Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants

作者：Jin‐Soo Kim, Carl O. Pabo · 发表于：Proceedings of the National Academy of Sciences · 年份：1998 · DOI：10.1073/pnas.95.6.2812 · 被引用次数：222 · 研究领域：RNA and protein synthesis mechanisms、RNA Interference and Gene Delivery、Monoclonal and Polyclonal Antibodies Research

Structure-based design was used to link zinc finger peptides and make poly-finger proteins that have dramatically enhanced affinity and specificity. Our studies focused on a fusion in which the three-finger Zif268 peptide was linked to a designed three-finger peptide (designated "NRE") that specifically recognizes a nuclear hormone response element. Gel shift assays indicate that this six-finger peptide, 268//NRE, binds to a composite 18-bp DNA site with a dissociation constant in the femtomolar range. We find that the slightly longer linkers used in this fusion protein provide a dramatic improvement in DNA-binding affinity, working much better than the canonical "TGEKP" linkers that have been used in previous studies. Tissue culture transfection experiments also show that the 268//NRE peptide is an extremely effective repressor, giving 72-fold repression when targeted to a binding site close to the transcription start site. Using this strategy, and linking peptides selected via phage display, should allow the design of novel DNA-binding proteins-with extraordinary affinity and specificity-for use in biological research and gene therapy.