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The modified RNA base acp(3)U is an attachment site for N-glycans in glycoRNA

Affiliations
  • 1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA.
  • 2Stem Cell Program and Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
  • 3Department of Chemistry and Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
  • 4Department of Chemistry and Sarafan ChEM-H, Stanford University, Stanford, CA, USA; Howard Hughes Medical Institute, Stanford, CA, USA.
  • 5Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA. Electronic address: bagarcia@wustl.edu.
  • 6Stem Cell Program and Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA. Electronic address: ryan.flynn@childrens.harvard.edu.

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August 22, 2024

Abstract

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although 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 RNA-optimized 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 (acpU) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.

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