A functional dual-color indicator is designed for in situ visualization of intracellular glycosylation. Using O-GlcNAcylation as model, the indicator is constructed on a poly-GlcNAc-coated gold nanoparticle (AuNP) by assembling dye labeled lectin (FSWGA) and then another dye-labeled GlcNAc (FGlcNAc) through the two opposite subunits of FSWGA. These dyes possess negligible overlapping emission and can be quenched by AuNP. In the presence of intracellular dissociated GlcNAc residue and O-GlcNAcylated proteins, the assembled FGlcNAc and the conjugate of FSWGA with FGlcNAc are released from AuNP through the dynamic competitive conjugation, which lights up the fluorescence of two dyes, respectively, and provides a simple technique for simultaneously monitoring the level of O-GlcNAcylated proteins and the total amount of GlcNAc groups in living cells. The practicality of the protocol for visually monitoring the biological pathway between intracellular O-GlcNAcylation and cell surface differentiation-related proteins demonstrates a convenient and powerful tool for research of glycosylation equilibrium and related biological processes.
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