Deoxyribozymes for Protein Phosphorylation and Dephosphorylation

描述（由申请人提供）：这是一个研究项目的更新申请，重点是用于蛋白质磷酸化和去磷酸化的脱氧核酶的开发和应用。脱氧核糖酶（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。作为一种催化剂，DNA相对于蛋白质和RNA具有许多概念上和实践上的优势。目前，许多努力都集中在用于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未被研究。将DNA催化扩展到磷酸化和去磷酸化关键反应的蛋白质底物将增加我们对这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的催化剂。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，我们和其他人之前的大部分努力都集中在寡核苷酸的底物上，其结合相互作用是由简单的沃森-克里克碱基配对提供的。在Aim 1中，我们将建立一种更通用的模块化方法，其中小分子底物由可通用的适体结构域结合，然后将其与催化结构域集成以提供功能性脱氧核酶。目标2和目标3将分别集中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。Aim 4将把新的激酶和磷酸酶脱氧核酶应用于生物学相关的环境中，如细胞表面修饰反应和与阿尔茨海默病有关的tau蛋白的研究。最后，Aim 5将使用生化方法、溶液态核磁共振光谱和x射线晶体学来表征新鉴定的激酶和磷酸酶脱氧核酶，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.