Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences.

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5'-GNN-3'. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5'-(GNN)6-3' family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.

[1]  A Klug,et al.  Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. , 1998, Biochemistry.

[2]  S H Kim,et al.  In vitro selection of zinc fingers with altered DNA-binding specificity. , 1994, Biochemistry.

[3]  C. Barbas,et al.  Phage display of combinatorial antibody libraries. , 1997, Current opinion in biotechnology.

[4]  D J Segal,et al.  Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[5]  D J Segal,et al.  Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[6]  John W. R. Schwabe,et al.  The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition , 1993, Nature.

[7]  N. Pavletich,et al.  Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A , 1991, Science.

[8]  A Klug,et al.  Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes. , 1985, The EMBO journal.

[9]  A Klug,et al.  Physical basis of a protein-DNA recognition code. , 1997, Current opinion in structural biology.

[10]  Toby J. Gibson,et al.  Base sequence discrimination by zinc-finger DNA-binding domains , 1991, Nature.

[11]  C. Pabo,et al.  Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. , 1994, Science.

[12]  C. Barbas,et al.  Combinatorial immunoglobulin libraries on the surface of phage (Phabs): Rapid selection of antigen-specific fabs , 1991 .

[13]  A Klug,et al.  Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J R Desjarlais,et al.  Toward rules relating zinc finger protein sequences and DNA binding site preferences. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[15]  P E Wright,et al.  Three-dimensional solution structure of a single zinc finger DNA-binding domain. , 1989, Science.

[16]  A Klug,et al.  Synergy between adjacent zinc fingers in sequence-specific DNA recognition. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. Pabo,et al.  High-resolution structures of variant Zif268-DNA complexes: implications for understanding zinc finger-DNA recognition. , 1998, Structure.

[18]  J. Berg,et al.  A 2.2 Å resolution crystal structure of a designed zinc finger protein bound to DNA , 1996, Nature Structural Biology.

[19]  J. Berg,et al.  Redesigning the DNA‐binding specificity of a zinc finger protein: A data base‐guided approach , 1992, Proteins.

[20]  M Gerstein,et al.  Stereochemical basis of DNA recognition by Zn fingers. , 1994, Nucleic acids research.

[21]  S H Kim,et al.  A zinc finger directory for high-affinity DNA recognition. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[22]  C. Barbas,et al.  Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[23]  T. Gibson,et al.  Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis. , 1992, Nucleic acids research.

[24]  C. Pabo,et al.  Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. , 1993, Science.

[25]  W. Stemmer,et al.  Construction and evolution of antibody–phage libraries by DMA shuffling , 1996, Nature Medicine.

[26]  A Klug,et al.  Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P E Wright,et al.  Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: determinants of affinity and sequence specificity. , 1997, Journal of molecular biology.

[28]  R. Kriwacki,et al.  Structures of Zinc Finger Domains from Transcription Factor Sp1 , 1997, The Journal of Biological Chemistry.

[29]  N. V. Vo,et al.  Designing zinc-finger ADR1 mutants with altered specificity of DNA binding to T in UAS1 sequences. , 1995, Biochemistry.

[30]  S. Harrison,et al.  Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Ptashne,et al.  Specific Binding of the λ Phage Repressor to λ DNA , 1967, Nature.

[32]  C. Pabo,et al.  Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. , 1996, Structure.

[33]  J. Milbrandt,et al.  DNA-binding specificity of NGFI-A and related zinc finger transcription factors , 1995, Molecular and cellular biology.

[34]  S K Burley,et al.  Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Barbas,et al.  Building zinc fingers by selection: toward a therapeutic application. , 1995, Proceedings of the National Academy of Sciences of the United States of America.