Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS

Current methods for the imaging of glycosylated RNAs (glycoRNAs) focus on limited types with identified RNA sequences, making the distribution of global glycoRNAs at single-cell and tissue levels largely unexplored. To address this gap, we developed a co-metabolic proximity labeling assay (COMPASS) for sequence-independent imaging of glycoRNAs. COMPASS employs metabolic chemical reporters (MCRs) to label glycans and RNAs, followed by click-chemistry-based dye conjugation to generate Forster resonance energy transfer (FRET) signals. The MCR-enabled RNA labeling allows for sequence-independent imaging, and FRET ensures accurate localization of glycoRNA distributions. COMPASS facilitates glycoRNA imaging across multiple cell lines, cerebral organoids, and mouse tissues. We observed the downregulation of glycoRNAs in metastatic lung nodules and upregulation in original regions of brain development in cerebral organoids, suggesting their potential association with carcinogenesis and neurogenesis. COMPASS illuminates previously invisible glycoRNAs and offers a comprehensive profile of glycoRNA distribution for studying their functions.