There is tremendous interest in investigating the intricate roles RNA play within cellular function beyond serving as translational templates. Despite this demand, few robust methods exist, and mainstream approaches utilize non-covalent interactions between reporting probes and the RNA of interest. Here, we demonstrate a covalent site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity handles of interest directly into a short RNA stem loop recognition motif by an enzyme mediated transglycosylation reaction. The RNA-TAG (transglycosylation at guanosine) labeling reaction is carried out by a bacterial tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated several large functional groups including biotin, BODIPY, and Cy7, as well a fluorogenic thiazole orange probe, through a short linker attached to PreQ1. RNAs of interest, including large endogenously transcribed and processed transcripts, can be site-specifically labeled, affinity purified, and visualized in cells when they possess the RNA-TAG recognition motif.

Site-specific covalent labeling of RNA with RNA-TAG: A robust technology applicable to a variety of biologically relevant investigations

251st National Meeting (2016)

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