Soft Matter Seminar: Interactions of a Riboswitch RNA With Fluorogenic Probes for Multiplexed RNA Detection in Live Cells With the Riboglow-FLIM Sensor
Prof. Esther Braselmann, Georgetown University
Abstract: Subcellular RNA localizations are closely linked to their function, motivating the need to visualize RNAs live. We redesigned a bacterial RNA riboswitch as a fluorescence sensor, Riboglow. Here, the riboswitch RNA is genetically tagged to an RNA of interest and its natural ligand Cobalamin was coupled to a synthetic fluorophore. We observed a change in fluorescence intensity and fluorescence lifetime of the probe upon binding to the RNA, leading us to explore fluorescence lifetime imaging microscopy (FLIM), yielding the Riboglow-FLIM sensor. First, we demonstrate that cellular contrast is superior for lifetime imaging compared with intensity imaging. Second, the intensity independence of FLIM makes it unnecessary to multiplex the RNA tag to achieve intensity-contrast, a common strategy for traditional RNA fluorescent tags. Finally, we exploit phylogenetic diversity of the riboswitch. Different RNA tag sequences change fluorescence lifetime to different extends. This feature leads us to explore Riboglow-FLIM as an RNA multiplexing sensor. Together, we demonstrate that FLIM is an advantageous approach for RNA sensing that addresses many current challenges in the field of RNA imaging tool development.