Identification of glycosylated nucleosides in small synthetic glyco‐RNAs

Abstract

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 glycoRNA 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.

In Situ Visualization of RNA-Specific Sialylation on Living Cell Membranes to Explore N-Glycosylation Sites

“…1 Recent research using blotting electrophoresis of RNA extracted from cells showed that small RNAs on the living cell surface were Nglycosylated and terminated with sialic acids (Sias). 7 Mass spectrometry, 8 high-performance anion-exchange chromatography, 9 and RNA sequencing 10 were also applied to the in vitro detection of glycoRNA. However, these in vitro analysis techniques discarded the location information on the sialylated RNA and could not provide the number of the glycosylation sites or the potential functions of the glycosylated RNAs (glycoRNAs).…”