High‐Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes

Abstract Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high‐fidelity determination of sEVs’ molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular‐Guided Hairpin‐Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single‐nucleotide discrimination as well as wash‐free fluorescence readout using a hairpin probe, the proposed approach enables high‐fidelity fluorescence visualization of sEVs’ microRNA without RNA extraction or leakage, demonstrating the potential of on‐site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV‐derived fundamental biological studies and molecular diagnostics.