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PMID: 10077584 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 96 ·No. 6 ·1999-03-16 ·Pages 2758-63

Segal DJ, Dreier B, Beerli RR, Barbas CF

Abstract

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.

MeSH Terms
Animals Binding Sites DNA/genetics DNA-Binding Proteins/chemistry,genetics Gene Library Mice Protein Engineering Sequence Analysis Zinc Fingers/genetics
Chemicals
DNA-Binding Proteins DNA
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Segal D J
The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Dreier B
Beerli R R
Barbas C F
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.96.6.2758
Published
1999-03-16
Pages
2758-63
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC15842
Subset
IM
Grants
NIGMS NIH HHS · GM 53910 · United States
External Links
PubMed source DOI: 10.1073/pnas.96.6.2758 PMC Full Text View journal details
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