Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA
Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA
复制标题
具有扩展和截短 β-发夹的修饰外膜 Protein-G 纳米孔用于识别双链 DNA
DOI：
10.1021/acsanm.1c04417
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发表时间：
2022
影响因子：
5.9
通讯作者：
Kamiya Koki
中科院分区：
文献类型：
--
作者：
Tosaka Toshiyuki;Kamiya Koki
通过生物纳米孔检测单链DNA等单分子和其他小分子是分析DNA序列和DNA形状以及疾病诊断的有力手段。生物纳米孔的固定直径限制了通过它们转移的生物分子的大小。尽管一些纳米孔，如ClyA、Frc、Phi29p和γ-溶血素已被证明可以移位双链DNA，但利用这些天然的生物纳米孔很难识别双链DNA和三向连接DNA之间的区别。OmpG是一种主要的外膜蛋白，它形成了一个由14条β链组成的纳米孔。在这里，我们创建了一种改进的OmpG，它扩展和截断β发夹，与野生型(WT)OmpG纳米孔相比，允许生成小的或大的纳米孔。在不同分子质量的聚乙二醇存在或不存在的情况下，测量了不同改性的OMPG的电流幅值的变化，以确定改性的OMPG的孔径。最后，我们演示了使用OmpG WT或突变的OmpG纳米孔来检测依赖于纳米孔大小的不同结构的DNA(分支DNA)。洞察孔直径的变化对于形成准确的孔直径将是至关重要的，该孔直径用于检测各种类型的单个生物分子以及对DNA、多肽和蛋白质进行测序。
The detection of single molecules such as single-stranded DNA (ssDNA) and other small molecules through biological nanopores is a powerful approach for analyzing DNA sequences and DNA shapes, as well as for disease diagnostics. The fixed diameter of biological nanopores restricts the size of biomolecules translocated through them. Although some nanopores such as ClyA, FraC, Phi29p, and γ-hemolysin have been shown to translocate double-stranded DNA (dsDNA), identifying the difference between dsDNA and three-way junction DNA is difficult using these native biological nanopores. OmpG, a major outer membrane protein, forms a nanosized pore with 14 β-strands. Here, we create a modified OmpG that expands and truncates β-hairpins, allowing the generation of small or large nanopores compared to that of wild-type (WT) OmpG nanopores. To determine the pore diameters of modified OmpGs, the change in the current amplitude of the various modified OmpGs was measured in the presence or absence of poly(ethylene glycol) at different molecular weights. Finally, we demonstrated the detection of various structures of DNA (branched DNA) depending on the nanopore size using OmpG WT or mutated OmpG nanopores. Insights into the changes in pore diameters will be crucial to form precise pore diameters for the detection of various types of single biomolecules and for sequencing DNA, peptides, and proteins.