Heteromeric pore-forming proteins often contain recognition patterns or stereospecific selection filters. However, the construction of heteromeric pore-forming proteins for single-molecule sensing is challenging due to the uncontrollability of producing position isomers and difficulties in purification of regio-defined products. To overcome these preparation obstacles, we present an in situ strategy involving single-molecule chemical modification of a heptameric pore-forming protein to build a stereo- and regio-specific heteromeric nanopore (hetero-nanopore) with a subunit stoichiometric ratio of 3:4. The steric hindrance inherent in the homo-nanopore of K238C aerolysin directs the stereo- and regio-selective modification of maleimide derivatives. Our method utilizes real-time ionic current recording to facilitate controlled voltage manipulation for stoichiometric modification and position-based side-isomer removal. Single-molecule experiments and all-atom molecular dynamics simulations revealed that the hetero-nanopore features an asymmetric stereo- and regio-defined residue structure. The hetero-nanopore produced was characterized by mass spectrometry and single-particle cryogenic electron microscopy. In a proof-of-concept single-molecule sensing experiment, the hetero-nanopore exhibited 95% accuracy for label-free discrimination of four peptide stereoisomers with single-amino-acid structural and chiral differences in the mixtures. The customized hetero-nanopores could advance single-molecule sensing.

异聚成孔蛋白通常包含识别模式或立体特异性选择过滤器。然而，由于产生位置异构体的不可控性和区域定义产物的纯化困难，用于单分子传感的异聚成孔蛋白的构建具有挑战性。为了克服这些制备障碍，我们提出了一种原位策略，涉及对七聚体成孔蛋白进行单分子化学修饰，以构建亚基化学计量比为 3：4 的立体和区域特异性异聚纳米孔（异质纳米孔）。K238C 溶血素同纳米孔中固有的空间位阻指导马来酰亚胺衍生物的立体和区域选择性修饰。我们的方法利用实时离子电流记录来促进受控电压操作，以进行化学计量修饰和基于位置的侧异构体去除。单分子实验和全原子分子动力学模拟表明，异质纳米孔具有不对称的立体和区域定义的残基结构。通过质谱和单颗粒低温电子显微镜对产生的异质纳米孔进行了表征。在概念验证单分子传感实验中，异纳米孔对混合物中具有单氨基酸结构和手性差异的四种肽立体异构体的无标记区分表现出 95% 的准确率。定制的异质纳米孔可以推进单分子传感。