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Related Experiment Videos

Single-molecule three-color FRET.

Sungchul Hohng1, Chirlmin Joo, Taekjip Ha

  • 1Physics Department, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.

Biophysical Journal
|August 10, 2004
PubMed
Summary
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Single-molecule fluorescence resonance energy transfer (FRET) now tracks three colors, revealing correlated movements in DNA Holliday junctions. This advancement offers a more complete picture of complex biomolecular dynamics.

Area of Science:

  • Biophysics
  • Molecular Biology
  • Biochemistry

Background:

  • Single-molecule fluorescence resonance energy transfer (FRET) is crucial for studying biomolecular dynamics under physiological conditions.
  • Current single-molecule FRET typically involves only two fluorophores, limiting the observation of complex conformational changes.
  • Observing changes in multiple distances simultaneously is essential for a comprehensive understanding of intricate molecular systems.

Purpose of the Study:

  • To extend single-molecule FRET to three spectrally distinct fluorophores.
  • To investigate the conformational dynamics of the DNA four-way (Holliday) junction.
  • To simultaneously measure distance changes between multiple points on a single molecule.

Main Methods:

  • Development and application of a three-color single-molecule FRET technique.

Related Experiment Videos

  • Utilizing the DNA four-way junction as a model system.
  • Labeling three arms of the junction with distinct fluorophores (Cy3, Cy5, Cy5.5) to serve as donor and two acceptors.

    • Main Results:

    • Simultaneous measurement of distance changes between the donor and two distinct acceptors on the DNA Holliday junction.
    • Observation of correlated movements: one arm moving away from the donor as another approaches, and vice versa.
    • Evidence suggesting that the DNA Holliday junction does not exist with unstacked helices or in detectable parallel conformations.

    Conclusions:

    • This study demonstrates the first observation of correlated movements of two different segments within a single molecule using three-color single-molecule FRET.
    • The findings provide new insights into the conformational states and dynamics of the DNA four-way junction.
    • The developed three-color FRET technique offers a powerful tool for studying complex biomolecular systems with greater detail.