The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e ., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis.

中文翻译：

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在蛋白质通过纳米孔的单列运输过程中形成斑点


生物聚合物穿过纳米孔的运输是目前正在研究的一个重要的生物过程，用于快速分析 DNA 和蛋白质。虽然 DNA 的运输已为人们所普遍了解，但诱导未折叠蛋白质易位的方法直到最近才被发现（Yu 等人，2023；Sauciuc 等人，2023）。在这里，我们发现，在电渗驱动的多肽易位过程中，纳米孔内通常会形成斑点状结构，通常会阻碍它们的运输并阻止寻址单个氨基酸。这与 DNA 的电泳运输形成鲜明对比，后者此类结构的形成尚未见报道。不同纳米孔尺寸和形状之间的比较以及不同表面化学的修饰允许制定斑点形成的机制。我们还表明，可以通过使用 1) 入口和内径小于聚合物持久长度的纳米孔，2) 具有非粘性（即非芳香族）内表面的纳米孔，以及 3 ）中等易位速度。这些实验为理解限制下的多肽运输以及改进用于蛋白质分析的纳米孔的设计和工程提供了基础。