Isolation and Analysis of Rare Enzymatic Events with Multiplex Flow Magnetic Tweezers

Our understanding of biomolecular dynamics has been revolutionized with the advent of techniques that enable the manipulation of forces and torques at the single-molecule level. However, the characterization of rare intermediates has proven challenging due to limited throughput. In this chapter, we present a method that dramatically enhances the throughput of force spectroscopy measurements with topological control. The method allows for routine imaging of tens of thousands of individual molecules undergoing millions of reaction cycles in parallel. The improvement in throughput enables the discovery of rare enzymatic events. Here, we describe the experimental procedures for the observation and analysis of supercoiling dynamics by DNA gyrase. To efficiently quantify diverse dynamic behaviors and rare events, we introduce a software platform with an incorporated automated feature classification pipeline. This method and accompanying software can be freely adapted for investigations into a wide array of complex, multistep enzymatic pathways where the characterization of rare intermediates has been hindered by limited throughput.