Department of Chemistry , Emory University , Atlanta , Georgia 30322 , United States.
Bioconjug Chem. 2018 Sep 19;29(9):2899-2903. doi: 10.1021/acs.bioconjchem.8b00541. Epub 2018 Aug 27.
Adenosine-to-inosine (A-to-I) RNA editing is a widespread and conserved post-transcriptional modification, producing significant changes in cellular function and behavior. Accurately identifying, detecting, and quantifying these sites in the transcriptome is necessary to improve our understanding of editing dynamics, its broader biological roles, and connections with diseases. Chemical labeling of edited bases coupled with affinity enrichment has enabled improved characterization of several forms of RNA editing. However, there are no approaches currently available for pull-down of inosines. To address this need, we explore acrylamide as a labeling motif and report here an acrylamidofluorescein reagent that reacts with inosine and enables enrichment of inosine-containing RNA transcripts. This method provides improved sensitivity in the detection and identification of inosines toward a more comprehensive transcriptome-wide analysis of A-to-I editing. Acrylamide derivatization is also highly generalizable, providing potential for the labeling of inosine with a wide variety of probes and affinity handles.
腺嘌呤到次黄嘌呤（A-to-I）RNA 编辑是一种广泛而保守的转录后修饰，可显著改变细胞功能和行为。准确识别、检测和定量转录组中的这些位点，对于提高我们对编辑动态、其更广泛的生物学作用以及与疾病的联系的理解是必要的。编辑碱基的化学标记与亲和富集相结合，已经能够更好地描述几种形式的 RNA 编辑。然而，目前还没有用于捕获次黄嘌呤的方法。为了解决这个需求，我们探索了丙烯酰胺作为标记基序，并在此报告了一种丙烯酰胺荧光素试剂，该试剂与次黄嘌呤反应，能够富集含有次黄嘌呤的 RNA 转录本。该方法在检测和鉴定次黄嘌呤方面提高了灵敏度，可更全面地分析 A-to-I 编辑的转录组。丙烯酰胺衍生化也具有高度的通用性，为用各种探针和亲和处理剂标记次黄嘌呤提供了潜力。
