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分子可以参与众多重要的生物学重要过程，包括许多与癌症等疾病有关的许多专门有关的过程。一些RNA也能够催化化学反应，这是蛋白质酶通常扮演的角色。研究RNA结构，折叠和催化是现代生物化学的主要努力。对于这些研究，希望在RNA的特定位置掺入修饰的核苷。修饰的核苷既有用，既可以允许对RNA结构 - 功能关系的详细原子水平理解，又可以使生物物理探针（例如荧光标签）掺入。但是，当前技术不允许可靠的大型位点特定修改的RNA合成。在此提案中，将开发一种用于连接较小的RNA碎片的综合方法，该方法本身可能包含修改。连接反应将连接两个在连接连接处获得官能团的RNA（例如2'，3'-循环磷酸盐和5'-羟基）。该反应将通过二价金属依赖性脱氧核酶（DNA酶）催化。这些将通过一种新的体外选择（从随机DNA池或从偏向RNA裂解DNA酶序列的DNA池开始）来识别。预计所选的脱氧核酶将是有效，一般和可靠的RNA序列连接的工具。新的脱氧核酶将在生化和结构上充分表征。这将允许它们用于实用的RNA连接，它将扩大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学靶标的治疗性核酸酶有影响。新鉴定的脱氧核酶将用于在RNA结构，折叠和催化中进行以前无法实现的实验，这些实验需要掺入改良的核苷。