Deoxyribozymes for Protein Phosphorylation and Dephosphorylation

描述（申请人提供）：这是一个研究项目的延续申请，该项目的重点是开发和应用脱氧核酶用于蛋白质的磷酸化和去磷酸化。脱氧核酶（DNA酶）是具有特定催化活性的特定DNA序列，就像蛋白质酶是氨基酸的催化序列一样。相对于蛋白质和RNA，DNA作为催化剂具有许多概念和实际优势。目前，许多努力都集中在脱氧核酶的RNA切割和连接，但更广泛的反应化学仍然在很大程度上未经审查。将DNA催化扩展到包括磷酸化和去磷酸化关键反应的蛋白质底物，将增加我们对此类催化过程的基本理解，并为涉及这些重要生物分子底物的应用提供实用催化剂。 我们以前的工作已经确定，脱氧核酶可以有非常高的速率增强具有挑战性的反应，如DNA磷酸二酯水解。然而，我们和其他人以前的大多数努力都集中在寡核苷酸底物上，其结合相互作用由简单的沃森-克里克碱基配对提供。在目标1中，我们将建立一个更普遍的，模块化的方法，其中小分子底物结合的通用适体结构域，然后将与催化结构域整合，以提供功能性脱氧核酶。目的2和3将集中于鉴定催化蛋白质磷酸化和去磷酸化的脱氧核酶（即，激酶和磷酸酶活性）。目的4将应用新的激酶和磷酸酶脱氧核酶在生物学相关的背景下，如细胞表面修饰反应和tau蛋白的研究，这是牵连在阿尔茨海默病。最后，目标5将使用生物化学方法，溶液状态NMR光谱学和X射线晶体学来表征新鉴定的激酶和磷酸酶脱氧核酶，提供重要的基础信息，这将有助于合理重新设计选择策略和催化活性。

DESCRIPTION (provided by applicant): This is a renewal application for a research program that focuses on the development and application of deoxyribozymes for phosphorylation and dephosphorylation of proteins. Deoxyribozymes (DNA enzymes) are specific DNA sequences that have particular catalytic activities, just like protein enzymes are catalytic sequences of amino acids. DNA has many conceptual and practical advantages as a catalyst relative to proteins and RNA. At present, much effort has focused on deoxyribozymes for RNA cleavage and ligation, but a broader range of reaction chemistries remains largely unexamined. Expanding DNA catalysis to include protein substrates for the key reactions of phosphorylation and dephosphorylation will increase our fundamental understanding of such catalytic processes and provide practical catalysts for applications involving these important biomolecular substrates. Our previous work has established that deoxyribozymes can have very high rate enhancements for challenging reactions such as DNA phosphodiester hydrolysis. However, most previous efforts by us and others have focused on substrates that are oligonucleotides, for which binding interactions are provided by simple Watson-Crick base pairing. In Aim 1, we will establish a more general, modular approach in which small-molecule substrates are bound by generalizable aptamer domains, which will then be integrated with catalytic domains to provide functional deoxyribozymes. Aims 2 and 3 will focus on identification of deoxyribozymes that catalyze protein phosphorylation and dephosphorylation (i.e., kinase and phosphatase activities), respectively. Aim 4 will apply the new kinase and phosphatase deoxyribozymes in biologically relevant contexts such as cell-surface modification reactions and studies of tau protein, which is implicated in Alzheimer's disease. Finally, Aim 5 will use biochemical methods, solution-state NMR spectroscopy, and X-ray crystallography to characterize the newly identified kinase and phosphatase deoxyribozymes, providing important fundamental information that will assist the rational redesign of selection strategies and catalytic activities.