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

FEBS Lett. 2014 Oct 1;588(19):3539-46. doi: 10.1016/j.febslet.2014.05.021. Epub 2014 May 21.


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.

Keywords: DNA curtains; Force spectroscopy; Particle tracking.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • DNA / metabolism*
  • Protein Binding
  • Proteins / metabolism*
  • Spectrum Analysis


  • Proteins
  • DNA