A Dual-Nanopore Instrument for Single DNA Measurements and Control

用于单 DNA 测量和控制的双纳米孔仪器

• 批准号: 8229881
• 负责人: William Bruce Dunbar
• 金额: $ 23.07万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-11 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8229881
• 关键词: Address; Amplifiers; Architecture; Bacteriophages; Binding; Binding Proteins; Biological; Caliber; Coupling; DNA; DNA Binding; DNA delivery; Detection; Devices; Drops; Electronics; Environment; Enzymes; Evaluation; Exonuclease; Filament; Genome; Genomics; Government; Hydrolysis; Individual; Kinetics; Left; Length; Liquid substance; Measurement; Measures; Methods; Microfluidic Microchips; Microfluidics; Molecular Analysis; Motion; Noise; Nucleic Acids; Nucleotides; Pattern; Persons; Polymerase; Polynucleotides; Protein Binding; Proteins; RNA; Rec A Recombinases; Research; Research Infrastructure; Resolution; Screening procedure; Signal Transduction; Silicon; Source; Speed; Structure; System; Thick; Time; Variant; War; cost; improved; instrument; instrumentation; mutant; nanopore; novel; single molecule; solid state; success; tool; transcription factor; voltage

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.

描述（由申请人提供）：生物和固态纳米孔已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广阔的前景。拟议研究的总体目标是开发一种集成双纳米孔仪器，该仪器将为结合或修饰核酸的分子种类提供单分子分析的新模式，并促进 DNA 纳米孔测序。有两个目标： 目标 1：（第一年）开发双孔微流控芯片，并演示使用单个放大器电压源捕获两个孔中的单个 DNA。同时，开发一个集成双放大器系统，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，但所提出的芯片成功的可能性很高。我们开发了一种针对纳米孔进行了优化的集成放大器，提供了一种占用空间小且成本低廉的模块，该模块提供了在单个芯片中实现多个孔功能化的可扩展方法。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 2：（第 2 年）双孔芯片和双放大器系统将同时具有两个重点应用：（a）沿着捕获并固定在两个孔中的 DNA 测量酶通过纳米孔的存在和易位时间， 在高时间分辨率下。 (b) 通过电泳拔河（即通过竞争电压）展示 DNA 通过两个孔的受控运动，并以高空间分辨率检测与 DNA 结合的蛋白质。意义：(a) 作为单分子仪器，双孔装置将允许检测和测量（约 10 kHz 带宽）许多结合 DNA 或 RNA 并沿其移动的酶，包括核酸外切酶和聚合酶。 (b) 虽然许多研究小组正在提高纳米孔测序的灵敏度，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一种纯粹的电泳运动控制方法，为每个孔提供解耦的高信噪比电流测量，同时通过电泳“拔河”实现 DNA 通过每个孔的缓慢输送。独立的电流测量可以相互关联，以识别受控递送过程中 DNA 的结构变化。沿单个 DNA 检测和定位单个蛋白质可以促进沿基因组筛选转录因子的工作。作为支持纳米孔测序的基础设施，支持运动控制的架构可用于任何可集成到芯片中的孔对，因此可以适应针对单核苷酸敏感性优化的孔/基底的进步。 公共健康相关性：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仪器，并为廉价的基因组测序提供了巨大的前景。所提出的仪器将通过提供一种新方法来控制DNA运动和通过两个纳米孔的速度来推进测序工作并促进单分子分析的新模式，这两个纳米孔主导着随机DNA运动，提供高信噪比检测电流，并且完全是电泳的（即不需要操纵DNA附件的辅助手段）。将开发用于控制双孔装置的专用集成电路，代表专用纳米孔控制仪器的进步。