Engineering covalent small molecule–RNA complexes in living cells

Abstract Covalent labeling of RNA in living cells poses many challenges. Here we describe a structure-guided approach to engineer covalent RNA aptamer–ligand complexes. The key is to modify the cognate ligand with an electrophilic handle that allows it to react with a guanine at the RNA binding site. We illustrate this for the preQ 1 -I riboswitch, in vitro and in vivo. Further, we demonstrate the versatility of the approach with a covalent fluorescent light-up aptamer. The coPepper system maintains strong fluorescence in live-cell imaging even after washing, can be used for super-resolution microscopy and, most notably, is uniquely suited for fluorescence recovery after photobleaching to monitor intracellular RNA dynamics. In addition, we have generated a Pepper ligand with a second handle for bioorthogonal chemistry to allow easily traceable pull-down of the covalently linked target RNA. Finally, we provide evidence for the suitability of this tethering strategy for drug targeting.