Xie, Y.; Hemberger, H.; Till, N. A.; Chai, P.; Watkins, C.; Lebedenko, C. G.; Caldwell, R. M.; George, B.; Bertozzi, C. R.; Garcia, B. A.; Flynn, R. A.

We recently identified glycoRNA-a previously undescribed glycoconjugate-which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.