Deoxyribozyme-catalyzed covalent labeling of RNAs

描述（由申请人提供）：脱氧核糖酶是催化多种反应的DNA寡核苷酸。通过体外选择，已经鉴定出脱氧核酶能够通过2‘,5’键连接（连接）两个RNA底物，形成分支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。本提案的目的是开发脱氧核酶，通过2‘,5’支链键将衍生RNA底物（“标记”RNA）共价连接到目标RNA。这些标记脱氧核酶将通过体外选择鉴定，目的是鉴定具有最小序列要求并耐受有用的生物物理标记（如荧光素和生物素）的脱氧核酶。这些标记脱氧核酶被设想为通用工具，可用于标记模型rna，用于通过FRET等方法进行体外研究。通过改变标记RNA的功能也可以实现其他用途。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有很大的用处。

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.