Toward controlling gene expression at will:: Selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences
Toward controlling gene expression at will:: Selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences
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DOI：
10.1073/pnas.96.6.2758
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发表时间：
1999-03-16
影响因子：
11.1
通讯作者：
Barbas, CF
中科院分区：
文献类型：
--
作者：
Segal, DJ;Dreier, B;Barbas, CF
我们已经采取了一种全面的方法来产生具有确定特异性的新的DNA结合锌指结构域。在此，rye描述了一个锌指结构域家族的产生和表征，该锌指结构域家族被开发用于识别具有序列5 ′-GNN-3 ′的16个可能的3-bp DNA结合位点中的每一个。在有利于富集序列特异性蛋白质的条件下创建和选择锌指蛋白质的噬菌体展示文库。锌指结构域识别的一些序列需要通过定点诱变，这是由噬菌体选择数据和结构信息的指导下进行细化。在许多情况下，预计不会进行碱基特异性接触的残基对特异性有影响。这些结构域中的许多表现出精确的特异性，并区分具有>100倍亲和力损失的单个碱基差异的序列。我们得出结论，锌指结构域的三个螺旋位置-1，3和6不足以允许。用于待预测的DNA结合域的精细特异性。这些结构域在功能上是模块化的，并且可以彼此重组以产生能够以亚纳摩尔亲和力结合18-bp序列的多指蛋白。本文描述的锌指结构域家族足以构建1700万种结合DNA的5 '-（GNN）（6）-3'家族的新蛋白。序列的这些材料和方法应允许快速构建新的基因位点，并为基因控制的通用系统提供基础。
We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity, Herein rye describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5'-GNN-3'. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity, We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow. for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5'-(GNN)(6)-3' family of DNA. sequences. These materials and methods should allow for the rapid construction of novel gene sa itches and provide the basis for a universal system for gene control.