Spatial Imaging of GlycoRNA in single Cells with ARPLA

Little is known about the biological roles of GlycoRNAs, a recently discovered class of glycosylated molecules, because of a lack of methods for visualizing them. We report Sialic Acid Aptamer and RNA in situ Hybridization-mediated Proximity Ligation Assay (ARPLA), to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts, as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggests that the surface glycoRNA is inversely associated with the tumor malignance and metastasis. Investigation of the relationship between glycoRNAs and monocyte-endothelial cell interactions suggested that glycoRNAs may mediate cell-cell interactions during the immune response. 

In this project, we provided all the raw images from the manuscript.