Novel Methods For In Situ Analysis Of Cell Surface Glycans

Glycans cover on all cell surface of eukaryotes,which mediate a wide variety of important biological processes,such as cell adhesion,receptor activation,immune response,intercellular and intracellular signaling events.Glycan expression change on cell surface has been demonstrated to be associated with many diseases,such as cancers.So in situ and specific analysis of cell surface glycans show great importance for uncovering the relationship between diseases and glycans,understanding the role of glycan in physiological and pathological states of cells,and clinical diagnosis and treatment of cancer.In this dissertation,which is based on materials science,chemical biology,cytology and molecular biology and combined the technology of microarray,fluorescence microscopy and Raman spectroscopy,a series of novel nanoprobes and analytical methods have been developed for in situ detection of multiple glycans on cell surface,analysis of glycan morphology and monitoring of protein-specific glycosylation.The dissertation contains the following five parts:1.In situ tracing of cell surface sialic acid by chemoselective recognition to unload gold nanocluster probe from density tunable dendrimeric arrayA density tunable dendrimeric array to electrostatically assemble 3-aminophenylboronic acid(APBA)functionalized gold nanoclusters(AuNCs)as luminescent probes.The probe could bind SA groups on the cell surface by a chemoselective covalent linkage between APBA and SAs.Using BGC-823 cells as the model,through covalently binding to the cells,the electrostatically adsorbed APBA-AuNC probe could be unloaded from the dendrimer-modified slide,leading to a decrease of fluorescence intensity of the probe on the slide.The dendrimer density determined the spatial matching extent between SA epitopes and the probe-adsorbed dendrimer,which could be reflected by the unloading efficiency.SA density on the cell surface could be estimated by monitoring the change of unloading efficiency.2.Arrayed profiling of multiple glycans on whole living cell surfacesAn 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 6.8×107,3.8×107,2.1 ×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.3.Micro-competition system for Raman quantification of multiple glycans on intact cell surfaceA micro-competition system is designed for simultaneous quantification of multiple glycans on intact cell surfaces,by integrating two-surface-one-molecule competition with surface enhanced Raman scattering(SERS).The micro-competition is achieved among multiple-polysaccharide-coated gold nanostars functionalized silica bubbles,target cells and gold nanoprobes at a micron scale.The gold nanoprobes are prepared by coating distinct Raman molecules and lectins on gold nanoparticles for signal resolution and glycan recognition,respectively.The silica bubble surface serves as an artificial glycan surface and a SERS substrate.Upon the competitive recognition of lectin to the corresponding glycan,the gold nanoprobes can be specifically captured by the bubbles and cells in a homogeneous system,and the amounts of different gold nanoprobes on bubbles are simultaneously detected by SERS to reflect the corresponding glycan amounts on the cell surface.This micro-competition system with multiple quantification capability provides a powerful tool for investigation of the complex glycan-related biological processes.4.Zone-controllable SERS effect for Raman imaging of protein-specific glycanA zone-controllable SERS effect is presented for Raman imaging of protein-specific glycan on cell surface using two types of newly designed nanoprobes.The signal probe,prepared using a Raman signal molecule and dibenzocyclooctyne-amine to functionalize 10-nm Au nanoparticle,exhibits negligible SERS effect and can recognize and link the azide-tagged glycan via a click reaction.The substrate probe,an aptamer modified 30 or 40-nm Au nanoparticle,can specifically recognize the target protein to create efficient SERS zone on the target protein.By controlling the size of the substrate probe to match the expression zone of protein-specific glycan,the efficient SERS signal can be generated.This method has been successfully used to in situ image sialic acids on the target protein EpCAM on MCF-7 cell surface and monitor the expression variation of protein-specific glycans during drug treatment.In addi-tion,the concept of zone control can be used for in situ measuring the space distance of glycoproteins on cell surface.This protocol shows its prospect in uncovering glycan-related biological processes.5.Liberation of protein-specific glycan signal by exonuclease ?-aided recycling hybridizationA strategy for signal liberation of protein-specific glycan on whole cell surface is designed via exonuclease ?-aided recycling hybridization.This strategy achieves in situ homogeneous quantification of specific glycan by recognizing the protein and glycan with different probes.The protein probe is composed of matching and spacer DNA sequences and an aptamer specific to target protein.The glycan probe contains a complementary sequence modified with neighboring fluorescein and its quencher,a spacer sequence and a dibenzocyclooctyne-amine end to bind azide-tagged glycan formed by metabolic labeling.Upon their sequential recognition and binding to target protein and specific glycan,two probes approach enough for hybridization.In the presence of exonuclease III the hybridized glycan probe is cleaved to release the fluorescein,which leads to recycling hybridization of the protein probe with other adjacent glycan probes for signal liberation of protein-specific glycan.This protocol has been used to in situ quantify the target EpCAM-specific sialic acid on MCF-7 cell surface and monitor its expression variation during drug treatment,which demonstrates a powerful quantification tool for research of glycosylation of protein in biological processes.