Shu Dan, Huang Lisa, Guo Peixuan
Department of Pathobiology and Purdue Cancer Research Center, B-36 Hansen Life Science Research Building, Purdue University, West Lafayette, IN 47907, USA.
J Virol Methods. 2004 Jan;115(1):19-30. doi: 10.1016/j.jviromet.2003.08.015.
In nanotechnology, biomolecular assemblies serve not only as model systems for the construction of nanodevices, but they can also be used directly as templates for the formation of nanostructures. Biological nano-building blocks can either be isolated as complete functional units from living cells or viruses (biological "Top down" approach) or formed by biomolecular assembly from recombinant or synthetic components ("Bottom up" approach). In both cases, rational design of nanostructures requires knowledge of the stoichiometry of the biological structures, which frequently occur as multimers, i.e., the morphological complex is composed of multiple copies of one or more macromolecules. In this paper, a method is described for the stoichiometric quantification of molecules in bio-nanostructures. The method is based on using dilution factors and relative concentrations rather than absolute quantities, which are often difficult to determine, especially in short-lived assembly intermediates. The approach exploits the fact that the larger the stoichiometry of the component is, the more dramatic is the influence of the dilution factor (decrease in concentration) on the reaction. We established and used the method to determine the stoichiometry of components of bacterial virus phi29. The log of dilution factors was plotted against the log of reaction yield. The stoichiometry Z was determined with the equation Z=-1.58+2.4193T-0.001746T(2) [T in (0,1000), or 90 degree angle alpha in (0 degrees, 89.9 degrees )], where T is the slope of the curve (tangent of 90 degree angle alpha, which is the angle between the x-axis and the concentration dependent curve). Z can also be determined from a standard table given in this report. With the bacteriophage phi29 in vitro assembly system, up to 5x10(8) infectious virions per ml can be assembled from 11 purified components, giving our method a sensitivity of nine orders of magnitude. We confirmed the stoichiometries of phi29 components that were determined previously with microscopic approaches. The described method also responded to programmed stoichiometry changes, which were generated by assembling the phi29 DNA packaging motor from modified pRNA (DNA-packaging RNA) molecules forming a trimer of dimers or a dimer of trimers, instead of the wild-type hexamer.
在纳米技术中，生物分子组装体不仅作为构建纳米器件的模型系统，还可直接用作纳米结构形成的模板。生物纳米构建块既可以作为完整的功能单元从活细胞或病毒中分离出来（生物“自上而下”方法），也可以由重组或合成成分通过生物分子组装形成（“自下而上”方法）。在这两种情况下，纳米结构的合理设计都需要了解生物结构的化学计量，生物结构经常以多聚体形式出现，即形态复合体由一个或多个大分子的多个拷贝组成。本文描述了一种用于生物纳米结构中分子化学计量定量的方法。该方法基于使用稀释因子和相对浓度，而不是绝对量，绝对量往往难以确定，尤其是在寿命较短的组装中间体中。该方法利用了这样一个事实，即组分的化学计量越大，稀释因子（浓度降低）对反应的影响就越显著。我们建立并使用该方法确定了细菌病毒phi29组分的化学计量。将稀释因子的对数与反应产率的对数作图。化学计量Z由方程Z = -1.58 + 2.4193T - 0.001746T² [T在(0,1000)范围内，或90度角α在(0度, 89.9度)范围内]确定，其中T是曲线的斜率（90度角α的正切值，α是x轴与浓度依赖曲线之间的夹角）。Z也可以从本报告给出的标准表中确定。利用噬菌体phi29体外组装系统，每毫升可从11种纯化组分中组装出高达5×10⁸个感染性病毒粒子，使我们的方法灵敏度达到九个数量级。我们证实了先前用显微镜方法确定的phi29组分的化学计量。所描述的方法还对程序化的化学计量变化有响应，这种变化是通过将phi29 DNA包装马达由形成三聚体二聚体或二聚体三聚体的修饰pRNA（DNA包装RNA）分子组装而成，而不是野生型六聚体。
