Cong Deyuan, Steinbuch Kfir B, Koyama Ryosuke, Lam Tyler V, Lam Jamie Y, Tor Yitzhak
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla California 92093-0358 USA
RSC Chem Biol. 2024 Mar 27;5(5):454-458. doi: 10.1039/d4cb00045e. eCollection 2024 May 8.
The templated enzymatic incorporation of adenosine and its analogs, including mA, A and A into RNA transcripts, has been explored. Enforced transcription initiation with excess free nucleosides and the native triphosphates generates 5'-end modified transcripts, which can be 5'-phosphorylated and ligated to provide full length, singly modified RNA oligomers. To explore structural integrity, functionality and utility of the resulting non-canonical purine-containing RNA constructs, a MazF RNA hairpin substrate has been synthesized and analyzed for its susceptibility to this endonuclease. Additionally, RNA substrates, containing a singly incorporated isomorphic emissive nucleoside, can be used to monitor the enzymatic reactions in real-time by steady state fluorescence spectroscopy.
人们已经探索了将腺苷及其类似物（包括mA、A和A）通过模板酶促掺入RNA转录本的方法。使用过量的游离核苷和天然三磷酸进行强制转录起始会产生5'端修饰的转录本，这些转录本可以进行5'磷酸化和连接，以提供全长的、单修饰的RNA寡聚物。为了探索所得含非经典嘌呤的RNA构建体的结构完整性、功能和实用性，已经合成了一种MazF RNA发夹底物，并分析了其对这种核酸内切酶的敏感性。此外，含有单掺入的同构发光核苷的RNA底物可用于通过稳态荧光光谱实时监测酶促反应。
