title: Characterization of a single molecule DNA switch in free solution.
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.
authors.affiliation: Department of Chemistry, University of Cambridge.
authors.forename: Samuel S
authors.initials: SS
authors.lastname: White
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Marsh
authors.forename: Joe D
authors.initials: JD
authors.lastname: Piper
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Leonczek
authors.forename: Nick
authors.initials: N
authors.lastname: Nicolaou
authors.forename: Angus J
authors.initials: AJ
authors.lastname: Bain
authors.forename: Liming
authors.initials: L
authors.lastname: Ying
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D012996:Solutions
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D012996:Solutions; C085321:cyanine dye 5; C067297:8-aminohexylamino cAMP; D004247:DNA; D000242:Cyclic AMP