A general approach to visualize protein binding and DNA conformation without protein labelling

Dan Song, Thomas G. W. Graham and Joseph J. Loparo ()
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Dan Song: Harvard Biophysics Program, Harvard Medical School
Thomas G. W. Graham: Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA
Joseph J. Loparo: Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA

Nature Communications, 2016, vol. 7, issue 1, 1-7

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms10976

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