5EU-Based Tracking of RNA Transfer Through Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-bound nanoparticles that mediate the intercellular transfer of diverse molecules. RNA species within EVs act as key regulators of gene expression and influence the phenotype in recipient cells. However, tracking RNA transfer via EVs remains technically challenging, as post-isolation fluorescent labeling can lead to artificial signals and genetic manipulation of recipient cells may disrupt endogenous regulatory pathways. Here, we detail a metabolic labeling approach with 5-ethynyl uridine (5-EU) that enables tracing of EV-mediated RNA transfer under physiological conditions. In this approach, donor cells incorporate 5-EU into nascent RNAs, which are naturally packaged into EVs. Following delivery to recipient cells, the transferred RNAs are selectively enriched via biotin-based click chemistry for downstream analysis. This method preserves native RNA loading into EVs and enables tracing their delivery to recipient cells, providing a reliable and minimally perturbative approach for studying EV-mediated RNA transport in intercellular communication.