BPS2026 – Live-cell visualization of endogenous RNAs with Riboglow-FLIM

RNA is one of nature’s most abundant macromolecules. Although commonly thought of as the mediatory between information storage as DNA and cellular activity through protein expression, RNAs indeed have diverse cell-critical functions in areas such as gene regulation, with carefully regulated subcellular localizations. Visualization of RNAs in live cellular contexts is essential to elucidate their dynamics and interactions spatiotemporally, especially in the context of multicellular systems. This is challenging because, in contrast to proteins, no fluorescent RNAs have been discovered in nature. A dye-quencher based system, Riboglow, has recently gained attention as an RNA visualization platform via fluorescence lifetime imaging microscopy (FLIM). In this system, target RNAs are genetically fused to a short cobalamin binding aptamer. Cobalamin, a natural quencher, is synthetically linked to a fluorescent dye that becomes spatially segregated upon aptamer binding, resulting in a quantifiable fluorescence lifetime “turn on.” Live-cell RNA visualization platforms commonly rely on RNA overexpression or lengthy tags to overcome background signal. However, these may disrupt natural cellular processes. Riboglow-FLIM can overcome these limitations because FLIM is a concentration independent signal. To test visualizing RNAs endogenously, we use CRISPR Cas9 mediated knockin to insert Riboglow into the 3’ UTR of the β-Actin-encoding gene. Ongoing work aims to these mRNA localization dynamics in real time.