Abstract：
Glycans play vital roles in nearly all life processes of multicellular organisms, and understanding these activities is inseparable from elucidating the biological significance of glycans. However, glycan research has lagged behind that of DNA and protein due to the challenges posed by structural heterogeneity and isomerism (i.e., structures with equal molecular weights) the lack of high-efficiency structural analysis techniques. Nanopore technology has emerged as a sensitive single-molecule biosensor, shining a light on glycan analysis. However, a significant number of glycans are small and uncharged, making it challenging to elicit identifiable nanopore signals. Here we introduce a R-binaphthyl tag into glycans, which enhances the cation-π interaction between the derivatized glycan molecules and the nanopore interface, enabling the detection of neutral glycans with an aerolysin nanopore. This approach allows for the distinction of di-, tri-, and tetrasaccharides with monosaccharide resolution and has the potential for group discrimination, the monitoring of enzymatic transglycosylation reactions. Notably, the aerolysin mutant T240R achieves unambiguous identification of six disaccharide isomers, trisaccharide and tetrasaccharide linkage isomers. Molecular docking simulations reveal that multiple noncovalent interactions occur between residues R282, K238, and R240 and the glycans and R-binaphthyl tag, significantly slowing down their translocation across the nanopore. Importantly, we provide a demonstration of the kinetic translocation process of neutral glycan isomers, establishing a solid theoretical foundation for glycan nanopore analysis. The development of our technology could promote the analysis of glycan structural isomers and has the potential for nanopore-based glycan structural determination and sequencing.
摘要：
聚糖在多细胞生物的几乎所有生命过程中发挥着至关重要的作用，理解这些活性与阐明聚糖的生物学意义是分不开的。然而,由于结构异质性和异构现象带来的挑战，聚糖研究落后于DNA和蛋白质的研究(即，具有相同分子量的结构）缺乏高效的结构分析技术。纳米孔技术已经成为一种敏感的单分子生物传感器，在聚糖分析上发光。然而,大量的聚糖是小的和不带电的，这使得引发可识别的纳米孔信号具有挑战性。在这里，我们将R-联萘标记引入聚糖中，这增强了衍生化聚糖分子与纳米孔界面之间的阳离子-π相互作用，使中性聚糖的检测与气溶素纳米孔。这种方法允许区分di-，三-,和四糖具有单糖分辨率，并具有群体歧视的潜力，酶促糖基化反应的监测。值得注意的是,气溶素突变体T240R实现了六种二糖异构体的明确鉴定，三糖和四糖连接异构体。分子对接模拟显示，残基R282、K238和R240与聚糖和R-联萘标记之间发生多种非共价相互作用，显著减缓它们跨纳米孔的易位。重要的是,我们提供了中性聚糖异构体的动力学转移过程的演示，为聚糖纳米孔分析奠定了坚实的理论基础。我们技术的发展可以促进聚糖结构异构体的分析，并具有用于基于纳米孔的聚糖结构测定和测序的潜力。
