Deoxyribozymes that ligate RNA

描述(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，这是蛋白质酶通常扮演的角色。研究RNA的结构、折叠和催化作用是现代生物化学的一项主要工作。对于这些研究，在RNA的特定位置加入修饰的核苷是可取的。修饰的核苷对于在原子水平上详细了解RNA的结构-功能关系以及引入生物物理探针(如荧光标记)都是有用的。然而，目前的技术不允许可靠地合成大的、定点修饰的RNA。在这项提案中，将开发一种全面的方法来连接较小的RNA片段，这些片段本身可能会包含修饰。连接反应将连接在连接连接处容易获得官能团的两个RNA(例如，2‘，3’-环磷酸和5‘-羟基)。该反应将由依赖于金属的二价脱氧核酶(DNA酶)催化。这些将通过一种新的体外选择策略来识别，要么从随机的DNA池开始，要么从偏向RNA裂解DNA酶序列的DNA池开始。所选择的脱氧核酶有望成为RNA序列特异性连接的有效、通用和可靠的工具。新的脱氧核酶将在生化和结构上得到充分的表征。这将使它们能够最有效地用于实际的RNA连接，并将扩大我们对核酸如何加速化学反应的知识。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具有指导意义。新发现的脱氧核酶将用于在RNA结构、折叠和催化方面进行以前无法实现的实验，这些实验需要掺入修饰的核苷。

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.