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

Author

Listed:
  • Dan Song

    (Harvard Biophysics Program, Harvard Medical School
    Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA)

  • Thomas G. W. Graham

    (Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA
    Harvard Medical School)

  • Joseph J. Loparo

    (Harvard Medical School, 250 Longwood Avenue, Seeley G. Mudd Room 204B, Boston, Massachusetts 02115, USA)

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.

Suggested Citation

  • Dan Song & Thomas G. W. Graham & Joseph J. Loparo, 2016. "A general approach to visualize protein binding and DNA conformation without protein labelling," Nature Communications, Nature, vol. 7(1), pages 1-7, April.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms10976
    DOI: 10.1038/ncomms10976
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    Cited by:

    1. Irina Shlosman & Elayne M. Fivenson & Morgan S. A. Gilman & Tyler A. Sisley & Suzanne Walker & Thomas G. Bernhardt & Andrew C. Kruse & Joseph J. Loparo, 2023. "Allosteric activation of cell wall synthesis during bacterial growth," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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