Institute of Organic Chemistry, Julius-Maximilians-Universität Würzburg, Würzburg, Germany.
Institute of Molecular Infection Biology (IMIB), Julius-Maximilians-Universität Würzburg, Würzburg, Germany.
Nat Chem. 2023 Nov;15(11):1523-1531. doi: 10.1038/s41557-023-01320-z. Epub 2023 Sep 4.
Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes.
转录后 RNA 修饰方法是实现 RNA 定点标记和分析 RNA 功能的迫切需求。体外筛选的核酶是 RNA 研究的有吸引力的工具，并且有可能克服重新利用甲基转移酶的化学酶方法的一些局限性。在这里，我们报告了一种烷基转移酶核酶，它使用合成的、稳定的 S-腺苷甲硫氨酸 (SAM) 类似物，并催化将炔丙基基团转移到靶 RNA 中特定的腺嘌呤上。在体外的广泛反应条件下，包括生理镁离子浓度，在 1 小时内几乎实现了定量转化。利用 SAM 类似物的基因编码核酶（SAMURI）在 HEK293T 细胞中表达，并通过特异性荧光标记证实了靶腺苷的细胞内炔丙基化。SAMURI 是用于叠氮-炔点击化学中最小标签的定点安装的通用工具，该标签可以进一步用荧光团、亲和标签或其他功能探针进行功能化。
