In situ tracking of glycoRNAs on single-cell surface to reveal RNA heterogeneity and transport mechanism

Abstract Cell-surface glycoRNA plays a crucial role in cellular behavior, yet its RNA substrate signatures and membrane transport mechanism remain unclear. Here, we developed GlycoRNA-Lighted In situ Nano-Tracking (GLINT), an approach for the visualization of distinct RNA-specific glycosignatures. GLINT employs a modular, localized concatenated DNA circuit based on a proximity ligation-mediated dual hierarchical hybridization chain reaction (HCR). Metabolically labeled sialic acid probe and RNA-specific probes are combined into a dual-recognition module through proximity ligation. The module subsequently initiates a dual hierarchical HCR cascade, enabling ultrasensitive in situ tracking of U1, U3, U35a, Y5, and U8 glycoRNAs at the single-cell level. Leveraging GLINT technology, glycoRNAs were confirmed to be transported intracellularly via a SNARE protein-mediated secretory extracellular mechanism. Furthermore, the identification of ten subtypes of breast cancer cells was achieved based on the level of distinct RNA-specific glycosignatures on the cell surface. GLINT demonstrates great potential for tracking RNA-specific glycosignatures, offering a powerful tool for in situ cell subtyping and exploration of RNA-related glycosylation processes.