High-Voltage Biomolecular Sensing Using a Bacteriophage Portal Protein Covalently Immobilized Within a Solid-State Nanopore
High-Voltage Biomolecular Sensing Using a Bacteriophage Portal Protein Covalently Immobilized Within a Solid-State Nanopore
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DOI：
10.1101/2022.08.07.503088
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发表时间：
2022-08
期刊：
影响因子：
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通讯作者：
Mehrnaz Mojtabavi;S. Greive;A. Antson;M. Wanunu
中科院分区：
文献类型：
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作者：
Mehrnaz Mojtabavi;S. Greive;A. Antson;M. Wanunu
纳米孔作为无标记的单分子生物传感器用于生物分子中结构特征的电或光探测的应用已经被广泛探索。虽然生物纳米孔（膜蛋白和噬菌体门户蛋白）和固态纳米孔（薄膜和二维材料）已被广泛使用，但由于实施中的挑战，第三类纳米孔被称为混合纳米孔，其中人工膜取代蛋白质的有机支撑膜，仅很少研究。G20 c门户蛋白含有天然的DNA孔，其被病毒用于用病毒基因组DNA填充其衣壳。我们先前已经通过将G20 c门户蛋白“塞住”到SiNx纳米孔中来开发了无脂质混合纳米孔。在本文中，我们证明了通过合成纳米孔的化学官能化，固态孔和G20 c门户蛋白之间的共价键大大提高了混合孔的稳定性、寿命和电压弹性。此外，我们证明了电场驱动和电机蛋白介导的DNA分子通过这个混合孔的运输。我们的集成蛋白质/固态混合纳米孔可以作为在高电压下进行传感和测序的鲁棒且耐用的框架，与更常规的膜嵌入蛋白质平台相比，可能提供更高的分辨率、更高的信噪比和更高的通量。
The application of nanopores as label-free, single-molecule biosensors for electrical or optical probing of structural features in biomolecules has been widely explored. While biological nanopores (membrane proteins and bacteriophage portal proteins) and solid-state nanopores (thin films and two-dimensional materials) have been extensively employed, the third class of nanopores known as hybrid nanopores, where an artificial membrane substitutes the organic support membrane of proteins, has been only sparsely studied, due to challenges in implementation. G20c portal protein contains a natural DNA pore that is used by viruses for filling their capsid with viral genomic DNA. We have previously developed a lipid-free hybrid nanopore by “corking” the G20c portal protein into a SiNx nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the G20c portal protein considerably improves the hybrid pore stability, lifetime, and voltage resilience. Moreover, we demonstrate electric-field-driven and motor protein-mediated transport of DNA molecules through this hybrid pore. Our integrated protein/solid-state hybrid nanopore can serve as a robust and durable framework for sensing and sequencing at high voltages, potentially providing higher resolution, higher signal-to-noise ratio, and higher throughput compared to the more conventional membrane-embedded protein platforms.