A Nanopore-based Instrument for Single Molecule Analysis of DNA-binding Proteins

基于纳米孔的 DNA 结合蛋白单分子分析仪器

• 批准号: 7940890
• 负责人: William Bruce Dunbar
• 金额: $ 20.45万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-27 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940890
• 关键词: Binding; Binding Proteins; Biochemical Reaction; Biological Process; Caliber; Catalysis; Clinical; Complex; DNA; DNA Binding; DNA Polymerase I; DNA Probes; DNA Sequence; DNA-Binding Proteins; DNA-Protein Interaction; Data; Detection; Dissociation; Enzymes; Equation; Escherichia coli; Evaluation; Event; Figs - dietary; Free Energy; Frequencies; Government; Height; Hour; Housing; Individual; Laboratories; Left; Lipid Bilayers; Logic; Macromolecular Complexes; Masks; Measurement; Measures; Mechanics; Modeling; Motion; Multienzyme Complexes; Nucleic Acids; Pattern; Persons; Polynucleotides; Protein Binding; Proteins; RNA-Binding Proteins; Reaction; Research; Research Personnel; Resolution; Shapes; Solutions; Time; Uncertainty; Variant; Width; authority; base; design; enzyme model; enzyme substrate; instrument; macromolecule; mathematical model; millisecond; nanopore; novel; novel strategies; nucleic acid binding protein; public health relevance; silicon nitride; single molecule; tool; voltage

DESCRIPTION (provided by applicant): Nanopores have shown great promise for DNA sequencing, and more recently as instruments for probing interactions between DNA and DNA-binding enzymes. The broad aim of the proposed research is to develop a nanopore-based instrument for single molecule analysis of polynucleotide-binding proteins, using the Klenow fragment (KF) of Escherichia coli DNA polymerase I as the model enzyme. There are two specific aims: Aim 1: Reduce the diameter of the lipid bilayer that houses the nanopore channel in steps, approaching a bilayer-free channel in a silicon nitride support. Significance: Bilayer diameter reduction will increase the stability and lifetime of the nanopore, from hours to days. Smaller bilayers will also shorten the duration of capacitive transients that are superimposed on the ionic current measurements following a change in applied voltage from milliseconds to microseconds. Reducing the transient settling time will in turn enable sub-millisecond detection of variations in the measured current, caused by changes in the captured macromolecular complex, that arise during or near a voltage change. Aim 2: Design and implement filtering and control logic to increase the control authority over the lifetime of the DNA-KF complex, regulating the availability of DNA for binding above the nanopore and the unbinding of KF from DNA by voltage-promoted dissociation. Implement the mathematical modeling framework of the Fokker-Planck equation with Bayesian statistical inference to construct the potential profile of dissociation for enzyme-DNA complexes. Significance: The proposed control approach will dramatically increase the throughput of DNA-KF dissociation time measurements under varying voltage patterns. The modeling framework uses the resulting distribution of dissociation time measurements to construct the shape of the free energy landscape as a function of the reaction coordinate, while permitting arbitrary voltage changing patterns. The framework is more general than Kramers' approximation. Kramers' approximation has been used to estimate the height, width and rate of escape of the potential profile, and assumes constant or slowly changing voltage patterns. Our framework does not assume constant or slowly changing voltage patterns, and uses statistical inference to assign uncertainty to profile model parameters. Advances in bilayer diameter reduction (Aim 1) will increase the range of voltage changing frequencies that can be used to dissociate the enzyme, while reliably detecting dissociation. Public Health Relevance Statement: Novel approaches for single molecule measurement, manipulation and modeling are required to uncover with high resolution the dynamics and function of biological macromolecules. The proposed instrument and modeling tools will provide details, previously not achieved, of the shape of the free energy curve as a function of the reaction coordinate describing the dissociation mechanics of the Klenow fragment of Escherichia coli DNA polymerase I from DNA. Using appropriately designed nucleic acid targets, this instrument has broad potential for reliable detection of DNA and RNA binding proteins, in both laboratory and clinical settings.

描述（由申请人提供）：纳米孔在DNA测序方面显示出巨大的前景，最近还显示出作为探测DNA和DNA结合酶之间相互作用的仪器的前景。拟议的研究的广泛目标是开发一种基于纳米孔的仪器，用于多核苷酸结合蛋白的单分子分析，使用大肠杆菌DNA聚合酶I的Klenow片段（KF）作为模型酶。有两个具体目标： 目标1：逐步减小容纳纳米孔通道的脂质双层的直径，接近氮化硅载体中的无双层通道。意义：双层直径减小将增加纳米孔的稳定性和寿命，从数小时到数天。较小的双层还将缩短电容瞬变的持续时间，所述电容瞬变在施加的电压从毫秒变化到微秒之后叠加在离子电流测量上。减少瞬态稳定时间又将使得能够亚毫秒检测由捕获的大分子复合物的变化引起的测量电流的变化，所述变化在电压变化期间或附近出现。 目标二：设计和实施过滤和控制逻辑，以增加DNA-KF复合物寿命期间的控制权限，调节DNA在纳米孔上方结合的可用性以及KF通过电压促进的解离从DNA中解结合。利用贝叶斯统计推断实现Fokker-Planck方程的数学建模框架，以构建酶-DNA复合物的潜在解离谱。重要性：所提出的控制方法将显着增加在不同电压模式下DNA-KF解离时间测量的吞吐量。建模框架使用解离时间测量结果的分布来构建作为反应坐标的函数的自由能景观的形状，同时允许任意的电压变化模式。该框架比Kramers近似更一般。Kramers的近似已被用来估计的高度，宽度和逃逸率的电位分布，并假定恒定或缓慢变化的电压模式。我们的框架不假设恒定或缓慢变化的电压模式，并使用统计推断来分配不确定性的配置文件模型参数。双层直径减小的进展（目标1）将增加可用于解离酶的电压变化频率的范围，同时可靠地检测解离。 公共卫生相关性声明：单分子测量、操纵和建模的新方法需要以高分辨率揭示生物大分子的动力学和功能。建议的仪器和建模工具将提供细节，以前没有实现，作为反应坐标的函数的自由能曲线的形状描述的大肠杆菌DNA聚合酶I的Klenow片段从DNA的解离力学。使用适当设计的核酸靶标，该仪器在实验室和临床环境中具有可靠检测DNA和RNA结合蛋白的广泛潜力。