Toward controlling gene expression at will

Selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences

David Segal, B. Dreier, R. R. Beerli, C. F. Barbas

Research output: Contribution to journalArticle

341 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2758-2763
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number6
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Zinc Fingers
Gene Expression
Bacteriophages
DNA
Switch Genes
Proteins
Site-Directed Mutagenesis
Protein Binding
Binding Sites
Genes

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

@article{604c5e46446142c399f62e71e55af470,
title = "Toward controlling gene expression at will: Selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences",
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.",
author = "David Segal and B. Dreier and Beerli, {R. R.} and Barbas, {C. F.}",
year = "1999",
doi = "10.1073/pnas.96.6.2758",
language = "English (US)",
volume = "96",
pages = "2758--2763",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "6",

}

TY - JOUR

T1 - Toward controlling gene expression at will

T2 - Selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences

AU - Segal, David

AU - Dreier, B.

AU - Beerli, R. R.

AU - Barbas, C. F.

PY - 1999

Y1 - 1999

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0032981699&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032981699&partnerID=8YFLogxK

U2 - 10.1073/pnas.96.6.2758

DO - 10.1073/pnas.96.6.2758

M3 - Article

VL - 96

SP - 2758

EP - 2763

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 6

ER -