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

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，最近作为探测DNA和DNA结合酶之间相互作用的工具。这项研究的主要目的是以大肠杆菌DNA聚合酶I的Klenow片段为模型酶，开发一种基于纳米孔的单分子分析多核苷酸结合蛋白的仪器。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，从几个小时增加到几天。较小的双层膜还将缩短在施加电压从毫秒变到微秒后叠加在离子电流测量上的电容瞬变的持续时间。减少瞬时建立时间将反过来实现亚毫秒级的测量电流变化的检测，该变化是由捕获的大分子络合物的变化引起的，这些变化在电压变化期间或附近出现。 目的2：设计和实现过滤和控制逻辑，以增加对DNA-KF复合体寿命的控制权威，调节DNA在纳米孔上方结合的可用性，以及通过电压促进解离KF与DNA的结合。用贝叶斯统计推论实现福克-普朗克方程的数学建模框架，以构建酶-DNA复合体的解离势能轮廓。意义：所提出的控制方法将极大地提高不同电压模式下DNA-KF解离时间测量的吞吐量。该建模框架使用所得到的解离时间测量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的近似更具一般性。Kramers近似被用来估计电势分布的高度、宽度和逃逸速率，并假定电压模式恒定或缓慢变化。我们的框架不假设电压模式恒定或缓慢变化，并使用统计推断为轮廓模型参数分配不确定性。在双层直径缩小方面的进展(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。 公共卫生相关声明：需要新的单分子测量、操作和建模方法，以高分辨率揭示生物大分子的动力学和功能。提出的仪器和建模工具将提供以前没有实现的关于自由能曲线形状的细节，作为描述大肠杆菌DNA聚合酶I的Klenow片段从DNA解离的反应坐标的函数。使用适当设计的核酸靶标，该仪器在实验室和临床环境中都具有可靠检测DNA和RNA结合蛋白的广泛潜力。

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.