Department of Chemistry, Emory University, 1515 Dickey Dr., Atlanta, GA, 30322, USA.
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA.
Chemistry. 2020 Aug 6;26(44):9874-9878. doi: 10.1002/chem.202001667. Epub 2020 Jun 17.
Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.
直截了当的方法来检测腺嘌呤到次黄嘌呤（A-to-I）RNA 编辑是更好地理解其调控、功能以及与疾病的关系的关键。我们通过开发一种新型试剂 N-(4-乙炔基苯基)丙烯酰胺（EPhAA）来满足这一需求，并展示了它选择性标记 RNA 中次黄嘌呤的能力。EPhAA 可通过一步合成得到，与次黄嘌呤快速反应，并且“点击”兼容，可在编辑位点灵活连接荧光探针。我们首先验证了 EPhAA 在核糖核苷和 RNA 底物中对次黄嘌呤的反应性和选择性，然后应用我们的方法直接监测使用重组 ADAR 酶的体外 A-to-I RNA 编辑活性。这种方法改进了现有的次黄嘌呤化学标记技术，提供了一种经济高效、快速且非放射性的方法来检测 RNA 中次黄嘌呤的形成。我们设想这种方法将改善 A-to-I 编辑的研究，并能够更好地描述不同环境下的 RNA 修饰模式。
