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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Single-molecule Dna Detection With An Engineeredmspa Protein Nanopore
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Single-molecule Dna Detection With An Engineeredmspa Protein Nanopore

机译：使用Engineeredmspa蛋白纳米孔的单分子Dna检测

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摘要

Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electro-phoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor ofrn20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibulern100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.
机译：纳米孔作为单分子分析设备和生物物理模型系统具有广阔的前景，因为它们产生的离子电流障碍物包含有关靶分子的身份，浓度，结构和动力学的信息。耻垢分枝杆菌的孔蛋白MspA对环境压力具有显着的稳定性，并可根据其晶体结构进行合理修饰。此外，MspA具有短而狭窄的通道收缩结构，有望用于DNA测序，因为它可以改善穿过孔的ssDNA分子短片段的表征。通过消除通道收缩中的负电荷，我们设计并构建了一个MspA突变体，该突变体能够以电子方式检测和表征通过电泳驱动的单分子ssDNA。前庭区域中带有负电荷残基与正电荷残基的额外交换的第二个突变体表现出高20的相互作用速率，只需要一半的电压即可观察相互作用，并且使ssDNA驻留在前庭中的时间比前者长100倍。第一个突变体。我们的结果将MspA用作核酸分析的纳米孔，并突出了其作为单分子检测和表征应用的可工程化平台的潜力。

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