Target DNA search by zinc-finger proteins

通过锌指蛋白进行目标 DNA 搜索

• 批准号: 8898128
• 负责人: Junji Iwahara
• 金额: $ 26.38万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8898128
• 关键词: Academia; Address; Affect; Affinity; Base Pairing; Binding; Biochemical; Biological Assay; Bypass; Cell Nucleus; Clinical; Clinical Trials; DNA; DNA Binding; DNA Repair; Defect; Disease; Drug Industry; Engineering; Enzymes; Equilibrium; Eukaryotic Cell; Exhibits; Fluorescence; Gene Activation; Goals; Health; Human; Kinetics; Knowledge; Location; Methods; Molecular; Mutagenesis; NMR Spectroscopy; Phase; Process; Property; Protein Binding; Protein Engineering; Proteins; Recording of previous events; Reporting; Research; Research Project Grants; Role; Scanning; Site; Specificity; Speed; Stimulus; Synthetic Genes; Technology; Testing; Therapeutic; Thermodynamics; Work; Zinc Fingers; base; density; gene therapy; human disease; improved; in vivo; knowledge base; nuclease; research study; success; targeted sequencing; therapeutic development; transcription factor

DESCRIPTION (provided by applicant): In the nuclei of eukaryotic cells, transcription factors and DNA repair/modifying enzymes locate particular sequences or structural signatures among billions of DNA base pairs and in the presence of many other proteins bound to DNA. The long-term goal for the research team of this project is to understand what dictates the efficiency in th processes whereby the proteins locate their target sites on DNA in order to apply this knowledge to the development of therapeutics for human diseases and disorders. The overall objective in the current project is to elucidate DNA-scanning mechanisms for zinc-finger proteins at both the molecular and atomic levels. Affinity-based engineering of Cys2-His2-type zinc fingers that exhibit desired DNA-binding specificity has gained popularity for artificial gene control/manipulation. In fact, human gene therapy based on the zinc-finger nuclease technology is in phase 1 and 2 clinical trials. However, there are an increasing number of reports that suggest kinetic defects in artificial zinc-finger proteins despite their high affinities for the taget DNA sites. This represents a bottleneck for successful therapeutic applications of artificial zinc-finger proteins. Preliminary studies by the research team suggest that this problem could be resolved via protein engineering based on knowledge of the DNA-scanning mechanisms. Recently the research team found that the Egr-1 zinc-finger protein undergoes two conformationally distinct states termed the search and recognition modes while the protein molecule scans DNA. In the current project, the research team will conduct research to test the central hypothesis that the balance between the search and recognition modes is a major determinant of the kinetic efficiency in target DNA search by zinc-finger proteins. The following three specific aims will be pursued in this project: 1) to delineate how zinc-finger proteins scan DNA; 2) to understand how zinc-finger proteins bypass obstacles on DNA; and 3) to understand how zinc-finger proteins displace other proteins from the target sites. For these specific aims, the research team will use biophysical and biochemical approaches along with mutagenesis to shift the equilibrium between the search and recognition modes. NMR spectroscopy will be used to investigate the dynamics of DNA scanning at an atomic level. Fluorescence and biochemical methods will be used to characterize the kinetic and thermodynamic properties of the zinc-finger proteins in the target DNA search process at a molecular level. The current project will substantially deepen our understanding of DNA scanning by proteins. This project will also enable improvement of kinetic properties of zinc-finger proteins, and thereby boost their applications to human therapeutics and other biomedical applications.

描述（由申请人提供）：在真核细胞的核中，转录因子和DNA修复/修饰酶在数十亿个DNA碱基对之间找到了特定的序列或结构特征，并且在许多其他与DNA结合的蛋白质存在下。该项目研究团队的长期目标是了解该过程中蛋白质在DNA上定位其目标位点的效率的原因，以便将此知识应用于针对人类疾病和疾病的治疗剂的发展。当前项目的总体目标是阐明分子和原子水平的锌指蛋白的DNA扫描机制。表现出所需的DNA结合特异性的CYS2-HIS2型锌指的基于亲和力的工程已在人工基因控制/操纵方面普及。实际上，基于锌指核酸酶技术的人类基因疗法在第1阶段和2阶段试验中。然而，尽管有较高的锌指DNA位点的亲和力，但仍有越来越多的报告表明人工锌指蛋白的动力学缺陷。这代表了人造锌指蛋白成功治疗应用的瓶颈。研究小组的初步研究表明，可以根据对DNA扫描机制的知识来通过蛋白质工程解决此问题。最近，研究小组发现，在蛋白质分子扫描DNA时，EGR-1锌指蛋白经历了两个构型不同的状态，称为搜索和识别模式。在当前项目中，研究团队将进行研究，以检验搜索模式和识别模式之间的平衡是锌指蛋白靶DNA搜索动力学效率的主要决定因素。该项目将实现以下三个特定目标：1）描绘锌指蛋白扫描DNA的方式； 2）了解锌指蛋白如何绕过DNA上的障碍； 3）了解锌指蛋白如何从靶位点移位其他蛋白质。对于这些具体目的，研究团队将使用生物物理和生化方法以及诱变，以改变搜索和识别模式之间的平衡。 NMR光谱法将用于研究原子水平的DNA扫描的动力学。荧光和生化方法将用于表征靶DNA搜索过程中锌指蛋白在分子水平上的动力学和热力学特性。当前的项目将大大加深我们对蛋白质扫描DNA扫描的理解。该项目还将能够改善锌指蛋白的动力学特性，从而提高其对人类疗法和其他生物医学应用的应用。