Identification of glycosylated nucleosides in small synthetic glyco‐RNAs

• Published in: Chembiochem : a European journal of chemical biology 2024-03, Vol.25 (5), p.e202300784-n/a
• Main Authors: Michael, Frank St, Hamouda, Maha Ben, Stupak, Jacek, Li, Jianjun, Pearson, Angela, Sauvageau, Janelle
• Format: Article
• Language: English
• Subjects: Alkynes; Azides; Cell culture; Cycloaddition Reaction; Glycan; Glyco-RNA; Glycoconjugates; Glycosylation; Humans; Immune response; Innate immunity; Interferon stimulated genes; Lactose; Mass Spectrometry; Nuclease; Nucleosides; Polysaccharides; Ribonucleic acid; RNA; RNA modification; Sialyllactose; Therapeutic targets; Y RNA
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300784

Recently, the post‐transcriptional modification of RNA with N‐glycans was reported, changing the paradigm that RNAs are not commonly N‐glycosylated. Moreover, glycan modifications of RNA are investigated for therapeutic targeting purposes. But the glyco‐RNA field is in its infancy with many challenges to overcome. One question is how to accurately characterize glycosylated RNA constructs. Thus, we generated glycosylated forms of Y5 RNA mimics, a short non‐coding RNA. The simple glycans lactose and sialyllactose were attached to the RNA backbone using azide‐alkyne cycloadditions. Using nuclease digestion followed by LC−MS, we confirmed the presence of the glycosylated nucleosides, and characterized the chemical linkage. Next, we probed if glycosylation would affect the cellular response to Y5 RNA. We treated human foreskin fibroblasts in culture with the generated compounds. Key transcripts in the innate immune response were quantified by RT‐qPCR. We found that under our experimental conditions, exposure of cells to the Y5 RNA did not trigger an interferon response, and glycosylation of this RNA did not have an impact. Thus, we have identified a successful approach to chemically characterize synthetic glyco‐RNAs, which will be critical for further studies to elucidate how the presence of complex glycans on RNA affects the cellular response. Sialyl lactose was conjugated to Y5 RNA, and its product was characterized after nuclease digestion via liquid‐chromatography mass‐spectrometry. The conjugation products were also evaluated for their ability to trigger an interferon response in Human Foreskin Fibroblasts.