Mapping GlycoRNAs on an Exosomal Surface

This study introduces an innovative periodate oxidation and oxime ligation (OL) method in combination with RNA-binding dyes or molecular beacons (MB) to detect glycoRNAs on endosome-derived exosome surfaces, addressing the challenges posed by exosomes nanoscopic dimensions and the lack of suitable detection techniques. This method enabled the identification of a direct glycan-RNA linkage, potentially advancing our understanding of glycoRNA biology. The method's specificity and sensitivity were validated using a sequential dual-labeling approach that distinguished exosome-surface-bound glycoRNAs, which was confirmed by flow cytometry and direct stochastic optical reconstruction microscopy (dSTORM). Furthermore, enzymatic treatments with ribonucleases and peptide-N-glycosidase F (PNGase F) elucidated the location and stability of glycan modifications, suggesting a significant role of glycoRNAs in cellular uptake for effective intercellular communication. The approach not only bypasses the need for metabolic chemical reporters but also lays the groundwork for exploring unknown glycoRNA sequences and expanding the investigation beyond sialic acids, thereby broadening the scope of epitranscriptomics and providing potential therapeutic insights.