Nanopore sequencing of DNA with MspA

使用 MspA 进行 DNA 纳米孔测序

• 批准号: 8314137
• 负责人: Aleksei Aksimentiev
• 金额: $ 74.34万
• 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: 2009-09-23 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8314137
• 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; public health relevance; reconstitution; reconstruction; research study; response; simulation; single molecule

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.

描述（由申请人提供）：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成为理想的，廉价的新型纳米孔测序开发平台。我们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传递电流来分解单个核苷酸。MspA分子动力学模拟结果与实验结果吻合较好。为纳米孔测序实验设计了一种快速、低噪声电流放大器。