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

Nucleic Acids Res. 2007;35(11):e81. doi: 10.1093/nar/gkm385. Epub 2007 May 30.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Bacteria / genetics
  • Binding Sites
  • DNA / chemistry
  • DNA / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Gene Library
  • Protein Engineering / methods*
  • Transcription Factors / chemistry
  • Two-Hybrid System Techniques*
  • Zinc Fingers*

Substances

  • DNA-Binding Proteins
  • Transcription Factors
  • DNA