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FRET imaging of glycoRNA on small extracellular vesicles enabling sensitive cancer diagnostics

Ren Tingju,Zhang Yingzhi,Tong Yuxiao,Zhang Qi,Wang Tianhao,Wang Yue,Yang Chunguang,Xu Zhangrun

Glycosylated RNAs (glycoRNAs), a recently discovered class of membrane-associated glyco-molecules, remain poorly understood in function and clinical value due to limited detection methods. Here, we show a dual recognition Förster resonance energy transfer (drFRET) strategy using nucleic acid probes to detect N-acetylneuraminic acid-modified RNAs, enabling sensitive, selective profiling of glycoRNAs on small extracellular vesicles (sEVs) from minimal biofluids (10 μl initial biofluid). Using drFRET, we identify 5 prevalent sEV glycoRNAs derived from 7 cancer cell lines. In a 100-patient cohort (6 cancer types and non-cancer controls), sEV glycoRNA profiles achieve 100% accuracy (95% confidence interval) in distinguishing cancers from non-cancer cases and 89% accuracy in classifying specific cancer types. Furthermore, drFRET reveal that sEV glycoRNAs specifically interact with Siglec proteins and P-selectin, which is critical for sEV cellular internalization. The drFRET strategy provides a versatile and sensitive platform for the imaging and functional analysis of sEV glycoRNAs, with promising implications for clinical applications.