Nanopore sequencing of DNA with MspA

描述(申请人提供)：该项目的目标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和对突变的耐受性使该蛋白质成为理想的、廉价的和新颖的纳米孔测序开发平台。我们已经取得了令人振奋的结果，证明了我们的建议的可行性。我们设计并制造了可以通过DNA的MSPA突变体。重要的是，突变的MSPA已经几乎可以单独使用同传电流来解析单核苷酸了。MSPA的分子动力学模拟与实验符合得很好。专门为纳米孔测序实验建造了一个快速、低噪声的电流放大器原型。 我们的具体目标是：(I)合理设计、生产和测试MSPA突变体，以提高DNA碱基识别和降低移位速度；(Ii)利用分子动力学模拟来了解DNA如何与MSPA相互作用，并优化MSPA用于纳米孔测序；(Iii)构建单链蛋白质，以进一步提高DNA碱基的敏感性，并控制DNA在不对称MSPA孔中的运动；(Iv)构建高灵敏度的电子放大器和实用的双层仪器。 我们组建了一支由三个优秀实验室组成的团队，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，以实现这些目标。我们的目标是开发一种能够以低于1000美元的价格对人类基因组进行测序的系统。 与公共卫生相关：这三个大学团队正在从分枝杆菌中设计出一种新的孔，MSPA，用于纳米孔DNA测序。MSPA是纳米孔测序的理想形状。蛋白质孔对突变具有显着的耐受性，因此当DNA穿过它时，它可以被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.