title: A general approach to visualize protein binding and DNA conformation without protein labelling.
abstract: Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions.
authors.affiliation: Harvard Biophysics Program, Harvard Medical School, Boston, Massachusetts 02115, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Song
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA.|Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Thomas G W
authors.initials: TG
authors.lastname: Graham
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Loparo
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D009690:Nucleic Acid Conformation; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins