Profiling extracellular vesicle glycoRNAs defines their function in vesicular communication.

Glycosylated RNAs (glycoRNAs) reside on the cell surface with their functions largely unexplored. Here, we show that extracellular vesicles (EVs) carry glycoRNAs with distinct sequence diversity, vesicle type specificity and transfer dynamics. By metabolically tagging glycoRNAs with bioorthogonal handles, we selectively enrich and sequence EV-associated glycoRNAs, identifying specific glycoRNA species and expanding the EV glycoRNA landscape. We further develop a proximity-based imaging strategy to track glycoRNAs, revealing that glycoRNA repertoires in large EVs vary with cell type and passage, whereas small EVs display distinct profiles indicative of vesicle-specific sorting. Functionally, EVs deliver glycoRNAs into recipient cells and glycoRNAs are subsequently detected in recipient cell-derived EVs, reflecting contributions from newly synthesized glycoRNAs and others that may be associated with donor EVs. Notably, hybridization-based glycoRNA blockade can heavily reduce EV uptake, highlighting glycoRNAs as potentially actionable targets for modulating EV-cell communication. These findings expand the glycoRNA landscape and suggest EV glycoRNAs are biomarkers of cell state and regulators of vesicle tropism.