University of Colorado Boulder Background
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. Researchers at Colorado Boulder have developed 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. The researchers demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding U RNA in live mammalian cells.
Researchers at the University of Colorado have introduced a new riboswitch-based RNA imaging platform that the researchers call ‘Riboglow’ characterized by several features that distinguish it from previous RNA-detection systems. First, the RNA tag is small and exploits the selective and high affinity (KD in nM range) 15 binding between a riboswitch and its cognate ligand, Cbl. Second, because the RNA tag binds the fluorescence quencher, Cbl, the system is compatible with a wide range of synthetic fluorophores spanning the green to far-red spectral range. The Riboglow-ATTO 590 and Riboglow-Cy5 probes used for live cell studies retain favorable photophysical properties, including slow photobleaching, of the parent dyes when bound to RNA. Third, the RNA tag is not subjected to undesired processing and hence does not require the tRNA-like scaffold used for other dye-binding aptamers. Fourth, cobalamin riboswitches include a large family of RNA sequences that all bind Cbl57 that can be exploited for future optimization and customization of the Riboglow platform. Fifth, the fluorescence lifetime of ATTO 590 and Cy5 in the context of the probes varies when the RNA binds, raising the possibility of using this system for fluorescence lifetime imaging. Finally and perhaps most importantly, the researchers were able to visualize recruitment of RNA polymerase II-dependent transcripts (mRNA and snRNA) to RNP granules, where the short size of the RNA tag (~80 nts for RNA tag AT) enabled tagging and visualization of U1 snRNA in live cells for the first time.
Stage of Development
A direct side-byside 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.
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