Ribonucleic acids (RNAs) are closely linked to fundamental cellular processes in healthy and disease states. Despite their importance, tools to probe subcellular RNA dynamics in live cells remain limited. Fluorescent labeling offers a promising strategy, yet real-time imaging of endogenous RNAs is technically challenging. To address this, we developed the Riboglow-FLIM platform, a small-molecule-based system for RNA tagging. Here, a bacterial riboswitch-derived RNA tag is fused to the RNA of interest and a cobalamin-ligand is added, where a fluorophore and quencher are covalently linked (Braselmann et al., NCB. 2018). Cobalamin quenches fluorescence, but in the fluorescent probe, both fluorescence lifetime and intensity increase upon RNA-binding to Cobalamin (Sarfraz et al., Nat. Comm . 2023). Traditional RNA visualization involves transiently transfecting plasmid DNA that expresses tagged RNAs of interest. A fundamental challenge with this approach is that gene expression is very high after transient transfection but decreases substantially with cell division and eventually disappears as the gene is not integrated into the cell's genome. To overcome this issue and mimic endogenous conditions, it is advantageous to produce a tagged gene from the genome. Here, we used the PiggyBac transposon system to stably express tagged mRNAs from the mammalian cell genome. Our findings demonstrate that a single RNA tag copy is sufficient to provide fluorescence lifetime contrast for the detection of endogenous RNAs. Fluorescence lifetime readouts are comparable to those from transient transfection models, confirming genomic integration as a stable and minimally disruptive method for gene delivery. Overall, our findings advance Riboglow-FLIM as a robust and versatile platform for concentration-independent, long-term, and real-time visualization of endogenous RNA dynamics in living mammalian cells.
Bindu et al. (Sun,) studied this question.