Deoxyribozymes for Bioorganic Chemistry

用于生物有机化学的脱氧核酶

• 批准号: 8069587
• 负责人: Scott K Silverman
• 金额: $ 34.09万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069587
• 关键词: Active Sites; Address; Amino Acids; Architecture; Binding; Biochemical; Biology; Biophysics; Catalysis; Catalytic DNA; Catalytic RNA; Chemicals; Chemistry; Cleaved cell; Complex; DNA; DNA Sequence; Development; Elements; Enzyme Precursors; Enzymes; Factor VIIa; Funding; Future; Generations; Goals; Grant; Health; Hemorrhage; In Vitro; Introns; Investigation; Knowledge; Learning; Ligation; Mediating; Modeling; Modification; Motivation; NMR Spectroscopy; Nature; Nucleic Acids; Oligonucleotides; Peptide Hydrolases; Peptides; Play; Post-Translational Protein Processing; Protein Biosynthesis; Protein Fragment; Proteins; Proteomics; RNA; RNA Ligase (ATP); RNA Splicing; RNA-Protein Interaction; Reaction; Research; Resolution; Ribosomes; Roentgen Rays; Role; Spliceosomes; Structure; Techniques; Testing; Trypsin; X-Ray Crystallography; aptamer; catalyst; chemical property; chemical reaction; comparative; design; gel electrophoresis; improved; in vivo; insight; lariat debranching enzyme; member; novel strategies; nucleoside triphosphate; polypeptide; practical application; programs; research study; single-molecule FRET; small molecule; structural biology

DESCRIPTION (provided by applicant): This is a renewal application for a research program that focuses on the development and application of deoxyribozymes for bioorganic chemistry. Deoxyribozymes (DNA enzymes) are DNA molecules that have particular catalytic activities. Long-term objectives of this research are to expand the limits of DNA catalysis by deoxyribozymes and to understand and control this catalysis. Achieving these goals should enable practical application of deoxyribozymes, which is another long-term objective of this research. Nature uses ribozymes (RNA enzymes) to catalyze biologically relevant reactions such as protein synthesis in the ribosome and RNA splicing in the spliceosome. Many other natural catalytic RNAs have been identified. Although no natural deoxyribozymes are known, the chemical properties of DNA suggest that it has catalytic potential when in largely single-stranded form. We and others have shown that catalytically active DNA sequences can be identified by in vitro selection from large pools of random DNA sequences. We hypothesize that DNA can catalyze reactions not only of oligonucleotide substrates (as explored extensively by us and others) but also small molecules and proteins. We propose to test this hypothesis by systematic in vitro selection experiments designed to probe the limits of DNA catalysis. We also propose to use a variety of approaches to characterize a specific deoxyribozyme that we previously identified and to apply this deoxyribozyme to address several fundamental biochemical questions related to RNA. Aim 1 systematically explores small molecules as deoxyribozyme substrates. This is a key step in applying DNA more widely to small-molecule bioorganic chemistry. Aim 2 seeks deoxyribozymes that catalyze reactions of amino acid sidechains, which will improve our understanding of how to achieve DNA-catalyzed modifications of proteins. Aim 3 targets cleavage of peptide linkages as a specific example of DNA catalysis applied to protein substrates. Practical downstream applications of protease deoxyribozymes include (a) as an alternative to trypsin and other proteases in proteomics for generation of large protein fragments without further degradation, and (b) as an approach for in vivo zymogen activation, such as formation of coagulation factor VIIa as a novel approach to treatment of bleeding. Aim 4 is directed towards detailed biophysical and structural characterizations of a deoxyribozyme that creates branched RNA, which is the natural intermediate of RNA splicing. These studies will increase our fundamental understanding of nucleic acid catalysis, which is crucial if we are to improve deoxyribozyme function and incorporate rational design elements alongside in vitro selection. Finally, Aim 5 applies deoxyribozymes to three important biochemical problems that involve or use branched RNA. We anticipate that all of these investigations will broaden our basic understanding of the catalytic potential of DNA as well as expand the practical biochemical utility of deoxyribozymes. PUBLIC HEALTH RELEVANCE: Catalytic DNA molecules (deoxyribozymes) are an intriguing new form of catalyst that can be used for increasing our basic understanding of nucleic acids and for practical applications. The proposed research expands the chemical scope of DNA catalysis and applies deoxyribozymes to fundamental biochemical questions involving RNA, which plays a central role throughout biology.

描述（由申请人提供）：这是研究计划的续签应用，侧重于脱氧核酶在生物有机化学中的开发和应用。脱氧核酶（DNA酶）是具有特定催化活性的DNA分子。这项研究的长期目标是扩大脱氧核酶DNA催化的极限，并了解和控制该催化。实现这些目标应该可以实现脱氧核酶的实际应用，这是这项研究的另一个长期目标。大自然使用核酶（RNA酶）来催化与生物学相关的反应，例如核糖体中的蛋白质合成和剪接体中的RNA剪接。已经确定了许多其他天然催化RNA。尽管尚无天然脱氧核酶，但DNA的化学特性表明它在很大程度上单链形式时具有催化潜力。我们和其他人表明，可以通过从大的随机DNA序列中的体外选择来鉴定催化活性的DNA序列。我们假设DNA不仅可以催化寡核苷酸底物的反应（正如我们和其他人广泛探索的），还可以催化小分子和蛋白质的反应。我们建议通过系统的体外选择实验来检验这一假设，该实验旨在探测DNA催化的极限。我们还建议使用各种方法来表征我们先前鉴定出的特定脱氧核糖核物，并应用此脱氧核酶来解决与RNA相关的几种基本生化问题。 AIM 1系统地探索小分子作为脱氧核酶底物。这是将DNA更广泛地应用于小分子生物有机化学的关键步骤。 AIM 2寻求脱氧核酶，以催化氨基酸侧链的反应，这将提高我们对如何实现DNA催化蛋白质修饰的理解。 AIM 3靶向肽键的切割，作为应用于蛋白质底物的DNA催化的特定例子。蛋白酶脱氧核酸酶的实际下游应用包括（a）作为胰蛋白酶和其他蛋白酶在蛋白质组学中的替代方案，用于生成大型蛋白质片段而不进一步降解，以及（b）作为体内Zymogen激活的方法，例如形成凝结因子VIIA作为一种新型治疗的方法。 AIM 4针对脱氧核酶的详细生物物理和结构特征，该脱氧核酶会产生分支的RNA，这是RNA剪接的天然中间体。这些研究将增加我们对核酸催化的基本理解，如果我们要改善脱氧核酶功能，并将合理的设计元素与体外选择同时结合在一起，这至关重要。最后，AIM 5将脱氧核酶应用于涉及或使用分支RNA的三个重要的生化问题。我们预计，所有这些研究都将扩大我们对DNA催化潜力的基本理解，并扩大脱氧核酶的实际生化实用性。公共卫生相关性：催化DNA分子（脱氧核酶）是一种有趣的新形式的催化剂，可用于增加我们对核酸和实际应用的基本理解。拟议的研究扩大了DNA催化的化学范围，并将脱氧核酶应用于涉及RNA的基本生化问题，RNA在整个生物学中起着核心作用。