Deoxyribozyme-catalyzed covalent labeling of RNAs

描述(申请人提供)：脱氧核酶是催化各种反应的DNA寡核苷酸。通过体外筛选，已经鉴定出能够通过2‘，5’键将两个RNA底物连接(连接)在一起的脱氧核酶，形成分支的RNA产物。这种催化活性为RNA衍生化和RNA标记的新方法打开了大门。这项建议的目的是开发脱氧核酶，通过2‘，5’-支链将衍生的RNA底物(标签RNA)共价连接到靶向RNA。这些标记的脱氧核酶将通过体外选择进行鉴定，目的是鉴定对序列要求最低且对荧光素和生物素等有用的生物物理标记具有耐受性的脱氧核酶。这些标记脱氧核酶被设想为通用工具，可用于通过FRET等方法标记用于体外研究的模型RNA。还可以通过改变标记RNA的功能来实现其他用途。最终，这些脱氧核酶将作为mRNA标记剂在体内进行测试。在细胞环境中将含有探针的标签共价连接到感兴趣的RNA上的能力将对发育和疾病的研究大有裨益。

DESCRIPTION (provided by applicant): Deoxyribozymes are DNA oligonucleotides that catalyze a variety of reactions. Through in vitro selection, deoxyribozymes have been identified that are able to join (ligate) two RNA substrates together via 2',5' linkages, forming branched RNA products. This catalytic activity opens the door to new methods of RNA derivatization and RNA labeling. The objective of this proposal is to develop deoxyribozymes that covalently link a derivatized RNA substrate (a "tagging" RNA) to target RNAs via 2',5'-branched linkages. These labeling deoxyribozymes will be identified by in vitro selection, with the goal of identifying deoxyribozymes that have minimal sequence requirements and are tolerant of useful biophysical labels such as fluorescein and biotin. These labeling deoxyribozymes are envisioned as general tools that could be used to label model RNAs for in vitro studies by methods such as FRET. Other uses are also possible by changing the functionality of the tagging RNA. Ultimately, these deoxyribozymes will be tested as mRNA labeling agents in vivo. The ability to covalently attach probe-containing tags to RNAs of interest in a cellular setting will be of great use for studies of development and disease.