Nanopore sequencing of DNA with MspA

描述（由申请人提供）：本项目的目的是设计一种新的蛋白质孔MspA，用于纳米孔DNA测序。MspA的短而窄的收缩，其对变性的极端稳定性及其对突变的耐受性使这种蛋白质成为理想的，廉价的和新型的纳米孔测序开发平台。 我们已经获得了令人兴奋的结果，证明了我们的建议的可行性。我们设计并制造了能传递DNA的MspA突变体重要的是，突变的MspA已经几乎可以单独使用共通过电流解析单个核苷酸。 MspA的分子动力学模拟结果与实验结果吻合较好。原型快速，低噪声电流放大器是专门为纳米孔测序实验。 我们的具体目标是（i）合理设计、生产和测试MspA突变体以改善DNA碱基识别并降低易位速度;（ii）使用分子动力学模拟来理解DNA与MspA的相互作用并优化用于纳米孔测序的MspA;（iii）构建单链蛋白以进一步改善DNA碱基敏感性和控制DNA在不对称MspA孔中的运动;（iv）构建单链蛋白以进一步改善DNA碱基敏感性和控制DNA在不对称MspA孔中的运动。（iv）构造高灵敏度的电子放大器和实用的双层装置。 为了实现这些目标，我们组建了三个优秀的实验室，他们在蛋白质科学、蛋白质模拟、单通道实验、分子生物学和仪器仪表方面具有互补的专业知识。我们的目标是开发一个系统，可以测序人类基因组低于1000美元。 公共卫生相关性：这个由三所大学组成的团队正在设计一种来自分枝杆菌的新型孔MspA，用于纳米孔DNA测序。MspA具有用于纳米孔测序的理想形状。蛋白质孔对突变具有显著的耐受性，因此当DNA通过它时，它可以精确地定制为对单个核苷酸敏感。

DESCRIPTION (provided by applicant): The objective of this project is to engineer a new protein pore, MspA, for nanopore DNA sequencing. MspA's short and narrow constriction, its extreme stability against denaturation and its tolerance to mutations make this protein an ideal, inexpensive and novel nanopore sequencing development platform. We have obtained exciting results that demonstrate the feasibility of our proposal. We designed and made MspA mutants that pass DNA. Importantly, mutated MspA can already nearly resolve single nucleotides using co-passing current alone. Molecular dynamics simulation of MspA agrees excellently with experiment. A prototype fast, low-noise current amplifier was built specifically for nanopore sequencing experiments. Our specific aims are to (i) rationally design, produce and test MspA mutants to improve DNA base recognition and reduce translocation speed; (ii) use molecular dynamics simulation to understand how DNA interacts with MspA and to optimize MspA for nanopore sequencing; (iii) construct a single chain protein to further improve DNA base sensitivity and control of DNA motion in an asymmetric MspA pore; (iv) construct a highly sensitive electronic amplifier and a practical bilayer apparatus. We have formed a team of three outstanding labs with complementary expertise in protein science, protein simulation, single-channel experiments, molecular biology, and instrumentation to realize these aims. It is our goal to develop a system that can sequence a human genome for under $1000. PUBLIC HEALTH RELEVANCE: This three university team is engineering a novel pore from mycobacteria, MspA, for nanopore DNA sequencing. MspA has an ideal shape for nanopore sequencing. The protein pore is remarkably tolerant of mutations so that it can be exactly tailored to be sensitive to individual nucleotides when DNA passes through it.