Riboglow: a multicolor riboswitch-based platform for live cell imaging of mRNA and small non-coding RNA in mammalian cells

RNAs directly regulate a vast array of critical cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular ‘Riboglow’ platform leverages different color fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding U RNA in live mammalian cells. A direct side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the current gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches. Graphical abstract