A Spatial Coding Strategy for In Situ Visualization of RNA-Specific Glycoforms

Visualization of site specific glycoforms of RNA on live cell surfaces remains technically challenging due to glycoform heterogeneity and the covalently linked structural motifs of glycoRNA. Here, a spatial coding strategy termed SpaceFRET (Spatial coded Förster resonance energy transfer) is developed for in situ visualization of RNA-specific glycoforms. SpaceFRET enables the identification of covalent conjugation of glycan and RNA by integrating chemoenzymatic glycan labeling (CeGL) with proximity induced dual encoding system. Glycoforms of glycoRNAs are incorporate bioorthogonal chemical groups via CeGL, which are further encoded with FRET donor probes by click chemistry. Specific RNA sequences are encoded by hairpin-shaped FRET acceptor probes through sequence specificity. The information on glycoRNAs is subsequently decoded by the dynamics of strand displacement reactions. SpaceFRET has been successfully employed to visualize the sia-glycoRNA, Gal/GalNAc-glycoRNA and LacNAc-glycoRNA. Furthermore, SpaceFRET elucidates the presence of glycoRNAs on small extracellular vesicles and delineates the spatial distributions of glycoRNAs on the cell surface, including their colocalization with lipid rafts. The SpaceFRET provides a versatile platform for profiling RNA-specific glycoforms, holding great promise for validating the mechanism underlying their function.