title: High-throughput single-molecule studies of protein-DNA interactions.
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.
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, United States.
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Robison
authors.affiliation: 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.
authors.forename: Ilya J
authors.initials: IJ
authors.lastname: Finkelstein
mesh_terms: D004247:DNA / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D011506:Proteins; D004247:DNA
keywords: DNA curtains; Force spectroscopy; Particle tracking