Fluorogenic Covalent Probes for RNA

Sequence-generalized fluorescent labels and stains for RNA can enable imaging, tracking, and analysis of the biopolymer. However, current noncovalent RNA dyes are poorly selective for RNA over DNA, interact weakly with their target, and can show limited utility in cellular RNA staining due to poor selectivity and high background signals. Here, we report a fluorogenic covalent labeling approach based on acylimidazole-mediated reaction of donor-acceptor fluorophores with 2'-hydroxyl (2'-OH) groups of RNA, providing a wavelength-tunable, sequence-independent strategy for selective labeling of the biopolymer. This reactive probe design enables labeling and imaging under mild aqueous conditions, providing up to 390-fold fluorescence enhancement and 970-fold selectivity for RNA over DNA, with four emission colors documented. The covalent fluorophore platform enables improved new tools for RNA-specific analysis and imaging in gels, in solution, and in living cells.