【摘要】 <正>肝素（HP）和硫酸乙酰肝素（HS）是一种高度柔性的直链多糖^[1]，其基本结构单元由交替重复的二糖组成，通常包含一种己糖胺（如N-乙酰-D-葡萄糖胺，GlcNAc）和一种糖醛酸（如D-葡萄糖醛酸，GlcA）^[2].值得注意的是，HP/HS中普遍存在多样的磺酸化修饰，包括GlcNAc的N-磺酸化、 GlcA的2-O-磺酸化以及GlcNAc的6-O-磺酸化^[3].这些修饰赋予了HP/HS高度负电性，因而在与金属离子结合时，不同链段负电荷的空间排列可导致多种局部构象，甚至同一金属离子也可能结合于不同位点，使其能够与多种蛋白质发生特异性相互作用，从而在细胞生长^[4]、炎症反应^[5]和肿瘤抑制^[6]等过程中发挥关键作用.因此，揭示HP/HS与不同金属离子的相互作用机制，对于深入理解其生物学功能具有重要意义^[7～9].更多还原

【Abstract】 Heparin（HP） and heparan sulfate（HS） are highly anionic glycosaminoglycans that play essential roles in diverse biological processes through the metal ion-mediated interactions with proteins. However, direct characterization of HS-metal ion interactions at the single-molecule level in solution remains challenging. Nanopore electrochemistry is a label-free and single-molecule technique that enables direct analysis of individual molecular interactions. In this study, a T232K/K238Q Aerolysin nanopore featuring an enhanced electrostatic repelling barrier was utilized to probe the interactions between HS and different metal ions. By systematically varying the electrolyte cations(Na⁺, K⁺, and Ca²⁺), we have found that the metal ions significantly regulate HS translocation behavior by modulating its conformation, charge screening, and HS-nanopore interactions. Notably, in addition to Ca²⁺, which exhibits strong binding affinity to HS, the monovalent cations Na + and K + with similar physicochemical properties and weaker binding also induce distinct single-molecule signal signatures. Our results demonstrate that the nanopore-based single-molecule analysis holds strong potential to resolve the fine structural features of HS, enabling the characterization of sulfation site distributions, repeat-unit lengths, and related sequence features, and thereby providing a new avenue for high-resolution analysis of complex glycans.更多还原