Structure and Dynamics of Molecular Machines, Max Planck Institute of Biochemistry, Martinsried, Germany.
Physik Department, Technische Universität München, Garching, Germany.
Nat Commun. 2020 Sep 18;11(1):4714. doi: 10.1038/s41467-020-18456-y.
The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.
在单分子水平上施加力和扭矩已经改变了我们对生物分子动态特性的理解，但由于通量有限，很少有中间产物能够被准确描述。在这里，我们描述了一种方法，通过拓扑控制，使力谱测量的通量提高了 100 倍，从而能够同时对 50000 个单分子和 1 亿个反应循环进行常规成像。这种改进使得检测细胞生命周期中的罕见事件成为可能。作为一个演示，我们对拓扑异构酶的超螺旋动力学和药物诱导的 DNA 断裂中间产物进行了表征。为了快速量化不同类别的动态行为和罕见事件，我们开发了一个带有自动特征分类管道的软件平台。该方法和软件可以很容易地应用于广泛的复杂、多步骤的酶促途径的研究，在这些途径中，由于通量有限，很少有中间产物能够被分类。
