Nanopore sequencing of DNA with MspA

内容提要 该项目目标是为纳米孔DNA设计一种新的蛋白质孔MSPA 测序。MSPA的短而窄的收缩，其极高的稳定性 变性和对突变的耐受性使这种蛋白质成为理想的、廉价的和 新型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 设计并制造了能通过DNA的MSPA突变体。重要的是，突变的MSPA可以 仅用共吸电流就几乎可以分辨出单核苷酸。分子 MSPA的动力学仿真与实验结果吻合较好。一种快速、低成本的原型- 噪声电流放大器是专门为纳米孔测序实验而设计的。 我们的具体目标是：(I)合理设计、生产和测试MSPA突变体，以改进 DNA碱基识别和降低转位速度；(Ii)使用分子动力学 模拟以了解DNA如何与MSPA相互作用并优化MSPA 纳米孔测序；(Iii)构建单链蛋白以进一步完善DNA碱基 DNA在不对称MSPA孔中运动的敏感性和控制性；(Iv)构建 高灵敏度电子放大器和实用的双层器件。 我们已经组建了一支由三个优秀实验室组成的团队，在 蛋白质科学、蛋白质模拟、单通道实验、分子生物学和 实现这些目标的手段。我们的目标是开发一种能够 为人类基因组测序，花费不到1000美元。

SYNOPSIS 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 copassing 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.