Deoxyribozymes for Protein Phosphorylation and Dephosphorylation

描述(申请人提供)：这是一个研究项目的续期申请，该项目专注于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上和实践上的优势。目前，许多工作都集中在用于RNA切割和连接的脱氧核酶上，但更广泛的反应化学仍在很大程度上尚未被研究。将DNA催化扩展到包括蛋白质底物的磷酸化和去磷酸化的关键反应，将增加我们对这种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的催化剂。我们以前的工作已经证明，脱氧核酶可以对具有挑战性的反应有非常高的增强作用，例如DNA磷酸二酯水解。然而，我们和其他人之前的大多数努力都集中在寡核苷酸的底物上，其结合作用是通过简单的Watson-Crick碱基配对提供的。在目标1中，我们将建立一种更通用的、模块化的方法，其中小分子底物通过可泛化的适配子结构域结合，然后与催化结构域整合以提供功能脱氧核酶。AIMS 2和AIMS 3将分别侧重于鉴定催化蛋白质磷酸化和去磷酸化(即激酶和磷酸酶活性)的脱氧核酶。目的4将在细胞表面修饰反应和研究与阿尔茨海默病有关的tau蛋白等生物学相关环境中应用新的激酶和磷酸脱氧核酶。最后，Aim 5将使用生化方法、溶液状态核磁共振光谱和X射线结晶学来表征新鉴定的激酶和磷酸脱氧核酶，为合理重新设计选择策略和催化活性提供重要的基础信息。

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.