Minmin Li, Yuting Xiong, Yucheng Cao, Chen Zhang, Yuting Li, Hanwen Ning, Fan Liu, Han Zou, Xiaonong Li, Xianlong Ye, Yue Peng, Jiaming Zhang, Xinmiao Liang* and Guangyan Qing*

Nature Communications 2023, 14, 1737.

Glycans perform varied and crucial functions in numerous cellular activities. Diverse roles of glycans are matched by their highly complex structures, which derive from differences in composition, branching, regio- and stereochemistry, and modification. This incomparable structural diversity presents a huge challenge to the structural analysis of glycans. Recently, a research group led by Prof. Guangyan Qing and Prof. Xinmiao Liang from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS) has developed a glycan identification method based on nanopore single-molecule sensing through a glycan derivatization strategy. The study was published in Nature Communications(https://doi.org/10.1038/s41467-023-37348-5) on March 28th, 2023.

    Identifying and sequencing glycans using nanopore single-molecule techniques has sparked potential interests. However, nanopore sensing of glycans has achieved little progress over the past dozen years. Only a handful of cases that focused on either high molecular weight polysaccharides or some monosaccharides were reported. For smaller but structurally more diverse glycans with greater biological significance, single molecule detection with nanopore has not yet been achieved, largely because fast passage of glycan through nanopore cannot be sensed because of the small size and weak affinity of glycan with nanopore.

    To address the challenge, the researchers in this work introduced a derivatization strategy by linking an aromatic-type tag group to small glycans via a high-efficiency and facile reductive amination reaction. The resulting tagged glycan was successful sensed with a wild-type aerolysin nanopore by presenting strong nanopore blockage signals. The obtained scatter plot based on blockage current and dwell time as the fingerprint map by processing the nanopore single-molecule blockage events allows us to identify different glycan isomers, glycans with varying lengths, and branched simple glycans, either independently or with the use of machine learning methods. Site-direct mutagenesis of aerolysin and molecular docking studies revealed that multiple cation-π interactions between the aromatic tag of glycan with K238 residues of nanopore interface retard the translocation of tagged glycan and contributed to the sensing.

    “This study pushes the boundary of nanopore sensing beyond its traditional focus on nucleic acid and protein and activates its power in the glycomics and glycoscience field, which might pave the way towards nanopore glycan sequencing”, Prof. Qing said.

    This work was supported by the National Natural Science Foundation of China and the Innovation Grant of DICP.