A dual-nanopore platform for sensing and control of polynucleotides

用于多核苷酸传感和控制的双纳米孔平台

• 批准号: 8593029
• 负责人: Trevor Justin Morin
• 金额: $ 15万
• 依托单位: TWO PORE GUYS, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8593029
• 关键词: Address; Binding; Binding Proteins; Budgets; Caliber; Characteristics; Chemistry; Complement; Coupled; Coupling; DNA; DNA Sequence; DNA Sequence Analysis; Detection; Devices; Emerging Technologies; Ensure; Enzymes; Filament; Funding; Genome; Genome Mappings; Genomics; Head; Housing; Immobilization; Individual; Label; Legal patent; Length; Manufacturer Name; Maps; Measurement; Measures; Medicine; Membrane; Methods; Microfluidics; Motion; National Human Genome Research Institute; Nucleotides; Optics; Pattern; Peptide Nucleic Acids; Performance; Phase; Polynucleotides; Positioning Attribute; Protein Binding; Proteins; Reading; Rec A Recombinases; Research; Resistance; Resolution; Sampling; Scheme; Single-Stranded DNA; Small Business Innovation Research Grant; Solutions; Technology; Testing; Variant; Vision; War; Work; cost; design; experience; genome sequencing; imaging modality; improved; instrument; lambda repressor; nanopore; next generation sequencing; particle; prototype; public health relevance; sensor; success; voltage

DESCRIPTION (provided by applicant): Nanopores are emerging technologies that offer the prospect of long-read (>100 kb) next-generation sequencing without the need for sample amplification. Such technology can complement existing short-read and high throughput sequencing platforms to reduce errors in de novo genome assembly and structural variant analysis, or entirely replace this technology if comparable error rates can be achieved; in either case, nanopores are positioned to make a considerable impact in the growing application of genomics in medicine. A long-standing challenge for nanopore sequencing has been to develop a method for controlling the rate of DNA through the pore to ensure accurate sequencing during nucleotide sensing. While leading research and commercial methods address this by using enzymes on top of each pore to control DNA motion through the pore, our patented method of DNA control eliminates the need for enzymes or chemistry, offering a considerable reduction in cost and instrument complexity. Our patented method involves the use of two nanopores to capture and control each DNA molecule. Independent voltage control across each pore permits electrophoretic "tug-of-war" to pull the DNA in competing directions, and thereby control the rate and direction of each DNA through the pores during sensing. Phase I funding will develop a prototype dual-nanopore device to demonstrate capture and rate control of individual dsDNA through both pores, and mapping of grosser features (i.e., binding proteins) using the two ionic nanopore current measurements. The long-term objective is to couple the control method with a single-nucleotide sensor for a reusable and chemistry-free platform for sequencing long single-stranded DNA. There are three aims: Aim 1. Develop a dual-pore microfluidic chip and housing. The proposed work leverages expertise in the fabrication of high performance nanopore-bearing membranes and devices. The chip design minimizes access resistances, which is critical to preserving sensing during control. Pores are sufficiently close (200 nm) for dual- pore capture of >1 kbp dsDNA, and sized (20-30 nm diam) for current sensing of dsDNA and bound proteins. Aim 2. Demonstrate capture and control of individual dsDNA. Preliminary analysis provides conditions under which the two ionic currents can sense DNA in each pore, and supports that the likelihood of second-pore capture following first-pore capture is high for the proposed geometry. Demonstrations of competing voltage control following capture will next be established, leveraging our expertise in voltage-control design. Aim 3. Demonstrate detection and localization of proteins bound to a single dsDNA molecule (2-50 kbp), achieving single protein resolution. We will build on the precedent for detecting RecA filaments formed on dsDNA using single nanopore devices, and also map the presence of phage lambda repressor which binds to specific sequences. The demonstrations support that the method has immediate commercial relevance, since mapping individual proteins (or, comparably, bound particle labels) on long dsDNA can be used for genome mapping, and without cameras or high resolution imaging methods.

描述（由申请人提供）：纳米孔是新兴技术，可提供长阅读（> 100 kb）下一代测序的前景，而无需样品放大。这种技术可以补充现有的短读和高通量测序平台，以减少从头基因组组装和结构变体分析中的错误，或者如果可以达到可比的错误率，则完全替换了该技术；无论哪种情况，纳米孔都可以对医学中基因组学不断增长的应用产生重大影响。纳米孔测序的长期挑战是开发一种通过孔控制DNA速率的方法，以确保在核苷酸传感期间进行准确的测序。尽管领先的研究和商业方法通过在每个孔顶上使用酶通过孔控制DNA运动来解决这一问题，但我们获得的DNA控制专利方法消除了对酶或化学的需求，从而大大降低了成本和仪器的复杂性。我们的专利方法涉及使用两种纳米孔捕获和控制每个DNA分子。跨每个孔的独立电压控制允许电泳“拖船”在竞争方向上拉动DNA，从而在传感过程中控制每个DNA的速率和方向。第一阶段的资金将开发一种原型双纳米孔设备，以通过两个毛孔来证明单个dsDNA的捕获和速率控制，并使用两个离子纳米孔电流测量值来映射毛（即结合蛋白）。长期目标是将控制方法与单核苷酸传感器搭配，以使用可重复使用的无化学平台来测序长的单链DNA。有三个目标：目标1。开发双孔微流体芯片和外壳。拟议的工作利用了高性能纳米孔膜和设备的专业知识。芯片设计可最大程度地减少访问电阻，这对于在控制过程中保留感应至关重要。毛孔足够接近（200 nm），用于双孔捕获> 1 kbp dsDNA，并且大小（直径为20-30 nm），用于电流的dsDNA和结合蛋白。 AIM 2。证明对单个DSDNA的捕获和控制。初步分析提供了两个离子电流可以在每个孔中感知DNA的条件，并支持提出的几何形状对第一孔捕获后第二孔捕获的可能性很高。接下来将建立捕获后竞争电压控制的演示，以利用我们在电压控制设计方面的专业知识。 AIM 3。证明与单个dsDNA分子（2-50 kbp）结合的蛋白质的检测和定位，可实现单蛋白分辨率。我们将建立在使用单个纳米孔设备在DSDNA上检测RECA丝的先例，并绘制与特定序列结合的噬菌体Lambda抑制剂的存在。该演示支持该方法具有立即商业相关性，因为可以将长dsDNA上的单个蛋白（或相当的结合粒子标签）映射用于基因组映射，并且没有摄像头或高分辨率成像方法。