Hanswillemenke Alfred, Stafforst Thorsten
Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
Methods Enzymol. 2019;624:47-68. doi: 10.1016/bs.mie.2019.06.004. Epub 2019 Jul 4.
The SNAP-tag technology offers a convenient way to assemble guideRNA-protein conjugates for transcript-specific RNA editing in vitro, in cell culture and in vivo. In contrast to other methods, including CRISPR/Cas-based, the SNAP-tag is small, well expressed and of human origin. Furthermore, the SNAP-ADAR approach enables the ready inclusion of photo control by caging/decaging of the benzylguanine moiety required for the conjugation reaction with the SNAP-tag. Beyond site-directed RNA editing, the method has high potential for various applications in the field of RNA targeting. However, the generation of the required guideRNAs includes some basic chemistry. Here, we provide step-by-step protocols for (a) conduction of photo controlled RNA editing reaction, (b) the generation of photo activatable guideRNAs, and (c) the synthesis of the caged benzylguanine moiety. With this we hope to foster a broader application of these attractive methods to researchers with less experience in chemistry.
SNAP标签技术为在体外、细胞培养和体内组装用于转录本特异性RNA编辑的向导RNA-蛋白质缀合物提供了一种便捷方法。与其他方法（包括基于CRISPR/Cas的方法）相比，SNAP标签体积小、表达良好且源自人类。此外，SNAP-ADAR方法通过对与SNAP标签缀合反应所需的苄基鸟嘌呤部分进行笼蔽/去笼蔽，能够方便地引入光控。除了定点RNA编辑外，该方法在RNA靶向领域的各种应用中具有很大潜力。然而，所需向导RNA的生成涉及一些基础化学知识。在此，我们提供了分步方案，用于（a）光控RNA编辑反应的进行，（b）光激活向导RNA的生成，以及（c）笼蔽苄基鸟嘌呤部分的合成。我们希望借此促进这些有吸引力的方法在化学经验较少的研究人员中得到更广泛的应用。
