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

Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605, USA.

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.

PMID: 17537811 [PubMed - indexed for MEDLINE]

PMCID: PMC1920264