A Dual-Nanopore Instrument for Single DNA Measurements and Control

描述（由申请人提供）：生物和固态纳米孔已经成为在单分子水平上分析DNA和结合或修饰DNA的酶的结构和动力学的可行工具，并为从头基因组测序提供了巨大的希望。拟议研究的广泛目标是开发一种集成的双纳米孔仪器，该仪器将提供结合或修饰核酸的分子种类的单分子分析的新模式，并将促进DNA纳米孔测序。有两个目标：目标1：（第1年）开发双孔微流控芯片，并使用单个放大器电压源演示在两个孔中捕获单个DNA。同时，开发一个集成的双放大器系统，该系统将允许对双孔芯片中的每个孔进行独立的电压控制和电流测量。意义：该仪器为耦合两个纳米孔测量一个DNA分子提供了一种新方法。将单个DNA捕获到两个孔中尚未得到证实，但对于所提出的芯片来说成功的可能性很高。我们已经开发出一种集成放大器，该放大器针对纳米孔进行了优化，提供了一种小尺寸和低成本的模块，该模块提供了在单个芯片中功能化多个孔的可扩展方法。由所提出的双放大器系统提供的独立电压控制和电流测量也是目标2中提出的双孔应用的先决条件。目标二：（第2年）双孔芯片和双放大器系统将并行地具有两个集中的应用：（a）以高时间分辨率测量酶通过纳米孔、沿着捕获并固定在两个孔中的DNA的存在和移位时间。(b)通过电泳拔河证明DNA通过两个孔的受控运动（即，通过竞争电压），以及以高空间分辨率检测与DNA结合的蛋白质。重要性：（a）作为单分子仪器，双孔设置将允许检测和测量（在~ 10 kHz带宽下）结合并沿着DNA或RNA移动的许多酶，包括核酸外切酶和聚合酶。(b)虽然许多研究小组正在改进用于测序的纳米孔灵敏度，但DNA通过纳米孔的受控运动仍然是一个普遍的技术挑战。所提出的仪器将提供运动控制的纯电泳方法，该方法为每个孔提供解耦的高信噪比电流测量，同时通过电泳“拔河”实现DNA通过每个孔的缓慢递送。“独立的电流测量可以互相关，以确定在受控输送过程中DNA的结构变化。沿着单个DNA检测和定位单个蛋白质可以促进沿着基因组筛选转录因子的努力。作为支持纳米孔测序的基础设施，运动控制使能架构可以用于可以集成到芯片中的任何一对孔，并且因此可以适应针对单核苷酸灵敏度优化的孔/基底的进步。 公共卫生相关性：生物和固态纳米孔是用于多核苷酸结合蛋白的单分子分析的可行工具，并为廉价的基因组测序提供了巨大的希望。所提出的仪器将通过提供一种控制DNA运动和速度通过两个纳米孔的新方法来推进测序工作并促进单分子分析的新模式，所述纳米孔主导否则随机的DNA运动，提供高信噪比检测电流，并且是完全电泳的（即，不需要操纵与DNA连接的辅助手段）。将开发用于控制双孔装置的专用集成电路，这代表了针对特定应用的纳米孔控制仪器的进步。

DESCRIPTION (provided by applicant): Biological and solid-state nanopores have emerged as viable tools for analyzing the structure and kinetics of DNA and enzymes that bind or modify DNA, at the single molecule level, and offer great promise for de novo genomic sequencing. The broad objective of the proposed research is to develop an integrated dual-nanopore instrument that will offer new modes of single molecule analysis of molecular species that bind or modify nucleic acids, and will facilitate DNA nanopore sequencing. There are two aims: Aim 1: (Year 1) Develop a dual-pore microfluidic chip and demonstrate capture of a single DNA in both pores using a single amplifier voltage source. In parallel, develop an integrated dual-amplifier system that will permit independent voltage control and current measurement for each pore in the dual-pore chip. Significance: The instrument provides a new method for coupling two nanopores to measure one DNA molecule. Capture of a single DNA into two pores has not been demonstrated, but has high likelihood of success for the proposed chips. We've developed an integrated amplifier that is optimized for nanopores, providing a small-footprint and low-cost module that provides a scalable means of functionalizing multiple pores in a single chip. Independent voltage control and current measurement afforded by the proposed dual-amplifier system is also a prerequisite for the dual-pore applications proposed in Aim 2. Aim 2: (Year 2) The dual-pore chip and dual-amplifier system will have two focused applications in parallel: (a) Measure the presence and translocation time of an enzyme through a nanopore, along a DNA captured and immobilized in both pores, at high temporal resolution. (b) Demonstrate controlled motion of a DNA through both pores, by electrophoretic tug-of-war (i.e., by competing voltages), and detection of proteins bound to the DNA at high spatial resolution. Significance: (a) As a single molecule instrument, the dual-pore setup will permit detection and measurement (at ~ 10 kHz bandwidth) of numerous enzymes that bind and move along DNA or RNA, including exonucleases and polymerases. (b) While many research groups are refining nanopore sensitivity for sequencing, controlled motion of the DNA through a nanopore remains a universal technical challenge. The proposed instrument will provide a purely electrophoretic method of motion control that provides decoupled high signal-to-noise current measurements for each pore, while achieving slow delivery of the DNA through each pore by electrophoretic "tug-of-war." The independent current measurements can be cross-correlated to identify structural variations in the DNA during controlled delivery. Detection and localization of individual proteins along a single DNA could facilitate efforts to screen for transcription factors along a genome. As an infrastructure to support nanopore sequencing, the motion control-enabling architecture can be employed for any pair of pores that can be integrated into a chip, and so can accommodate advances in pores/substrates that are optimized for single nucleotide sensitivity. PUBLIC HEALTH RELEVANCE: Biological and solid-state nanopores are viable instruments for single molecule analysis of polynucleotide-binding proteins and offer great promise for inexpensive genomic sequencing. The proposed instrument will advance sequencing efforts and facilitate new modes of single molecule analysis, by providing a new method to control DNA motion and speed through two nanopores that dominates the otherwise stochastic DNA motion, provides high signal-to-noise detection currents, and is entirely electrophoretic (i.e., does not require auxiliary means of manipulating an attachment to the DNA). A dedicated integrated circuit for control of the dual-pore device will be developed, representing an advance for application-specific nanopore control instrumentation.