Haque Farzin, Zhang Hui, Wang Shaoying, Chang Chun-Li, Savran Cagri, Guo Peixuan
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
Department of Physiology and Cell Biology, Dorothy M Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2018;1805:423-450. doi: 10.1007/978-1-4939-8556-2_21.
Bacteriophage phi29 DNA packaging motor consists of a dodecameric portal channel protein complex termed connector that allows transportation of genomic dsDNA and a hexameric packaging RNA (pRNA) ring to gear the motor. The elegant design of the portal protein has facilitated its applications for real-time single-molecule detection of biopolymers and chemicals with high sensitivity and selectivity. The robust self-assembly property of the pRNA has enabled biophysical studies of the motor complex to determine the stoichiometry and structure/folding of the pRNA at single-molecule level. This chapter focuses on biophysical and analytical methods for studying the phi29 motor components at the single-molecule level, such as single channel conductance assays of membrane-embedded connectors; single molecule photobleaching (SMPB) assay for determining the stoichiometry of phi29 motor components; fluorescence resonance energy transfer (FRET) assay for determining the structure and folding of pRNA; atomic force microscopy (AFM) for imaging pRNA nanoparticles of various size, shape, and stoichiometry; and bright-field microscopy with magnetomechanical system for direct visualization of viral DNA packaging process. The phi29 system with explicit engineering capability has incredible potentials for diverse applications in nanotechnology and nanomedicine including, but not limited to, DNA sequencing, drug delivery to diseased cells, environmental surveillance, and early disease diagnosis.
噬菌体phi29 DNA包装马达由一个被称为连接体的十二聚体门户通道蛋白复合体组成，该复合体允许基因组双链DNA运输，还有一个六聚体包装RNA（pRNA）环来驱动马达。门户蛋白的精妙设计促进了其在生物聚合物和化学物质的实时单分子检测中的应用，具有高灵敏度和选择性。pRNA强大的自组装特性使得对马达复合体进行生物物理研究，以在单分子水平确定pRNA的化学计量以及结构/折叠情况成为可能。本章重点介绍在单分子水平研究phi29马达组件的生物物理和分析方法，例如膜嵌入连接体的单通道电导测定；用于确定phi29马达组件化学计量的单分子光漂白（SMPB）测定；用于确定pRNA结构和折叠的荧光共振能量转移（FRET）测定；用于对各种大小、形状和化学计量的pRNA纳米颗粒成像的原子力显微镜（AFM）；以及使用磁机械系统的明场显微镜用于直接观察病毒DNA包装过程。具有明确工程能力的phi29系统在纳米技术和纳米医学的各种应用中具有令人难以置信的潜力，包括但不限于DNA测序、向患病细胞递送药物、环境监测和疾病早期诊断。
