first, second, and third adjacent zinc fingers, wherein the sequence of the second zinc finger is identical for each entry in the database, and wherein the database comprises at least ten entries. |
| Claim: |
17. A method of creating a set of multi-zinc-finger array sequences, the method comprising: providing a parent zinc finger polypeptide comprising at least first, second, and third adjacent zinc fingers, wherein the zinc finger polypeptide binds to a known parental target sequence comprising at least first, second, and third adjacent subsites; producing a library of zinc finger polypeptides based on the parent zinc finger polypeptide sequence, wherein each member of the library comprises the parental second zinc finger sequence and the sequence of either or both of the first and third fingers are varied; and selecting members of the library of zinc finger polypeptides that bind to one or more target sequences comprising the parental second subsite and either or both of a non-parental first and third subsite, thereby providing a set of multi-zinc-finger array sequences with common second finger sequences. |
| Claim: |
18. The method of claim 17, wherein the library is expressed in vitro. |
| Claim: |
19. The method of claim 17, wherein the library is expressed in an expression system selected from the group consisting of eukaryotic, prokaryotic and viral expression systems. |
| Claim: |
20. The method of claim 19, wherein the library is expressed in bacteria. |
| Current U.S. Class: |
506/9 |
| Current International Class: |
40; 12; 07; 12; 12; 12; 12; 12; 40; 12; 12 |
| Accession Number: |
edspap.20120178647 |
| Database: |
USPTO Patent Applications |
