GETTING A HANDHOLD ON DNA - DESIGN OF POLY-ZINC FINGER PROTEINS WITH FEMTOMOLAR DISSOCIATION-CONSTANTS

Structure-based design was used to link zinc finger peptides and make poly-finger proteins that have dramatically enhanced affinity and spec ificity. Our studies focused on a fusion in which the three-finger Zif 268 peptide was linked to a designed three-finger peptide (designated ''NRE'') that specifically recognizes a nuclear hormone response eleme nt. Gel shift assays indicate that this six-finger peptide, 268//NRE, binds to a composite 18-bp DNA site with a dissociation constant in th e femtomolar range. We find that the slightly longer linkers used in t his fusion protein provide a dramatic improvement in DNA-binding affin ity, working much better than the canonical ''TGEKP'' linkers that hav e been used in previous studies, Tissue culture transfection experimen ts also show that the 268//NRE peptide is an extremely effective repre ssor, giving 72-fold repression when targeted to a binding site close to the transcription start site. Using this strategy, and linking pept ides selected via phage display, should allow the design of novel DNA- binding proteins-with extraordinary affinity and specificity-for use i n biological research and gene therapy.