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PMID: 17537811 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Validation Study

Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method.

Nucleic acids research ·Vol. 35 ·No. 11 ·2007-00-00 ·Pages e81

Meng X, Thibodeau-Beganny S, Jiang T, Joung JK, Wolfe SA

Abstract

The C2H2 zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities.

MeSH Terms
Bacteria/genetics Binding Sites DNA/chemistry,metabolism DNA-Binding Proteins/chemistry,genetics,metabolism Gene Library Protein Engineering/methods Transcription Factors/chemistry Two-Hybrid System Techniques Zinc Fingers
Chemicals
DNA-Binding Proteins Transcription Factors DNA
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Meng Xiangdong
Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605, USA.
Thibodeau-Beganny Stacey
Jiang Tao
Joung J Keith
Wolfe Scot A
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkm385
Published
2007-00-00
Epub
2007-00-30
Pages
e81
Language
English
Region
England
NLM ID
0411011
PMCID
PMC1920264
Subset
IM
Grants
NIGMS NIH HHS · R01 GM072621 · United States
NIGMS NIH HHS · R01 GM068110 · United States
NIGMS NIH HHS · R01 GM069906 · United States
NIGMS NIH HHS · 1R01GM068110 · United States
NIH HHS · DP1 OD006862 · United States
External Links
PubMed source DOI: 10.1093/nar/gkm385 PMC Full Text View journal details
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