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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

基本信息

批准号：
8458100
负责人：
William Bruce Dunbar
金额：
$ 17.94万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-11 至 2015-01-31
项目状态：
已结题

项目摘要

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.

描述(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，并为从头开始的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综合的双纳米孔仪器，该仪器将提供结合或修饰核酸的分子物种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)开发一种双孔微流控芯片，并演示使用单个放大电压源在两个孔中捕获单个DNA。同时，开发一个集成的双放大系统，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捕获到两个毛孔中还没有得到证实，但拟议中的芯片成功的可能性很高。我们开发了一款针对纳米孔进行优化的集成放大器，提供了一个占地面积小、成本低的模块，提供了一种在单个芯片中实现多个气孔功能化的可扩展手段。目标2：(第2年)双孔芯片和双放大孔系统将有两个并行的重点应用：(A)以高时间分辨率测量通过纳米孔的酶的存在和转移时间，以及在两个孔中捕获和固定DNA。(B)通过电泳拉锯战(即通过竞争电压)和以高空间分辨率检测与DNA结合的蛋白质，展示DNA通过两个毛孔的受控运动。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带宽下)结合并沿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纳米孔对测序的敏感性，但DNA在纳米孔中的受控运动仍然是一个普遍的技术挑战。拟议的仪器将提供一种纯粹的电泳式运动控制方法，为每个毛孔提供分离的高信噪比电流测量，同时通过电泳式“拉锯战”实现DNA通过每个毛孔的缓慢输送。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。检测和定位单个DNA上的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，支持运动控制的架构可用于可集成到芯片中的任何一对孔，因此可适应针对单核苷酸敏感性进行优化的孔/底物的进步。