AbstractGlycans are essential building blocks of life that are located at the outermost surface of all cells from mammals to bacteria, and even viruses. Cell surface glycans mediate multicellular communication in diverse biological processes and are useful as “surface markers” to identify cells. Various single-cell sequencing technologies have already been emerged that enable the high-throughput analysis of omics information, such as transcriptome and genome profiling on a cell-by-cell basis, which has advanced our understanding of complex multicellular interactions. However, there has been no robust technology to analyze the glycome in single cells, mainly because glycans with branched and heterogeneous structures cannot be readily amplified by polymerase chain reaction like nucleic acids. Therefore, in this study, we hypothesized that the generation of lectins conjugated with DNA barcodes (DNA-barcoded lectins) would enable the conversion of glycan information to gene information, which may be amplified and measured using DNA sequencers. This technology will enable the simultaneous analysis of glycan and RNA in single cells. Based on this concept, we began to develop a technology to analyze glycans and RNA in single cells in 2016. We first demonstrated that cell surface glycans of bulk cells can be profiled by DNA-barcoded lectins using next-generation sequencing. This method, designated Glycan-seq, was then extended to the profiling of single cells (scGlycan-seq). The resulting data were fully consistent with that obtained by flow cytometry as a gold standard. scGlycan-seq was then combined with scRNA-seq for the joint analysis of glycan and RNA in single cells, which was later referred to as scGR-seq. Using scGR-seq, we could acquire glycan and gene expression profiles of individual cells constituting heterogeneous cell populations, such as tissues. We further extended Glycan-seq to the profiling of the surface glycans of bacteria and even gut microbiota. Glycan-seq and scGR-seq are new technologies that enable us to elucidate the function of glycans in cell-cell and cell-microorganism communication, which extends glycobiology to the level of single cells and microbiomes.Concept of glycan profiling by sequencingHigh-throughput single-cell sequencing has been transformative for the identification and study of complex cell populations. Recently, simultaneous profiling of multiple types of molecules within a single cell has been developed in order to establish a more comprehensive molecular view of the cell. However, there have been no techniques to simultaneously analyze the glycome and transcriptome in single cells. One reason is that, unlike DNA and RNA, glycans cannot be amplified by methods such as the polymerase chain reaction (PCR). Therefore, we hypothesized that the generation of lectins conjugated with a DNA sequence (DNA-barcoded lectin) would enable the transformation of glycan information to gene information, which may be amplified and further measured using DNA sequencer, such as next-generation sequencer (Figure 1A). In addition, the simultaneous analysis of the glycome with other molecular profiles, such as the transcriptome, may be realized.Development of Glycan-seqWe began developing a technology to analyze glycans in single cells based on the above concept. Lectins with known specificity were conjugated to DNA oligonucleotides containing a barcode sequence for the identification of the specific lectin, thus enabling the lectins to be specifically identified by sequence analysis (Figure 1A)1). The lectins were conjugated via their amino groups with the photocleavable dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS), which allowed the efficient conjugation with 5’-azide-modified oligonucleotides. The oligonucleotides were released from the lectin following ultraviolet (UV) exposure2). We prepared a panel of 39 DNA-barcoded lectins that covered various glycans, such as sialylated, galactosylated, GlcNAcylated, mannosylated, and fucosylated glycans that are present in glycoconjugates1), whereas DNA-barcoded mouse and goat IgG were used as negative controls. In total, 41 DNA-barcoded proteins were incubated with 1 × 105 cells, and the unbound lectins were removed by washing (Figure 1B)2). Then, the bulk or single cells were separated into a PCR tube and exposed to UV light. After centrifugation, the supernatants containing the released DNA barcodes were 55Development of a nondestructive system to analyze disease-associated glycomesHiroaki TatenoCellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology
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