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Related Experiment Video

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Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

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Characterization of a single molecule DNA switch in free solution.

Samuel S White¹, Haitao Li, Richard J Marsh

¹Department of Chemistry, University of Cambridge.

Journal of the American Chemical Society

|August 31, 2006

Summary

Single molecule FRET studies reveal Alexa-647 dye photoisomerization in DNA switches. This photoinduced switching mechanism explains observed FRET signals and impacts dye-DNA interactions.

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Area of Science:

Single-molecule biophysics
Photochemistry
Molecular dynamics

Background:

Donor-acceptor fluorophore-labeled DNA switches are used to study molecular mechanisms.
Alexa-647, a carbocyanine dye, is a common acceptor in these systems.
Understanding fluorescence switching is crucial for interpreting experimental data.

Purpose of the Study:

To elucidate the fluorescence switching mechanism of Alexa-647 on a DNA switch at the single molecule level.
To investigate the photoisomerization pathways of the dye.
To correlate dye dynamics with FRET signal changes.

Main Methods:

Single molecule fluorescence spectroscopy.
Time-resolved single molecule FRET measurements.
Analysis of FRET signal dynamics to probe fluorophore-DNA interactions.

Main Results:

Alexa-647 undergoes photoisomerization into nonfluorescent triplet trans and cis states during transit.
These states are populated rapidly, occurring faster than the probe transit time.
Dye-DNA interactions influence isomerization rates, affecting FRET signal changes.
Observed phenomena explain the zero peak in proximity ratio in FRET experiments.

Conclusions:

Photoinduced effects are significant in single molecule FRET experiments with carbocyanine dyes.
Preventing dye-DNA and dye-surface interactions is recommended for fast photoinduced switching studies.
The study provides insights into fluorophore-DNA intramolecular dynamics.