Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants
Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants
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DOI：
10.1073/pnas.95.6.2812
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发表时间：
1998-03-17
影响因子：
11.1
通讯作者：
Pabo, CO
中科院分区：
文献类型：
--
作者：
Kim, JS;Pabo, CO
基于结构的设计用于连接锌指肽，并使多指蛋白具有显着增强的亲和力和特异性。我们的研究集中在一个融合，其中三指Zif 268肽连接到一个设计的三指肽（命名为“NRE”），特异性识别核激素反应元件。凝胶位移分析表明，这六指肽，268//NRE，结合到一个复合的18 bp的DNA位点的解离常数在飞摩尔范围内。我们发现，在这种融合蛋白中使用的稍长的接头提供了DNA结合亲和力的显着改善，工作比在以前的研究中使用的典型的“TGEKP”接头好得多。组织培养转染实验还表明，268//NRE肽是一个非常有效的阻遏物，当靶向接近转录起始位点的结合位点时，产生72倍的阻遏。使用这种策略，并通过噬菌体展示选择的连接肽，应该允许设计新的DNA结合蛋白质-具有非凡的亲和力和特异性-用于生物学研究和基因治疗。
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.