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

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

Author information

1
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, United States.
2
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, United States; Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, United States. Electronic address: ifinkelstein@cm.utexas.edu.

Abstract

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

PMID:
24859086
PMCID:
PMC4163502
DOI:
10.1016/j.febslet.2014.05.021
[Indexed for MEDLINE]
Free PMC Article

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