Modeling macromolecular transport through protein and solid-state nanopores

描述(由申请人提供)：迫切需要开发低成本和高质量的革命性技术来对哺乳动物大小的基因组进行测序，这激发了许多实验策略。其中最主要的是基于纳米孔的电泳法。虽然这项技术不断取得卓越的进展，但在实现非常高质量的测序和制造大规模并行测序设备的目标方面仍存在许多挑战。这些挑战源于基于纳米孔的DNA电泳的物理学，这需要从基本的科学观点来理解。这项拟议的研究涉及对纳米孔环境中DNA在电动力和流体动力以及酶的棘轮力影响下的行为的基本理解。我们将调查几个关键系统组件背后的挑战，以降低成本，提高速度，并提高哺乳动物大小基因组测序的准确性。主要的挑战涉及通过纳米孔减缓DNA的速度，特定离子的影响，DNA的构象波动，流体动力学产生的流场的影响，盐浓度梯度和电渗流，以及酶的加工性的波动。我们将使用聚合物物理、统计力学理论、计算机模拟和耦合非线性方程的数值计算的组合概念来研究DNA移位现象中的聚电解质统计和动力学、静电学和流体动力学。这项拟议的研究，虽然一般与所有基于纳米孔的实验有关，但将具体取决于：(A)减缓DNA和对由电压、温度、相同和数量的电解液、盐浓度梯度和孔表面图案介导的易位的基本了解，(B)控制酶棘轮易位的随机性和对酶活性波动、DNA构象波动和电泳漂移-扩散之间的耦合的基本了解，以及(C)设计数千个纳米孔的紧凑阵列的最佳配置，以便在单元之间没有串扰的情况下进行大规模并行DNA测序。

DESCRIPTION (provided by applicant): The urgent need to develop low-cost and high-quality revolutionary technologies for sequencing mammalian-sized genomes has inspired many experimental strategies. Chief among these is the nanopore-based electrophoresis. While excellent progress is continuously being made with this technique, there are many challenges in reaching the goals of very high quality sequencing and fabricating massively parallel sequencing devices. These challenges stem from the physics of nanopore-based electrophoresis of DNA which needs to be understood from a fundamental scientific point of view. The proposed research deals with fundamental understanding of the behavior of DNA in nanopore environments under the influence of electric and hydrodynamic forces, and ratcheting forces from enzymes. We will investigate the challenges underlying several key system components in the goal of reducing the cost, increasing the speed, and increasing the accuracy of sequencing mammalian-sized genomes. The major challenges deal with slowing down DNA through nanopore, effects of specific ions, conformational fluctuations of DNA, effects of flow fields arising from hydrodynamics, salt concentration gradients, and electroosmotic flow, and fluctuations in the processivity of enzymes. We will use a combination of concepts from polymer physics, statistical mechanics theory, computer simulations, and numerical computation of coupled nonlinear equations to address polyelectrolyte statistics and dynamics, electrostatics, and hydrodynamics in the phenomena of DNA translocation. The proposed research, while being generally relevant to all nanopore-based experiments, will be hinged specifically on: (a) slowing down DNA and fundamental understanding of translocation, mediated by voltage, temperature, identity and amount of electrolyte, salt concentration gradient, and patterns on pore surface, (b) controlling the stochasticity in enzyme-ratcheted translocation and fundamental understanding of coupling among fluctuations in enzyme processivity, DNA conformational fluctuations, and electrophoretic drift-diffusion, and (c) designing optimum configuration of compact arrays of thousands of nanopores for massively parallel DNA sequencing without crosstalk between the units.