Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application,Chemical Science

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Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application
Chemical Science ( IF 7.6 ) Pub Date : 2021-11-12 , DOI: 10.1039/d1sc04342k
Zhijie Chen ₁ , Zhenqin Wang ₁ , Yang Xu ₁ , Xiaochun Zhang ₁ , Boxue Tian ₁ , Jingwei Bai ₁

Affiliation

The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone's size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies.

中文翻译：

通过解旋酶马达控制 ssDNA 缀合肽通过耻垢分枝杆菌孔蛋白 A (MspA) 纳米孔的运动，用于肽测序应用

缺乏高效、低成本的测序方法长期以来一直是蛋白质研究和应用的重大瓶颈。近年来，纳米孔平台已成为一种快速且廉价的单分子核酸测序方法，但将其应用于蛋白质/肽测序的尝试却取得了有限的成功。在这里，我们报告了一种通过 MspA 纳米孔控制肽易位的策略，这可以作为链肽测序的第一步。通过将目标肽与解旋酶调节的手柄 ssDNA 缀合，我们实现了高达 17 个氨基酸 (aa) 的读取长度，并证明了区分不同电荷的氨基酸残基或不同磷酸化位点之间的可行性。分辨率的进一步提高可能需要对 MspA-M2 进行工程改造，以减小其收缩区的尺寸并拉伸纳米孔内的目标肽，以最大限度地减少随机热运动。我们相信，我们在这项研究中的方法可以显着加速基于纳米孔的肽测序技术的开发和商业化。

更新日期：2021-11-24

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