Nanopore sequencing of DNA with MspA

使用 MspA 进行 DNA 纳米孔测序

• 批准号: 7938419
• 负责人: Aleksei Aksimentiev
• 金额: $ 27.61万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938419
• 关键词: Alabama; Amplifiers; Characteristics; Charge; Communities; Computers and Advanced Instrumentation; DNA; DNA Sequence; Data; Detection; Development; Devices; Electronics; Engineering; Ensure; Genes; Genus Mycobacterium; Goals; Human Genome; Hydrophobicity; Individual; Laboratories; Length; Lipid Bilayers; Location; Measures; Microfluidics; Modeling; Molecular Biology; Motion; Mutate; Mutation; Mycobacterium smegmatis; National Human Genome Research Institute; Noise; Nucleotides; Pore Proteins; Preparation; Proteins; Reader; Reading; Regulation; Research; Resolution; Resources; Sampling; Science; Shapes; Speed; Stream; Structure; System; Techniques; Technology; Testing; Time; Universities; Washington; Work; base; constriction; cost; design; design and construction; improved; instrumentation; link protein; molecular dynamics; mutant; nanopore; next generation; novel; nucleobase; porin; prototype; reconstitution; reconstruction; research study; response; simulation; single molecule

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.

概要 该项目的目标是为纳米孔DNA设计一种新的蛋白质孔MspA 测序MspA的短而窄的收缩，其极端的稳定性， 变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可以 已经几乎可以单独使用铜电流解析单个核苷酸。分子 MspA的动力学模拟与实验结果吻合较好。一个快速，低- 噪声电流放大器是专门为纳米孔测序实验而构建的。 我们的具体目标是：（i）合理设计，生产和测试MspA突变体，以改善 DNA碱基识别和降低易位速度;（ii）利用分子动力学 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改善DNA碱基 不对称MspA孔中DNA运动敏感性和控制;（iv）构建一个 高灵敏度电子放大器和实用的双层膜装置。 我们已经组建了一个由三个优秀实验室组成的团队，在以下方面具有互补的专业知识： 蛋白质科学，蛋白质模拟，单通道实验，分子生物学， 工具来实现这些目标。我们的目标是开发一个系统， 一个人类基因组的测序只要不到一千美元