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Free keywords:
ESCHERICHIA-COLI DNA; STRUCTURAL TRANSITIONS; FORCE; GYRASE; ELASTICITY;
COMPLEX; TOPOISOMERASES; COORDINATION; REPLICATION; CLEAVAGEScience & Technology - Other Topics;
Abstract:
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. Single molecule force measurements have shed light on dynamic biological events, but rare events escape notice owing to low throughput of the methods. Here, the authors combine an array of magnetic tweezers with lateral flow to increase throughput 100-fold, and detect rare DNA breaks induced by gyrase.
