Förster Resonance Energy Transfer Imaging of Cell-Surface GlycoRNAs: Elucidating GlycoRNA-Cell Migration Correlation and Profiling of Associated RNA-Binding Proteins

Abstract Cell‑surface glycosylated RNAs (glycoRNAs) are N‑glycosylated noncoding RNAs and associate with cell‑surface RNA‑binding proteins (csRBPs). Conventional detection methods suffer from destructive processing and diffraction-limited resolution, failing to resolve native distributions. To address this, we developed GlycoFRET‑PLIA (Proximity Labelling & Imaging Analysis), which integrates metabolic azido‑sialic acid labeling, strain‑promoted azide‑alkyne cycloaddition, N3‑kethoxal RNA tagging, and Förster resonance energy transfer (FRET) imaging. GlycoFRET‑PLIA revealed heterogeneous glycoRNA distributions across six cell lines and an inverse correlation between FRET intensity and migratory capacity, with highly metastatic cells showing reduced FRET compared to cells with low migratory capacity. We further engineered sequence-specific proximity labeling using azide-modified in situ hybridization probes to recruit horseradish peroxidase, enabling covalent tagging of csRBPss. Quantitative proteomics identified 196 nanodomain-associated csRBPs enriched in focal adhesion and chemokine signaling. Collectively, GlycoFRET‑PLIA enables in situ visualization and interactome mapping of glycoRNAs, supporting their role as spatial organizers of prometastatic signalosomes.