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（由申请人提供）：生物学和固态纳米孔已成为可行的工具，用于分析在单分子水平上结合或修饰DNA的DNA和酶的结构和动力学，并为从头开始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新模式，并将促进DNA纳米孔测序。有两个目标：AIM 1 ：（ 1年）开发双孔微流体芯片，并使用单个放大器电压源在两个孔中展示了单个DNA的捕获。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每个孔的独立电压控制和电流测量。意义：该仪器提供了一种偶联两个纳米孔以测量一个DNA分子的新方法。尚未证明将单个DNA捕获成两个孔，但对于拟议的芯片而言，成功的可能性很大。我们已经开发了一个集成的放大器，该放大器已针对纳米孔进行了优化，提供了一个小脚印和低成本模块，该模块提供了可扩展的单个芯片中多个毛孔功能功能化的方法。 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,在高时间分辨率下。（b）通过电泳拔河（即，通过竞争电压）和在高空间分辨率下结合与DNA结合的蛋白质的检测通过两种孔（即，通过两种孔）进行了控制。意义：（a）作为单个分子仪器，双孔设置将允许检测和测量（〜10 kHz带宽）沿DNA或RNA结合并移动的许多酶，包括外丝酶和聚合酶。（b）尽管许多研究小组都在提炼纳米孔灵敏度以进行测序，但通过纳米孔的DNA受控运动仍然是普遍的技术挑战。所提出的仪器将提供一种纯电泳的运动控制方法，为每个孔提供脱钩的高信号到噪声电流测量值，同时通过电泳“拖船战线”通过每个孔通过每个孔通过每个孔就能缓慢递送DNA。独立的电流测量值可以交叉相关，以识别受控递送过程中DNA中的结构变化。沿单个DNA的单个蛋白质的检测和定位可以促进沿基因组筛查转录因子的努力。作为支持纳米孔测序的基础架构，可以针对可以集成到芯片中的任何一对毛孔来采用运动控制结构，因此可以适应用于单核苷酸敏感性优化的孔/底物的进步。