GlycoRNAs have recently been discovered as a new class of molecules for glycosylation. However, compared to proteoglycans, glycoproteins, and glycosphingolipids, much less is known about the distributions, abundances, and roles of glycoRNAs in biological processes, mainly due to a lack of methods to visualize glycoRNAs in living organisms. To address this issue, we are developing an imaging method named Sialic Acid Aptamer and RNA in situ Hybridization-mediated Proximity Ligation Assay (ARPLA) to visualize glycoRNAs with high sensitivity and selectivity at subcellular levels. ARPLA allows in situ glycoRNA imaging across multiple cell lines for a variety of sequences without the need for metabolic labeling on glycans. The signal output of ARPLA occurs only when the dual recognition of glycan (by aptamer) and RNA (by hybridization) triggers in situ ligation and then rolling circle amplification (RCA) that generates signal outputs by binding fluorophore-labeled oligonucleotide reporters. Using this method, for the first time, we visualized the intracellular and membranal distribution of glycoRNAs. We validated colocalizations between glycoRNA and V-SNARE vesicles intracellularly as well as lipid rafts on the membrane surfaces, indicating the transportation of glycoRNA via exocytosis vesicles and functional relationship with lipid rafts. Moreover, we observed a decrease in glycoRNAs during breast cancer malignant transformation and innate immune cell differentiation; and an increase upon lipopolysaccharide (LPS) stimulation in the polarized macrophage. A glycoRNA function in mediating monocyte-endothelial cell interactions was also discovered, suggesting a role of glycoRNA in mediating cell-cell interactions during the immune response. The development of ARPLA expanded the toolbox for glycoRNA research and will pave the way to investigate the potential roles of glycoRNAs in cancer pathogenesis and cancer-immune interactions.

In situ GlycoRNA imaging through aptamer and RNA hybridization-based proximity ligation assay

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