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

Post-transcriptional tRNA modifications are regulators of gene expression, yet their spatial organization and dynamic regulation remain poorly understood, because methods to track tRNA modification status inside cells are lacking. Sequencing and biochemical approaches provide population-level readouts but typically require RNA extraction, eliminating spatial information and obscuring cell-to-cell heterogeneity. Here, we report a chemoenzymatic RNA fluorescent labeling strategy that enables the imaging of a specific tRNA modification state in mammalian cells. Our method relies on the catalytic selectivity of the bacterial enzyme tRNA guanine transglycosylase (TGT) to covalently incorporate fluorophore-conjugated preQ₁ 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.