Functional Dual-Color Indicator To Achieve in Situ Visualization of Intracellular Glycosylation

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

Arrayed Profiling of Multiple Glycans on Whole Living Cell Surfaces

An array-based method for profiling and quantification of multiple glycans on whole living cell surfaces was developed through combining DNA encoding technology with DNA microarray. Using four kinds of lectins as the model to recognize four types of cell surface glycans, the specific barcode-lectin probes that contained the endonuclease cutting site were designed. The barcode-lectin probes had the DNA sequences complementary to four sequences immobilized on a DNA microarray, respectively. After the living cells were incubated with the mixture of four barcode-lectin probes, these probes could bind to cell surface through the specific interaction between the lectins and corresponding glycans. Thus, the glycans and their amounts could be profiled by releasing the barcodes from cell surface with endonuclease cleaving, binding the barcodes to DNA microarray with specific hybridization, and then producing the amplified fluorescence signal with hybridization chain reaction (HCR). The HCR was performed with two kinds of Cy5 labeled hairpins. The average amount of mannose, <i>N</i>-acetylgalactosamine, <i>N</i>-acetylglucosamine, and <i>N</i>-acetylneuraminic acid on BGC cell was obtained to be 6.8 × 10⁷, 3.8 × 10⁷, 2.1 × 10⁸, and 1.1 × 10⁷ moieties per cell, respectively. The proposed method possessed whole cell surface accessibility, powerful distinguishing capability, fast recognition kinetics, easy miniaturization, and high throughput without need of cell pretreatment or labeling. It could become a powerful tool for elucidation of the complex glycan-related biological processes

Liberation of Protein-Specific Glycosylation Information for Glycan Analysis by Exonuclease III-Aided Recycling Hybridization

A strategy for information liberation of protein-specific glycosylation is designed via an exonuclease III-aided recycling “hybridization and cleavage” process with glycan and protein probes, which achieves homogeneous quantification of cell surface glycan. The protein probe contains matching and spacer DNA sequences and an aptamer specific to target protein. The glycan probe contains a complementary sequence modified with neighboring fluorescein and quencher, a spacer sequence, and a dibenzocyclooctyne-amine end to bind azide-tagged glycan. Upon sequential binding to their targets, the complementary sequences of two probes approach enough for their hybridization, which leads to the cleavage of hybridized glycan probe by exonuclease III and followed recycling “hybridization and cleavage” process of protein probe with other adjacent glycan probes to release the labeled fluorescein for obtaining the information on protein-specific glycosylation. This protocol has been used to in situ quantify EpCAM-specific sialic acid on MCF-7 cell surface and monitor its variation during drug treatment. This work demonstrates a powerful quantification tool for research of glycosylation