Deoxyribozymes for Protein Phosphorylation and Dephosphorylation

用于蛋白质磷酸化和去磷酸化的脱氧核酶

基本信息

批准号：
8723837
负责人：
Scott K Silverman
金额：
$ 34.08万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-05-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8723837
关键词：
Alzheimer&apos s Disease Amino Acid Sequence Amino Acids Base Pairing Binding Biochemical Biological Biology Catalysis Catalytic DNA Catalytic Domain Cell surface Cells Chemicals Chemistry Collaborations DNA DNA Ligation DNA Sequence Development Enzymes Goals Grant Guanosine Triphosphate Hydrolysis Hydroxyl Radical Illinois In Vitro Length Life Ligation Methods Michigan Modification NMR Spectroscopy Nucleic Acids Nucleotides Oligonucleotides Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Process Protein Dephosphorylation Protein Kinase Protein phosphatase Proteins RNA Reaction Relative (related person)Research Side Solutions Surface Universities Work X-Ray Crystallography aptamer catalyst design insight phosphodiester practical application programs public health relevance small molecule sugar tau Proteins

项目摘要

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.

描述（由申请人提供）：这是研究计划的续签应用，该计划的重点是脱氧核酸脂蛋白磷酸化和去磷酸化的开发和应用。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。与蛋白质和RNA相对于催化剂，DNA具有许多概念和实用的优势。目前，许多努力集中在脱氧核酶进行RNA的裂解和连接上，但更广泛的反应化学范围仍然在很大程度上尚未进行。扩展DNA催化以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的催化剂。我们以前的工作已经确定，脱氧核酶可以对诸如DNA磷酸二酯水解等有挑战性的反应具有很高的增强率。但是，我们和其他人以前的大多数努力都集中在寡核苷酸的底物上，而简单的Watson-Crick基础配对为此提供了结合相互作用。在AIM 1中，我们将建立一种更通用的模块化方法，其中小分子底物受到可推广的适体域的约束，然后将其与催化域集成以提供功能性的脱氧核酶。 AIM 2和3将集中在分别催化蛋白质磷酸化和去磷酸化（即激酶和磷酸酶活性）的脱氧核酶的鉴定上。 AIM 4将应用新的激酶和磷酸酶脱氧核酶在生物学相关的环境中，例如细胞表面修饰反应和tau蛋白的研究，这与阿尔茨海默氏病有关。最后，AIM 5将使用生化方法，溶液状态NMR光谱和X射线晶体学来表征新近鉴定的激酶和磷酸酶脱氧核酶，从而提供重要的基本信息，从而提供有助于选择策略和催化活性的理性重新设计。