Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants
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- Jin-Soo Kim
- Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
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- Carl O. Pabo
- Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
書誌事項
- 公開日
- 1998-03-17
- DOI
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- 10.1073/pnas.95.6.2812
- 公開者
- Proceedings of the National Academy of Sciences
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説明
<jats:p>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.</jats:p>
収録刊行物
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- Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences 95 (6), 2812-2817, 1998-03-17
Proceedings of the National Academy of Sciences