Modeling Macromolecular Transport through Protein Channels and Nanopores

描述(由申请人提供)：我们建议开发所需的大分子模型，以便对带电聚合物分子如何通过蛋白质通道和固态纳米孔进行基本的分子理解。这样的分子理解对于探索聚合物移位的基本过程和成功开发高速DNA序列检测是至关重要的。由于需要立即和廉价地对大量基因组进行测序，最近报道了非常令人兴奋的单分子电生理学实验。虽然这些实验是以测序技术为基础的，但它们是更复杂的生物易位过程的体外模拟。即使在这样简单的条件下，这些实验的结果也非常令人费解，需要了解聚合物物理，并结合化学特性。我们建议实现在大长度和时间尺度上有效的聚合物物理概念，并结合布朗动力学模拟在较小长度和时间尺度上考虑细节。本提案涉及对以下方面的基本理解：(1)二级结构对单链DNA/RNA通过α-溶血素孔的移动机制和对离子电流特征的影响，(2)酶调节的DNA在孔中的移位以优化孔中聚合物的速度以实现单碱基水平的同时询问，以及(3)dsDNA在固态纳米孔中的构象以实现测序策略所需的稳定运动并了解半柔性dsDNA分子在空间约束下的电动力学行为。我们独特的理论、模拟和与活跃的实验者合作的结合，将对了解聚合物易位、DNA/RNA和蛋白质的高速测序、信号转导、生物战剂的筛选、药物诊断以及疾病及其控制的大分子方面产生直接而深远的影响。

DESCRIPTION (provided by applicant): We propose to develop the macromolecular modeling needed for a fundamental molecular understanding of how electrically charged polymer molecules move through protein channels and solid-state nanopores. Such a molecular understanding is crucial for probing the fundamental process of polymer translocation and for a successful development of high-speed detection of DNA sequences. Stimulated by the need to sequence enormous number of genomes immediately and inexpensively, very exciting single-molecule electrophysiology experiments have recently been reported. Although couched in the technology of sequencing, these experiments are the in vitro analogs of the more complex biological translocation processes. Even under such simpler conditions, the results of these experiments are very puzzling and require an understanding of polymer physics, in combination with chemical specificities. We propose to implement polymer physics concepts valid at large length and time scales, in conjunction with Brownian Dynamics simulations accounting for details at smaller length and time scales. The present proposal addresses a fundamental understanding of (1) effects of secondary structures on the mechanism of movement of single stranded DNA/RNA through alpha-hemolysin pores and on the ionic current signatures, (2) enzyme-modulated DNA translocation through pores to optimize the speed of the polymer in the pore to enable simultaneous interrogation at a single-base level, and (3) conformations of dsDNA inside solid-state nanopores to enable a steady movement required for sequencing strategies and to understand the electrodynamical behavior of semiflexible dsDNA molecules under spatial constraints. Our unique combination of theory, simulations, and collaborations with active experimentalists, will have a direct and profound impact on understanding of polymer translocation, high-speed sequencing of DNA/RNA and proteins, signal transduction, screening of biological warfare agents, pharmaceutical diagnostics, and macromolecular aspects of diseases and their control.