Dr. Esther Braselmann
Georgetown University
Friday, December 5, 2025
12:00 Noon
Room 120 – Meyerhoff Chemistry Building
Host: Dr. Deepak Koirala
“Rational designs of the Riboglow platform for multiplexed imaging of RNA molecules in live cells”
Central roles for ribonucleic acids (RNAs) continue to be discovered across all domains of life. Subcellular RNA localizations in space and time are closely linked to their function, motivating the need to robustly visualize RNAs and their dynamics in live cells. In contrast to the protein world, no genetically encoded fluorescent RNAs were found in nature, leading to intense tool development efforts. We are redesigning a bacterial RNA riboswitch as a fluorescence sensor, called Riboglow1. The riboswitch RNA is genetically fused as a tag to an RNA of interest. The natural riboswitch ligand Cobalamin (Cbl, vitamin B12) is the basis of a small molecule probe that includes a synthetic fluorophore, connected to Cbl via a chemical linker. Binding of the probe to the RNA tag induces a change in probe fluorescence intensity and fluorescence lifetime. We exploit fluorescence lifetime changes of the probe upon binding the RNA tag to demonstrate that fluorescence lifetime imaging microscopy (FLIM) is an advantageous modality of Riboglow for RNA sensing in live cells.2 First, we demonstrate that cellular contrast is superior when using lifetime imaging compared with traditional intensity-based imaging. Second, the intensity independence of FLIM makes it unnecessary to attach many copies of the RNA tag to an RNA of interest to achieve intensity-contrast, a common strategy for traditional RNA fluorescent tags. Third, the versatility of Riboglow probes allows for using far-red fluorophores, enabling visualizing RNA dynamics in multicellular, complex environments.3,4 Finally, we show that varying the RNA tag sequence that binds the ligand changes fluorescence lifetime differently5 . This feature leads us to explore Riboglow-FLIM as an RNA multiplexing sensor with the goal of detecting different RNAs simultaneously in the same cell. 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.