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）：纳米孔是一种新兴技术，可以在不需要样品扩增的情况下进行长读（> 100kb）下一代测序。该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异分析中的错误，或者如果可以达到相当的错误率，则完全取代该技术；无论哪种情况，纳米孔都将对基因组学在医学上日益增长的应用产生相当大的影响。纳米孔测序的一个长期挑战是开发一种方法来控制DNA通过孔的速率，以确保在核苷酸传感过程中准确测序。虽然领先的研究和商业方法通过在每个孔顶部使用酶来控制DNA通过孔的运动来解决这个问题，但我们的DNA控制专利方法消除了对酶或化学的需求，大大降低了成本和仪器的复杂性。我们的专利方法包括使用两个纳米孔来捕获和控制每个DNA分子。每个孔的独立电压控制允许电泳“拔河”将DNA拉向竞争方向，从而在传感过程中控制每个DNA通过孔的速率和方向。第一阶段的资金将用于开发一个双纳米孔原型装置，以演示通过两个孔捕获和控制单个dsDNA的速率，并使用两个离子纳米孔电流测量来绘制更大的特征（即结合蛋白）。长期目标是将控制方法与单核苷酸传感器偶联，为长单链DNA测序提供可重复使用的无化学平台。有三个目标：研制一种双孔微流控芯片和外壳。提出的工作利用了制造高性能纳米孔膜和设备的专业知识。芯片设计最大限度地减少了访问电阻，这是在控制过程中保持传感的关键。孔足够接近（200 nm），可以捕获bbb10 - 1 kbp的双孔dsDNA，并且孔径（20-30 nm直径）可用于dsDNA和结合蛋白的电流传感。目标2。演示捕获和控制单个dsDNA。初步分析提供了两个离子电流可以在每个孔中感应DNA的条件，并支持在第一孔捕获之后捕获第二孔的可能性对于所提出的几何结构来说很高。接下来将利用我们在电压控制设计方面的专业知识，建立捕获后竞争电压控制的演示。目标3。展示结合在单个dsDNA分子（2-50 kbp）上的蛋白质的检测和定位，实现单个蛋白质的分辨率。我们将建立在使用单纳米孔设备检测dsDNA上形成的RecA细丝的先例的基础上，并绘制与特定序列结合的噬菌体λ抑制因子的存在。该演示支持该方法具有直接的商业意义，因为在长dsDNA上绘制单个蛋白质（或类似的结合粒子标签）可用于基因组绘制，而无需相机或高分辨率成像方法。