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

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. Addi...

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Bibliographic Details
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
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Summary: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.
ISSN:0014-5793
1873-3468
DOI:10.1016/j.febslet.2014.05.021