Enzyme-Mediated Covalent Labeling Enables In Situ Imaging of RNA Modification States

analogues into q-cognate tRNAs only when they lack queuine, a unique hypermodified base that influences translation, stress responses, and mammalian physiology. Because queuine-modified tRNAs are not substrates for transglycosylation, fluorophore incorporation directly reports the hypomodification status. Queuine-hypomodified tRNAs are selectively visualized with subcellular resolution in fixed cells. Using this approach, we track queuine incorporation and loss kinetics across cell lines, analyze genetic factors that control modification, and resolve differences between cytosolic and mitochondrial tRNA populations. Beyond queuine, this work demonstrates a strategy for converting the endogenous RNA modification state into a covalent fluorescent readout, providing a chemical framework for spatial analysis of RNA modifications in intact cells.