Untangling reaction pathways through modern approaches to high-throughput single-molecule force-spectroscopy experiments

•Multiplexing brings high-throughput to single-molecule force-spectroscopy studies.•Multiplexing captures molecular events previously obscured by averaging or limited statistics.•Large datasets are visualized in their entirety using dwell-time distributions.•Dwell-time distributions are used to understand pause dynamics of molecular motors. Single-molecule experiments provide a unique means for real-time observation of the activity of individual biomolecular machines. Through such techniques, insights into the mechanics of for example, polymerases, helicases, and packaging motors have been gleaned. Here we describe the recent advances in single-molecule force spectroscopy instrumentation that have facilitated high-throughput acquisition at high spatiotemporal resolution. The large datasets attained by such methods can capture rare but important events, and contain information regarding stochastic behaviors covering many orders of magnitude in time. We further discuss analysis of such data sets, and with a special focus on the pause states described in the general literature on RNA polymerase pausing we compare and contrast the signatures of different reaction pathways.