Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

中文翻译：

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tRNA鸟嘌呤转糖基酶修饰mRNA的位点特异性共价偶联

修饰的mRNA（mod-mRNA）由于具有较高的稳定性和较低的免疫应答，因此已作为可用于治疗应用的RNA形式进行了广泛的研究。在这里，我们扩展了最近建立的RNA-TAG（鸟苷中的转糖基化）方法的范围，这是一种通过使用mod-mRNA作为底物来对大mRNA转录本进行遗传编码的位点特异性标记的新方法。作为概念的证明，我们将荧光探针共价连接到mCherry编码的具有5甲基胞苷和/或伪尿苷取代标记的mod-mRNA转录物，具有较高的标记效率。为了提供具有广泛可能应用的通用标记方法，我们采用了两步策略对mod-mRNA进行功能化，以突出这种新方法的治疗潜力。