UMMS Affiliation

Program in Gene Function and Expression; Department of Biochemistry and Molecular Pharmacology

Date

6-1-2007

Document Type

Article

Subjects

Bacteria; Binding Sites; DNA; DNA-Binding Proteins; Gene Library; Protein Engineering; Transcription Factors; *Two-Hybrid System Techniques; *Zinc Fingers

Disciplines

Life Sciences | Medicine and Health Sciences

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.

Rights and Permissions

Citation: Nucleic Acids Res. 2007;35(11):e81. Epub 2007 May 30. Link to article on publisher's site

DOI of Published Version

10.1093/nar/gkm385

Related Resources

Link to Article in PubMed

Journal Title

Nucleic acids research

PubMed ID

17537811

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