Characterization of a single molecule DNA switch in free solution

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.