Zinc finger proteins with high affinity new DNA binding specificities

• US 5,789,538 A
• Filed: 04/18/1997
• Issued: 08/04/1998
• Est. Priority Date: 02/03/1995
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Rebar, Edward J., Pabo, Carl O.
• Primary Examiner(s): JACOBSON, DIAN COOK
• Application Number:
  US08/850,250
• Time in Patent Office: 473 Days
• Field of Search: 530/300, 530/324, 530/350, 530/358, 530/400, 435/69.1, 435/172.3
• US Class Current: 530/324
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

Described is a polypeptide comprising one or more zinc fingers. The polypeptide binds to new polynucleotide subsites with high affinity and consequently has a binding specificity that differs from wild type zinc finger proteins. The binding occurs through contacts between certain amino acid residues of the zinc fingers and the nucleic acids of the subsites. The polypeptide sequence of at least one zinc finger differs from wild type zinc fingers, and the difference involves at least one amino acid residue that contacts the bases of the polynucleotide during binding.

Claims

1. A polypeptide comprising three zinc fingers, each of said zinc fingers comprising a set of base-contacting amino acid residues, wherein:

a) at least one set of base-contacting amino acid residues in at least one zinc finger differs in sequence at one or more base-contacting amino acid residues as compared with a corresponding set of base-contacting amino acid residues in any known wild type zinc finger protein;

b) the polypeptide has a DNA base sequence specificity different from that of any known wild type zinc finger protein; and

c) the polypeptide binds a targeted polynucleotide with a dissociation constant of less than about 1.0 nanomolar.

2. The polypeptide of claim 1 wherein the three or more zing fingers bind the targeted polynucleotide with an dissociation constant of less than about 0.5 nanomolar.

3. The polypeptide of claim 2 wherein the wild type zinc finger is selected from the group consisting of EGR1, EGR2, GLI, Wilm'"'"'s tumor gene, Sp1, Hunchback, Kruppel, ADR1 and BrLA.

4. The polypeptide of claim 1 wherein the targeted polynucleotide is an HIV promoter.

5. The polypeptide of claim 1 wherein the amino acids at positions 3 and 6 of at least one zinc finger are asparagine and arginine, respectively, wherein the amino acid at position -1 is selected from the group consisting of serine, threonine and aspartic acid and wherein the targeted polynucleotide comprises GACC.

6. The polypeptide of claim 1 wherein the amino acids at positions -1, 3 and 6 of at least one zinc finger are arginine, aspartic acid and arginine, respectively, and the targeted polynucleotide comprises GCAC.

7. The polypeptide of claim 1 wherein the amino acids at positions 2 and 6 of at least one zinc finger are glycine and arginine, respectively, and wherein the amino acid at position 3 is serine or threonine and wherein the targeted polynucleotide comprises GCAC.

8. A polypeptide comprising three zinc fingers, wherein the polypeptide

a) has essentially the same amino acid sequence as a wild type zinc finger protein, further characterized by at least one or more point mutations at one or more base-contacting amino acid residues;

b) has a base sequence specificity different from the wild type zinc finger protein; and

c) binds a targeted polynucleotide with a dissociation constant of less than about 1.0 nanomolar.