Yunlong Chen

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, N-acetylgalactosamine, N-acetylglucosamine, and N-acetylneuraminic acid on BGC cell was obtained to be 6.8 × 10(7), 3.8 × 10(7), 2.1 × 10(8), and 1.1 × 10(7) 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.

Quantitative Screening of Cell‐Surface Gangliosides by Nondestructive Extraction and Hydrophobic Collection

A screening strategy involving designed extractors and collectors was used for the nondestructive quantitation of gangliosides on cell surfaces. The extractors were constructed by functionalizing maleimide silica bubbles with a DNA probe, which contains an endonuclease cleavage site and a boronic acid end to extract cell-surface sialic acid-containing compounds through simple centrifugation. After the extractors containing the extracted compounds were incubated with endonuclease, the released oligonucleotide-gangliosides were selectively collected by silanized collector bubbles through hydrophobic interactions. The in vitro fluorescent signals from the collectors were used for the quantitation of cell-surface gangliosides. By combining with sialidase cleavage, a protocol for the identification of ganglioside subtypes was developed. The successful monitoring of the regeneration of cell-surface gangliosides demonstrates the potential of this strategy in probing related biological processes.

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.

In Situ Cellular Glycan Analysis

Glycan decorates all mammalian cell surfaces through glycosylation, which is one of the most important post-modifications of proteins. Glycans mediate a wide variety of biological processes, including cell growth and differentiation, cell-cell communication, immune response, pathogen interaction, and intracellular signaling events. Besides, tumor cells aberrantly express distinct sets of glycans, which can indicate different tumor onsets and progression processes. Thus, analysis of cellular glycans may contribute to understanding of glycan-related biological processes and correlation of glycan patterns with disease states for clinical diagnosis and treatment. Although proteomics and glycomics have included great efforts for in vitro study of glycan structures and functions using lysis samples of cells or tissues, they cannot offer real-time qualitative or quantitative information, especially spatial distribution, of glycans on/in intact cells, which is important to the revelation of glycan-related biological events. Moreover, the complex lysis and separation procedures may bring unpredictable loss of glycan information. Focusing on the great urgency for in situ analysis of cellular glycans, our group developed a series of methods for in situ analysis of cellular glycans in the past 10 years. By construction of electrochemical glycan-recognizable probes, glycans on the cell surface can be quantified by direct or competitive electrochemical detection. Using multichannel electrodes or encoded lectin probes, multiple glycans on the cell surface can be dynamically monitored simultaneously. Through design of functional nanoprobes, the cell surface protein-specific glycans and intracellular glycan-related enzymes can be visualized by fluorescence or Raman imaging. Besides, some biological enzymes-based methods have been developed for remodeling or imaging of protein-specific glycans and other types of glycoconjugates, such as gangliosides. Through tracing the changes of glycan expression induced by drugs or gene interference, some glycan-related biological processes have been deduced or proved, demonstrating the reliability and practicability of the developed methods. This Account surveys the key technologies developed in this area, along with the discussion on the shortages of current methodology as well as the possible strategies to overcome those shortages. The future trend in this topic is also discussed. It is expected that this Account can provide a versatile arsenal for chemical and biological researchers to unravel the complex mechanisms involved in glycan-related biological processes and light new beacons in tumor diagnosis and treatment.