Deoxyribozymes for Bioorganic Chemistry

描述（由申请人提供）：这是一个研究项目的更新申请，重点是生物有机化学中脱氧核酶的开发和应用。脱氧核糖酶（DNA酶）是具有特殊催化活性的DNA分子。本研究的长期目标是扩大脱氧核酶催化DNA的限制，并了解和控制这种催化。实现这些目标应该使脱氧核酶的实际应用成为可能，这是本研究的另一个长期目标。自然界利用核糖酶（RNA酶）催化生物相关反应，如核糖体中的蛋白质合成和剪接体中的RNA剪接。许多其他的天然催化rna已经被鉴定出来。虽然没有已知的天然脱氧核酶，但DNA的化学性质表明，当DNA以单链形式存在时，它具有催化潜力。我们和其他人已经证明，催化活性DNA序列可以通过从大量随机DNA序列中进行体外选择来鉴定。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那样），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验证这一假设，旨在探索DNA催化的极限。我们还建议使用各种方法来表征我们之前确定的特定脱氧核酶，并应用这种脱氧核酶来解决与RNA相关的几个基本生化问题。目的1系统地探索作为脱氧核酶底物的小分子。这是将DNA更广泛地应用于小分子生物有机化学的关键一步。目的2寻找催化氨基酸侧链反应的脱氧核酶，这将提高我们对如何实现dna催化蛋白质修饰的理解。目的3针对肽键的裂解作为DNA催化应用于蛋白质底物的具体例子。蛋白酶脱氧核酶的实际下游应用包括(a)作为蛋白质组学中胰蛋白酶和其他蛋白酶的替代品，用于产生大的蛋白质片段而不进一步降解，以及(b)作为体内酶原激活的方法，如凝血因子VIIa的形成，作为治疗出血的新方法。目标4是针对产生分支RNA的脱氧核酶的详细生物物理和结构特征，分支RNA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的功能，并在体外选择中纳入合理的设计元素，这是至关重要的。最后，Aim 5将脱氧核酶应用于涉及或使用分支RNA的三个重要生化问题。我们期望所有这些研究将扩大我们对DNA催化潜力的基本认识，并扩大脱氧核酶的实际生化用途。公共卫生相关性：催化DNA分子（脱氧核酶）是一种有趣的新型催化剂，可用于增加我们对核酸的基本了解和实际应用。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。

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.