Deoxyribozymes that ligate RNA

连接 RNA 的脱氧核酶

• 批准号: 6890873
• 负责人: Scott K Silverman
• 金额: $ 25.7万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890873
• 关键词: DNA; chemical cleavage; conformation; fluorescent dye /probe; ligands; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; nucleoside analog; ribozymes

DESCRIPTION (provided by applicant): RNA molecules that adopt specific three-dimensional structures can participate in numerous biologically important processes, including many specifically related to diseases such as cancer. Some RNAs are also capable of catalyzing chemical reactions, a role typically played by protein enzymes. Investigating RNA structure, folding, and catalysis is a major effort of modern biochemistry. For these studies, it is desirable to incorporate modified nucleosides at particular positions of RNA. Modified nucleosides are useful both to allow a detailed atomic-level understanding of RNA structure-function relationships and to enable incorporation of biophysical probes such as fluorescent labels. However, current techniques do not allow dependable synthesis of large, site-specifically modified RNAs. In this proposal, a comprehensive approach will be developed for ligation of smaller RNA fragments, which may themselves incorporate modifications. The ligation reaction will join two RNAs that have readily obtained functional groups at the ligation junction (e.g., 2',3'-cyclic phosphate and 5'-hydroxyl). The reaction will be catalyzed by divalent metal-dependent deoxyribozymes (DNA enzymes). These will be identified by a new in vitro selection .strategy, starting either from random DNA pools or from DNA pools biased towards sequences of RNA-cleaving DNA enzymes. The selected deoxyribozymes are anticipated to be efficient, general, and reliable tools for sequence-specific ligation of RNA. The new deoxyribozymes will be fully characterized biochemically and structurally. This will allow their optimal use for practical RNA ligations, and it will expand our knowledge of how nucleic acids can accelerate chemical reactions. The results of this research will have implications for designing therapeutic nucleic acid enzymes directed towards specific biological targets in vivo. The newly identified deoxyribozymes will be applied to perform previously unachievable experiments in RNA structure, folding, and catalysis that require incorporation of modified nucleosides.

描述（由申请人提供）：采用特定三维结构的RNA分子可以参与许多重要的生物学过程，包括许多与癌症等疾病特别相关的过程。一些 RNA 还能够催化化学反应，这一作用通常由蛋白酶发挥。研究 RNA 结构、折叠和催化是现代生物化学的一项重大工作。对于这些研究，需要在 RNA 的特定位置掺入修饰的核苷。修饰核苷可用于在原子水平上详细了解 RNA 结构-功能关系，并可掺入荧光标记等生物物理探针。然而，目前的技术不允许可靠地合成大的、位点特异性修饰的RNA。在该提案中，将开发一种用于连接较小 RNA 片段的综合方法，这些片段本身可能包含修饰。连接反应将连接两个已在连接点轻松获得功能基团（例如 2',3'-环状磷酸盐和 5'-羟基）的 RNA。该反应将由二价金属依赖性脱氧核酶（DNA 酶）催化。这些将通过新的体外选择策略来鉴定，从随机DNA库或从偏向RNA切割DNA酶序列的DNA库开始。所选脱氧核酶预计将成为有效、通用且可靠的 RNA 序列特异性连接工具。新的脱氧核酶将在生化和结构上得到全面表征。这将使它们能够在实际 RNA 连接中得到最佳应用，并将扩展我们对核酸如何加速化学反应的了解。这项研究的结果将对设计针对体内特定生物靶点的治疗性核酸酶产生影响。新鉴定的脱氧核酶将用于执行以前无法实现的、需要掺入修饰核苷的 RNA 结构、折叠和催化实验。