A dual-nanopore platform for sensing and control of polynucleotides

描述（由申请人提供）：纳米孔是新兴技术，其提供了无需样品扩增的长读段（>100 kb）下一代测序的前景。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异分析中的错误，或者如果可以实现相当的错误率，则完全取代这种技术;在任何一种情况下，纳米孔都可以对基因组学在医学中日益增长的应用产生相当大的影响。纳米孔测序的长期挑战是开发一种用于控制DNA通过孔的速率的方法，以确保在核苷酸感测期间的准确测序。虽然领先的研究和商业方法通过在每个孔的顶部使用酶来控制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要，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。跨每个孔的独立电压控制允许电泳“拔河”以在竞争方向上拉动DNA，从而在感测期间控制每个DNA通过孔的速率和方向。第一阶段的资金将开发一个原型双纳米孔装置，以证明通过两个孔捕获和控制单个dsDNA的速率，以及绘制更大的特征（即，结合蛋白）。长期目标是将控制方法与单核苷酸传感器耦合，用于对长单链DNA进行测序的可重复使用和无化学品的平台。有三个目标：目标1。研制了双孔微流控芯片及外壳。拟议的工作利用了高性能纳米孔轴承膜和设备制造的专业知识。芯片设计最大限度地减少了接入电阻，这对于在控制期间保持检测至关重要。孔足够接近（200 nm）以用于>1 kbp dsDNA的双孔捕获，并且尺寸（20-30 nm直径）用于dsDNA和结合蛋白的电流感测。目标2.证明单个dsDNA的捕获和控制。初步分析提供的条件下，两个离子电流可以感测在每个孔中的DNA，并支持第二孔捕获的可能性第一孔捕获后，所提出的几何形状是高的。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。演示结合单个dsDNA分子（2-50 kbp）的蛋白质的检测和定位，实现单个蛋白质分辨率。我们将建立在使用单个纳米孔装置检测在dsDNA上形成的RecA细丝的先例的基础上，并且还绘制了与特定序列结合的噬菌体λ阻遏物的存在。该演示支持该方法具有直接的商业相关性，因为在长dsDNA上映射单个蛋白质（或，不，结合的颗粒标记）可用于基因组映射，并且无需相机或高分辨率成像方法。

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.