Meng, XiangdongThibodeau-Beganny, StaceyJiang, TaoJoung, J. KeithWolfe, Scot A.2022-08-232022-08-232007-06-012009-03-16Nucleic Acids Res. 2007;35(11):e81. Epub 2007 May 30. <a href="http://dx.doi.org/10.1093/nar/gkm385">Link to article on publisher's site</a>1362-4962 (Electronic)10.1093/nar/gkm38517537811https://hdl.handle.net/20.500.14038/38472The 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.en-USBacteriaBinding SitesDNADNA-Binding ProteinsGene LibraryProtein EngineeringTranscription Factors*Two-Hybrid System Techniques*Zinc FingersLife SciencesMedicine and Health SciencesJournal Articlehttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=2342&amp;context=oapubs&amp;unstamped=1https://escholarship.umassmed.edu/oapubs/1343783022oapubs/1343