Abstract
We have studied a donor-acceptor fluorophore-labeled DNA switch where the acceptor is Alexa-647, a carbocyanine dye, in solution at the single molecule level to elucidate the fluorescence switching mechanism. The acceptor, which is in an initial high fluorescence trans state, undergoes a photoisomerization reaction resulting in two additional states during its sub-millisecond transit across the probe volume. These two states are assigned to a nonfluorescent triplet trans state that strongly quenches the donor emission and a singlet cis state that blocks the fluorescence resonance energy transfer (FRET) pathway and gives rise to donor-only fluorescence. The formation of these states is faster than the transit time, so that all three states are approximately equally populated under our experimental conditions. The acceptor dye can stick to the DNA in all these states, with the rate of unsticking determining the rate of isomerization into the other states. Measurement of the rate of change of the FRET signal therefore provides information about the fluorophore-DNA intramolecular dynamics. These results explain the large zero peak in the proximity ratio, often seen in single molecule FRET experiments, and suggest that photoinduced effects may be important in single molecule FRET experiments using carbocyanine dyes. They also suggest that for fast photoinduced switching the interactions of the acceptor dye with the DNA and other surfaces should be prevented.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.