DNA-binding specificities of Cys2His2 zinc fingers

描述(由申请人提供)：本申请是对计划公告PA-03-127(整合和协作研究方法)的响应，长期目标是开发预测Cys2His2锌指蛋白结合特异性的算法，Cys2His2锌指蛋白是人类基因组中编码的最常见的DMA结合结构域。为此，我们建议设计和表征一大系列人工合成的Cys2His2锌指蛋白的DMA结合特异性，并使用这个全面的数据集来开发和验证Cys2His2锌指蛋白的概率DMA识别代码，并大大提高预测能力。大量具有明确特异性的工程化Cys2His2锌指结构域的开发也应该对大量对工程定制的“设计型”锌指核酸酶感兴趣的研究人员普遍有用(最近被证明是在人类细胞中诱导位点特异性基因靶向的强大试剂)。我们将与联盟成员Gary Stormo博士(华盛顿大学医学院)和Scot Wolfe博士(马萨诸塞大学医学院)合作，开发与了解Cys2His2锌指的DMA结合特异性相关的方法、试剂、数据和算法。这些活动将增强R01资助的所有三个实验室研究项目的长期目标。具体地说，拟议的项目将提供重要的锌指试剂和结合特异性数据，有助于实现R01GM069906(NRSF/REST锌指-DMA相互作用研究；PI：Joung)的长期目标，以了解自然发生的扩展锌指-DMA接口，并设计能够识别哺乳动物基因组中唯一地址的高度特异的工程锌指结构域。同样，这些试剂和特异性数据也将有助于开发能够与真核细胞中的单个内源基因结合并调节单个内源基因的二聚体工程锌指蛋白(R01GM068110：用于基因靶向的二聚体Cys2His2锌指蛋白；PI：Wolfe)。最后，预测真核锌指转录因子的DMA结合特异性的算法的开发扩展了R01 HG000249(DMA模式识别和分析；PI：STORMO)的一个基本目标，即开发计算方法来确定DMA结合转录因子的特异性并确定它们在基因组序列中的调控位置。

DESCRIPTION (provided by applicant): This application is in response to Program Announcement PA-03-127 (Integrative and Collaborative Approaches to Research) and has the long-term goal of developing algorithms for predicting the binding specificities of Cys2His2 zinc finger proteins, the most common DMA-binding domain encoded in the human genome. To do this, we propose to engineer and characterize the DMA-binding specificities of a large series of artificial Cys2His2 zinc finger proteins and to use this comprehensive dataset to develop and validate probabilistic DMA-recognition codes for Cys2His2 zinc fingers with greatly improved predictive power. The development of a large archive of engineered Cys2His2 zinc finger domains with well-defined specificities should also be generally useful to a large number of researchers interested in engineering customized "designer" zinc finger nucleases (recently shown to be powerful reagents for inducing site- specific gene targeting in human cells). Working together with Consortium Members Dr. Gary Stormo (of the Washington University School of Medicine) and Dr. Scot Wolfe (of the University of Massachusetts Medical School), we will develop methodologies, reagents, data, and algorithms relevant to understanding the DMA-binding specificities of Cys2His2 zinc fingers. These activities will enhance the long-term goals of R01-funded research programs in all three laboratories. Specifically, the proposed project will provide important zinc finger reagents and binding specificity data that will facilitate the long-term goals of R01GM069906 (Studies of NRSF/REST Zinc Finger-DMA Interactions; PI: Joung) to understand naturally occurring extended zinc finger-DMA interfaces and to design highly specific engineered zinc finger domains capable of recognizing unique addresses within mammalian genomes. Similarly, these reagents and specificity data will also facilitate the development of dimeric engineered zinc finger proteins that can bind to and regulate a single endogenous gene in eukaryotic cells (R01GM068110: Dimeric Cys2His2 Zinc Finger Proteins for Gene Targeting; PI: Wolfe). Finally, the development of algorithms to predict DMA-binding specificities of eukaryotic zinc finger transcription factors extends a fundamental goal of R01 HG000249 (DMA pattern identification and analysis; PI: Stormo) to develop computational methods to determine the specificity of DMA-binding transcription factors and identify their regulatory sites within genome sequences.