Orthogonal Chemical Encoding Enables Sequence‐Independent GlycoRNA Imaging in Organoids and Tissues

ABSTRACT Current methods for imaging glycosylated RNAs (glycoRNAs) rely on sequence‐specific recognition or bulky DNA nanostructures, which compromise spatial fidelity and largely confine applications to simplified cell systems. Here, we report a Co valent M olecular P roximity Ass ay (COMPASS) that enables sequence‐independent visualization of glycoRNAs in complex physiological contexts. COMPASS integrates orthogonal encoding with proximity‐gated signal readout. Through chemoselective precursor uptake, azide groups are installed on terminal sialic acids and alkyne groups on nascent uridine residues, establishing a dual‐encoded chemical framework. Independent analysis confirmed the efficient installation of both functionalities. Subsequent dual‐color click labeling introduces donor and acceptor fluorophores to these orthogonal handles, producing sensitized acceptor emission when they are located in the same glycoRNA molecule. Accordingly, COMPASS reports the metabolically labeled, sialylated, uridine‐containing glycoRNA population generated during the labeling window. This design eliminates the need for bulky nucleic acid probes, reduces probe‐induced signal spreading, and enables glycoRNA imaging across diverse systems, including cell cultures, cerebral organoids, and mouse tissues. COMPASS revealed spatially heterogeneous glycoRNA‐associated signals, including relatively stronger signals near lumen‐adjacent VZ‐like regions of cerebral organoids and lower signals in pulmonary metastatic nodules. These observations establish spatial associations with neurodevelopmental and tumor‐associated states.