Zhang Hui, Guo Peixuan
Nanobiotechnology Center, Markey Cancer Center, and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, USA.
Methods. 2014 May 15;67(2):169-76. doi: 10.1016/j.ymeth.2014.01.010. Epub 2014 Jan 15.
Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described.
对生物复合物或纳米机器中的生物分子进行直接计数颇具难度。由于衍射极限，使用光学显微镜进行单分子计数具有挑战性。我们团队开发的用于直接计数的单分子光漂白（SMPB）技术（Shu等人，2007年[18]；Zhang等人，2007年[19]）提供了一种简单直接的方法，可在纳米尺度上确定生物复合物或纳米机器中分子或亚基的化学计量。化学计量通过实时观察单个荧光团光漂白产生的下降步数来确定。该技术现已广泛用于各种复合物或纳米结构中蛋白质、RNA和其他大分子的单分子计数。在此，我们以噬菌体phi29 DNA包装生物马达中RNA分子的计数为例，阐述SMPB技术。这里描述的方法可应用于其他系统中其他分子的单分子计数。描述了一种结合棱镜型和物镜型全内反射荧光（TIRF）构建简洁、简单且经济的单分子TIRF显微镜的方法。成像系统包括具有单荧光团检测灵敏度的深度冷却灵敏电子倍增电荷耦合器件（EMCCD）相机、用于同时双色激发的激光组合器以及基于波长将多个输出信号分离到不同探测器通道的双视场成像仪。描述了用于阐明phi29 DNA包装马达上RNA化学计量和蛋白质/RNA相互作用机制的单分子光漂白测定方法。综述了RNA分子单荧光团标记的不同方法。还描述了基于二项分布揭示生物分子真实拷贝数的统计建模过程。
