The more the merrier: high-throughput single-molecule techniques

Abstract

The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput. This review will discuss many developing techniques which address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments, but have also made improvements to ease-of-use, accessibility, and automation of data analysis.