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Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants
Cited 190 time in
Web of Science
Cited 202 time in Scopus
- Authors
- Issue Date
- 1998-03
- Publisher
- National Academy of Sciences
- Citation
- Proceedings of the National Academy of Sciences of the United States of America, Vol.95 No.6, pp.2812-2817
- Abstract
- 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.
- ISSN
- 0027-8424
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