Nanofluidic Devices for Studying Assembly of Single Virus Particles

描述（由申请人提供）：我们将建立通用方法来确定病毒和自组装纳米颗粒的组装途径。我们将使用B型肝炎病毒（HBV）衣壳组装来证明这些方法。电阻脉冲传感允许通过识别单个颗粒和组装中间体来对组装进行真实的时间监测。了解病毒组装的机制不仅需要了解前体和最终产物的结构，还需要获得中间体。在涉及许多稀有中间体的情况下，系综方法使它们变得模糊，使得病毒组装类似于双态反应。 HBV已经急性感染了超过20亿人;大约3.6亿人患有慢性HBV;每年有近100万人死于HBV相关的肝病。HBV二十面体衣壳的组装已被确定为抗病毒治疗的新靶点。HBV组装和抗病毒组装效应物的行为（和发展）相对较好地理解，主要源于Zlotnick实验室的工作，但尚未直接观察和表征溶液中的关键早期中间体。组装的计算模型表明，所观察到的动力学反映了早期建立的星座的中间体需要支持衣壳形成。成核步骤和早期中间体被认为在体内募集病毒组分中起作用。抗病毒组装效应物过度刺激成核，扭曲中间体的分布，并且经常扭曲它们的结构。在我们以前的工作中，我们建立了HBV组装作为一个定义明确的纳米流体实验系统，现在有基础询问组装和抗病毒组装效应。 与微流体装置集成的纳米流体组件为回答这些突出问题提供了一个独特的平台。这些装置上的电阻脉冲感测允许真实的时间、无标记的方法来监测生物相关浓度（nM至mM）下的组装。更具体地说，我们将开发通过纳米通道网络研究粒子传输特性的设备和方法。这些耦合的纳米通道可以以几乎任何二维格式排列，并在适度的外加电位下操作。如何影响颗粒输送强烈地取决于纳米级导管的尺寸和几何形状、施加的波形、导管的表面性质和输送介质的组成以及颗粒形状和组成。我们将优化这些设备参数，以发展一个基本的理解衣壳形成。本申请的具体目的是：（1）表征在各种反应条件下的衣壳组装;（2）制造和测试具有单个孔和串联的多个孔的平面内纳米通道，用于改进的连续脉冲感测;（3）计算模拟纳米流体装置中的（i）组装和（ii）颗粒运输;以及（4）开发耦合的纳米通道以感测不同尺寸的颗粒并与单个衣壳进行反应。

DESCRIPTION (provided by applicant): We will establish general methods to determine the assembly pathways of viruses and self- assembling nanoparticles. We will demonstrate these methods using Hepatitis B virus (HBV) capsid assembly. Resistive-pulse sensing allows real time monitoring of assembly by identifying single particles and assembly intermediates. Understanding the mechanism of virus assembly requires not only knowledge of precursors and final product structures, but also access to intermediates. Where many rare intermediates are involved, ensemble methods obscure them so that virus assembly resembles a two-state reaction. HBV has acutely infected more than 2B people; about 360M people have chronic HBV; every year nearly 1M will die of HBV-related liver disease. Assembly of HBV's icosahedral capsid has been identified as a new target for antiviral therapies. HBV assembly and the behavior (and development) of antiviral assembly effectors are relatively well understood, largely stemming from work in the Zlotnick lab, but there has been no direct observation and characterization of critical early intermediates in solution. Computational models of assembly suggest that the observed kinetics reflect early establishment of a constellation of intermediates needed to support capsid formation. The nucleation step and early intermediates are believed to play a role in recruiting viral components in vivo. Antiviral assembly effectors over-stimulate nucleation, distorting the distribution of intermediates and very often their structure. In our previous work, we established HBV assembly as a well-defined experimental system for nanofluidics and now have the foundation to interrogate assembly and antiviral assembly effectors. Nanofluidic components integrated with microfluidic devices offer a unique platform for answering these outstanding questions. Resistive-pulse sensing on these devices permits a real time, label-free approach to monitoring assembly at biologically relevant concentrations (nM to mM). More specifically, we will develop devices and methods to study particle transport properties through nanochannel networks. These coupled nanochannels can be arranged in virtually any two-dimensional format and operated with modest applied potentials. How particle transport is influenced depends strongly on the dimensions and geometries of the nanoscale conduits, applied waveforms, surface properties of the conduit, and composition of the transport medium, and particle shape and composition. We will optimize these device parameters in order to develop a fundamental understanding of capsid formation. The Specific Aims for this application are to: (1) characterize capsid assembly under various reaction conditions; (2) fabricate and test in-plane nanochannels with single pores and multiple pores in series for improved resistive-pulse sensing; (3) computationally simulate (i) assembly and (ii) particle transport in nanofluidic devices; and (4) develop coupled nanochannels to sense particles of different sizes and to perform reactions with single capsids.