A Proximity Hierarchical Template Adhesion Reaction for Tripartite Profiling of Glycosylation-Associated Biomolecular Information

Abstract Glycosylation of cell−surface biomolecules critically regulates cellular identity and function by modulating protein stability, receptor interactions, and immune signaling. However, existing proximity-based imaging strategies generally report composite glycosylation-associated signals, making it difficult to independently resolve signals from cell−surface glycans, total biomolecules, and glycosylation-associated biomolecules. Here, we develop a proximity hierarchical template adhesion reaction (PH-TAR) strategy to simultaneously amplify three independent molecular signals associated with glycosylation. In PH-TAR, an N-acetylneuraminic acid (Neu5Ac) recognition probe and a biomolecule-targeting probe independently report cell−surface Neu5Ac-associated signals and total biomolecule abundance, respectively. When glycan-associated and biomolecule-associated recognition modules achieve a productive spatial organization, DNA assembly is initiated to generate a third signal corresponding to glycosylation-associated biomolecular information through the TAR. PH-TAR enables simultaneous profiling of these three molecular features and reveals cellular heterogeneity that is masked by conventional composite glycosylation measurements. Using thyroid cell models, PH-TAR identified differential glycosylation-associated molecular signatures among normal and cancer cells, with altered glycoPD-L1-associated signals observed in thyroid cancer cells. Furthermore, PH-TAR was preliminarily extended to analyze glycosylation-associated RNA molecular information in HeLa cells. Collectively, PH-TAR provides a multiplexed molecular imaging strategy for resolving glycosylation-associated biomolecular heterogeneity and offers potential applications in immune checkpoint analysis, disease biomarker profiling, and glycosylation remodeling studies.