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（研究的综合和协作方法）的响应，其长期目标是开发预测Cys 2 His 2锌指蛋白结合特异性的算法，Cys 2 His 2锌指蛋白是人类基因组中编码的最常见的DMA结合域。要做到这一点，我们建议工程师和表征的DMA结合特异性的一个大系列的人工Cys 2 His 2锌指蛋白，并使用这个全面的数据集开发和验证概率DMA识别代码Cys 2 His 2锌指大大提高了预测能力。具有明确特异性的工程化Cys 2 His 2锌指结构域的大量档案的开发通常也应该对大量对工程化定制的“设计者”锌指核酸酶（最近显示为用于在人细胞中诱导位点特异性基因靶向的强大试剂）感兴趣的研究人员有用。与联盟成员加里斯托莫博士（华盛顿大学医学院）和斯科特沃尔夫博士（马萨诸塞州大学医学院）合作，我们将开发与理解Cys 2 His 2锌指的DMA结合特异性相关的方法，试剂，数据和算法。这些活动将加强R 01资助的研究计划在所有三个实验室的长期目标。具体而言，拟议的项目将提供重要的锌指试剂和结合特异性数据，这将有助于R 01 GM 069906（NRSF/REST锌指-DMA相互作用研究; PI：JAPAN）的长期目标，以了解天然存在的扩展锌指-DMA界面，并设计能够识别哺乳动物基因组内独特地址的高度特异性工程化锌指结构域。类似地，这些试剂和特异性数据也将促进可结合并调节真核细胞中单个内源性基因的二聚体工程化锌指蛋白的开发（R 01 GM 068110：用于基因靶向的二聚体Cys 2 His 2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R 01 HG 000249（DMA模式识别和分析; PI：Stormo）的基本目标，以开发计算方法来确定DMA结合转录因子的特异性并识别其在基因组序列内的调控位点。

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.