High-throughput single-molecule studies of protein–DNA interactions

• Published in: FEBS letters 2014-10, Vol.588 (19), p.3539-3546
• Main Authors: Robison, Aaron D., Finkelstein, Ilya J.
• Format: Article
• Language: English
• Subjects: DNA - metabolism; DNA curtains; Force spectroscopy; Particle tracking; Protein Binding; Proteins - metabolism; Spectrum Analysis
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2014.05.021

Fluorescence and force-based single-molecule studies of protein–nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell.