Enrichment of glycoRNAs using galactose oxidase, hydrazide chemistry, and glycosidase digestion

Ribonucleic acid (RNA), essential for protein production and immune function, undergoes glycosylation, a process that attaches carbohydrates to RNA, creating unique glycoRNAs. These sugar-coated RNA molecules regulate immune responses and may be related to immune disorders. However, studying them is challenging due to RNAs fragility. Therefore, a robust method for identifying glycosylated RNA is important. To address this, we optimized methods for enriching and identifying glycoRNAs, opening doors to explore their potential interactions with immune receptors and tumor suppression. Our approach involved investigating factors such as preservation solutions, enzyme buffers, digestion temperature, and incubation time. We successfully achieved efficient digestion of both N-linked and O-linked glycoRNAs at room temperature using 25 mM ammonium bicarbonate, demonstrating the effectiveness of this method. Additionally, RNA preservation in RNAlater at -80{degrees}C allows controlled release of glycoRNAs within hours. While sequential digestion of different glycoRNA types is possible, significant degradation occurs after the first enzyme step. Thus, we recommend separate harvesting for each type of glycoRNA. These optimized protocols, utilizing SPCgRNA and TnORNA methods, pave the way for further research on N- and O-glycoRNAs in health and disease.