Mojtabavi Mehrnaz, Greive Sandra J, Antson Alfred A, Wanunu Meni
Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States.
York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, U.K.
J Am Chem Soc. 2022 Dec 14;144(49):22540-22548. doi: 10.1021/jacs.2c08514. Epub 2022 Dec 1.
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. 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 portal protein into a SiN nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the 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 nanopore. Our integrated protein/solid-state device 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.
纳米孔作为无标记的单分子生物传感器，用于对生物分子的结构特征进行电学或光学探测，这一应用已得到广泛探索。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）已被广泛应用，但第三类纳米孔，即混合纳米孔，由于实施过程中的挑战，研究较少。在混合纳米孔中，人工膜替代了蛋白质的有机支撑膜。门户蛋白包含一个天然的DNA孔，病毒利用它将病毒基因组DNA装入衣壳。我们之前通过将门户蛋白“塞”入氮化硅纳米孔中，开发出了一种无脂质的混合纳米孔。在此，我们证明，通过对合成纳米孔进行化学功能化，固态孔与门户蛋白之间的共价连接显著提高了混合孔的稳定性、寿命和电压耐受性。此外，我们还展示了DNA分子通过这种混合纳米孔的电场驱动和马达蛋白介导的转运。我们的集成蛋白/固态装置可作为一个强大而耐用的框架，用于在高电压下进行传感和测序，与更传统的膜嵌入蛋白平台相比，可能提供更高的分辨率、更高的信噪比和更高的通量。
