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分析DNA和结合或修饰DNA的酶的结构和动力学的可行工具，在单分子水平上，并为从头基因组测序提供了巨大的希望。该研究的主要目标是开发一种集成的双纳米孔仪器，该仪器将为结合或修饰核酸的分子物种的单分子分析提供新的模式，并将促进DNA纳米孔测序。有两个目标：目标1:（1年）开发一个双孔微流控芯片，并演示捕获一个单一的DNA在两个毛孔使用一个放大器电压源。同时，开发一个集成的双放大器系统，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：该仪器提供了一种耦合两个纳米孔来测量一个DNA分子的新方法。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。我们开发了一种针对纳米孔进行优化的集成放大器，提供了一种占地面积小、成本低的模块，可以在单个芯片上实现多个孔的功能化。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Aim 2中提出的双孔应用的先决条件。目标2:（第2年）双孔芯片和双放大器系统将有两个并行的重点应用：(a)在高时间分辨率下，通过纳米孔测量酶的存在和易位时间，以及在两个孔中捕获和固定的DNA。(b)通过电泳拉锯战（即通过竞争电压）和在高空间分辨率下检测与DNA结合的蛋白质，演示DNA通过两个孔的受控运动。意义：(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）许多结合和沿着DNA或RNA移动的酶，包括外切酶和聚合酶。虽然许多研究小组正在改进纳米孔对测序的敏感性，但通过纳米孔控制DNA的运动仍然是一个普遍的技术挑战。该仪器将提供一种纯电泳的运动控制方法，为每个孔提供解耦的高信噪比电流测量，同时通过电泳的“拔河”实现DNA通过每个孔的缓慢递送。独立的电流测量可以相互关联，以确定DNA在受控递送过程中的结构变化。单个DNA上单个蛋白质的检测和定位有助于筛选基因组上的转录因子。作为支持纳米孔测序的基础设施，运动控制架构可以用于任何可以集成到芯片中的孔对，因此可以适应针对单核苷酸敏感性进行优化的孔/底物的进步。

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.