Profiling the DNA-Binding Specificities of Engineered Cys2His2 Zinc Finger Domains Using a Rapid Cell-Based Method
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CitationMeng, Xiangdong, Stacey Thibodeau-Beganny, Tao Jiang, J. Keith Joung, and Scot A. Wolfe. 2007. Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Research 35(11): e81.
AbstractThe 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.
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