在冷冻溶液中通过脱氧核酶切割生成 2'，3'-环磷酸化 RNA。

Generation of RNA with 2', 3'-Cyclic Phosphates by Deoxyribozyme Cleavage in Frozen Solutions.

机构信息

Biomimetic Systems, Max Planck Institute of Biochemistry, Martinsried, Germany.

Faculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany.

出版信息

Methods Mol Biol. 2022;2439:301-309. doi: 10.1007/978-1-0716-2047-2_19.

DOI:10.1007/978-1-0716-2047-2_19

PMID:35226329

Abstract

The generation of terminal 2', 3'-cyclic phosphates on RNA oligomers is an important process in the study of tRNA splicing and repair, ribozyme catalysis, and RNA circularization. Here, we describe a simple method for producing 2', 3'-cyclic phosphate functionalized RNA by the deoxyribozyme-catalyzed cleavage of a short 3'-RNA overhang in frozen solution. This method avoids the nonspecific modification and degradation of RNA and attached functional groups (e.g., fluorophores) inherent in other methods, and the use of frozen conditions enables cleavage at very low divalent metal ion concentrations, limiting RNA hydrolysis.

摘要

寡核苷酸上末端 2'，3'-环磷酸的生成是 tRNA 剪接和修复、核酶催化以及 RNA 环化研究中的一个重要过程。在这里，我们描述了一种通过脱氧核酶在冷冻溶液中切割短的 3'-RNA 突出端来产生 2'，3'-环磷酸功能化 RNA 的简单方法。该方法避免了其他方法中固有的 RNA 及其附着的官能团（例如荧光团）的非特异性修饰和降解，并且使用冷冻条件可以在非常低的二价金属离子浓度下进行切割，从而限制了 RNA 的水解。