中国科学院上海药物所高召兵小组近日取得一项新成果。他们开发出了基于糖苷酶辅助纳米孔传感的聚糖测序。2025年1月2日，国际知名学术期刊《美国化学会杂志》发表了这一成果。

该课题组研究人员提出了，结合纳米孔技术与糖苷酶水解反应的聚糖测序的概念验证。通过纳米孔连续监测聚糖水解产物易位，所产生的特征电流的变化，可以根据糖苷酶的特异性准确识别聚糖序列。通过机器学习，该课题组研究人员将测序精度提高到98%以上，可以可靠地确定连续构建块和聚糖链的糖苷键，同时减少对操作人员专业知识的需求。该方法通过亲水相互作用色谱-高效液相色谱法（HILIC-HPLC），和质谱法（MS）在实际聚糖样品上进行了验证。

该研究团队实现了天然聚糖链中10个连续单元的测序，首次为纳米孔糖苷酶兼容系统在聚糖测序中可行性提供了证据。与传统方法相比，该方法的测序效率提高了5倍以上。此外，研究团队引入了“逆测序”的概念，该概念侧重于电信号变化而不是单糖鉴定。这消除了在假定的“正向水解”策略中通常需要的对聚糖指纹库的依赖。当“正向和反向水解测序策略”的挑战都得到解决时，这种方法将为在单分子水平上建立聚糖测序技术铺平道路。

研究人员表示，纳米孔是一种很有前途的聚糖分析传感器，可以准确鉴定复杂聚糖，为纳米孔测序奠定基础。然而，它们对连续聚糖测序的适用性尚未得到证实。

附：英文原文

Title: Glycan Sequencing Based on Glycosidase-Assisted Nanopore Sensing

Author: Guangda Yao, Bingqing Xia, Fangyu Wei, Jiahong Wang, Yuting Yang, Shengzhou Ma, Wenjun Ke, Tiehai Li, Xi cheng, Liuqing Wen, Yi-tao Long, Zhaobing Gao

Issue&Volume: January 2, 2025

Abstract: Nanopores are promising sensors for glycan analysis with the accurate identification of complex glycans laying the foundation for nanopore-based sequencing. However, their applicability toward continuous glycan sequencing has not yet been demonstrated. Here, we present a proof-of-concept of glycan sequencing by combining nanopore technology with glycosidase-hydrolyzing reactions. By continuously monitoring the changes in the characteristic current generated by the translocation of glycan hydrolysis products through a nanopore, the glycan sequence can be accurately identified based on the specificity of glycosidases. With machine learning, we improved the sequencing accuracy to over 98%, allowing for the reliable determination of consecutive building blocks and glycosidic linkages of glycan chains while reducing the need for operator expertise. This approach was validated on real glycan samples, with accuracy calibrated using hydrophilic interaction chromatography-high-performance liquid chromatography (HILIC-HPLC) and mass spectrometry (MS). We achieved the sequencing of ten consecutive units in natural glycan chains, which provided the first evidence for the feasibility of a nanopore-glycosidase-compatible system in glycan sequencing. Compared to traditional methods, this strategy enhances sequencing efficiency by over 5-fold. Additionally, we introduced the concept of ‘inverse sequencing’, which focuses on electrical signal changes rather than monosaccharide identification. This eliminates the reliance on glycan fingerprint libraries typically required in putative ‘forward hydrolysis’ strategies. When the challenges in both ‘forward and inverse hydrolysis sequencing strategies’ are addressed, this approach will pave the way for establishing a glycan sequencing technology at a single-molecule level.

DOI: 10.1021/jacs.4c12940

Source: https://pubs.acs.org/doi/abs/10.1021/jacs.4c12940