Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation

We demonstrate the site-specific incorporation of nucleo­base derivatives bearing fluoro­phores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (trans­glycosyl­ation at guanosine) is carried out by a bacterial (<i>E. coli</i>) tRNA guanine trans­glycosyl­ase (TGT), whose natural substrate is the nitrogenous base PreQ₁. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a poly­ethylene glycol linker attached to the exocyclic amine of PreQ₁. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleo­bases using a simple hairpin recognition element