Enzymatic modification of 5'-capped RNA with a 4-vinylbenzyl group provides a platform for photoclick and inverse electron-demand Diels-Alder reaction.

Chemo-enzymatic strategies provide a highly selective means to label different classes of biomolecules in vitro, but also in vivo. In the field of RNA, efficient labeling of eukaryotic mRNA with small organic reporter molecules would provide a way to detect endogenous mRNA and is therefore highly attractive. Although more and more bioorthogonal reactions are being reported, they can only be applied to chemo-enzymatic strategies if a suitable (i.e., click compatible) modification can be introduced into the RNA of interest. We report enzymatic site-specific transfer of a 4-vinylbenzyl group to the 5'-cap typical of eukaryotic mRNAs. The 4-vinylbenzyl group gives access to mRNA labeling using the inverse electron-demand Diels-Alder reaction, which does not work with an enzymatically transferred allyl group. The 4-vinylbenzyl-modified 5'-cap can also be converted in a photoclick reaction generating a "turn-on" fluorophore. Both click reactions are bioorthogonal and the two step approach also works in eukaryotic cell lysate. Enzymatic transfer of the 4-vinylbenzyl group addresses the lack of flexibility often attributed to biotransformations and thus advances the potential of chemo-enzymatic approaches for labeling.