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改变了我们对生物分子动态特性的理解，但由于产量有限，稀有中间体仍然难以表征。在这里，我们描述了一种方法，该方法通过拓扑控制将力谱测量的吞吐量提高了 100 倍，从而能够对 50,000 个单分子和 1 亿个平行反应循环进行常规成像。这种改进能够检测细胞生命周期中的罕见事件。作为演示，我们表征了拓扑异构酶的超螺旋动力学和药物诱导的 DNA 断裂中间体。为了快速量化不同类别的动态行为和罕见事件，我们开发了一个具有自动特征分类管道的软件平台。