Nucleic Acids Research Advance Access published online on May 30, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm385
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Methods Online |
Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method
1Program in Gene Function and Expression, 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605 USA, 3Molecular Pathology Unit, Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, 149 13th Street, 7th floor, Charlestown, MA 02129 USA and 4Department of Pathology, Harvard Medical School, Boston, MA 02115 USA
*To whom correspondence should be addressed. Tel: 508 856 3953; Fax: 508 856 5460; Email: scot.wolfe{at}umassmed.edu
Received February 9, 2007. Revised April 28, 2007. Accepted April 30, 2007.
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.
Present address: Tao Jiang, Department of Biochemistry, Tufts University School of Medicine, Jaharis 601C, 150 Harrison Ave., Boston, MA 02111 USA