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Related Experiment Videos

Simultaneous, coincident optical trapping and single-molecule fluorescence.

Matthew J Lang1, Polly M Fordyce, Anita M Engh

  • 1Department of Biological Sciences, Stanford University, Stanford, California 94305, USA.

Nature Methods
|March 23, 2005
PubMed
Summary

We developed a novel instrument for simultaneous optical trapping and single-molecule fluorescence. This allowed us to study DNA strand separation under different forces, revealing distinct unbinding pathways.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Optical Instrumentation

Background:

  • Understanding DNA mechanics is crucial for molecular biology.
  • Previous methods lacked simultaneous force application and fluorescence detection.
  • Studying DNA unbinding pathways requires precise force control.

Purpose of the Study:

  • To develop a novel instrument for simultaneous optical trapping and single-molecule fluorescence.
  • To investigate force-induced DNA strand separation (unzipping and shearing).
  • To elucidate distinct unbinding pathways and thermodynamic properties of double-stranded DNA (dsDNA).

Main Methods:

  • Construction of a microscope-based instrument integrating optical trapping and fluorescence.
  • Application of controlled force (parallel and perpendicular) to a dye-labeled dsDNA segment.

Related Experiment Videos

  • Simultaneous monitoring of mechanical transitions and fluorescence emission during DNA rupture.

    • Main Results:

    • Demonstrated simultaneous optical trapping and single-molecule fluorescence capabilities.
    • Observed force-dependent DNA strand separation with distinct rupture forces for unzipping and shearing.
    • Correlated mechanical transitions with discontinuous fluorescence changes, indicating DNA hybrid rupture.
    • Determined thermodynamic transition state distances and thermal off-rates for both force-loading modes.

    Conclusions:

    • The developed instrument enables simultaneous mechanical and optical studies of single molecules.
    • DNA unbinding pathways are distinct and highly dependent on the direction of applied force.
    • Quantitative thermodynamic parameters of DNA rupture were obtained, providing insights into molecular interactions.