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，它提供了无需样本扩增即可进行长读长（> 100 kb）下一代测序的前景。此类技术可以补充现有的短读长和高通量测序平台，以减少从头基因组组装和结构变异分析中的错误，或者如果可以实现可比较的错误率，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中不断增长的应用产生相当大的影响。纳米孔测序面临的长期挑战是开发一种控制 DNA 通过孔的速率的方法，以确保核苷酸传感过程中的准确测序。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物质的需求，从而大大降低了成本和仪器复杂性。我们的专利方法涉及使用两个纳米孔来捕获和控制每个 DNA 分子。每个孔上的独立电压控制允许电泳“拔河”将 DNA 拉向竞争的方向，从而在传感过程中控制每个 DNA 通过孔的速率和方向。第一阶段资金将开发原型双纳米孔装置，以展示通过两个孔对单个双链 DNA 的捕获和速率控制，以及使用两个离子纳米孔电流测量来绘制更粗大的特征（即结合蛋白）。长期目标是将控制方法与单核苷酸传感器结合起来，形成一个可重复使用且无需化学物质的平台，用于对长单链 DNA 进行测序。共有三个目标： 目标 1. 开发双孔微流控芯片和外壳。拟议的工作利用了高性能纳米孔膜和设备制造方面的专业知识。芯片设计最大限度地减少了访问阻力，这对于在控制过程中保持传感至关重要。孔足够近（200 nm），可以双孔捕获>1 kbp dsDNA，并且尺寸（20-30 nm直径）可以检测dsDNA和结合蛋白的电流。目标 2. 展示个体 dsDNA 的捕获和控制。初步分析提供了两个离子电流可以感测每个孔中 DNA 的条件，并支持对于所提出的几何结构来说，在第一孔捕获之后第二孔捕获的可能性很高。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. 展示与单个 dsDNA 分子 (2-50 kbp) 结合的蛋白质的检测和定位，实现单个蛋白质分辨率。我们将利用单纳米孔装置检测 dsDNA 上形成的 RecA 细丝的先例，并绘制与特定序列结合的噬菌体 lambda 阻遏物的存在图谱。这些演示表明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（或类似的结合颗粒标签）可用于基因组作图，并且无需相机或高分辨率成像方法。