Photophysical properties of the hemicyanine Dy-630 and its potential as a single-molecule fluorescent probe for biophysical applications

Abstract

Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction coefficient, and has a moderate fluorescence quantum yield that increases significantly when the dye is in close proximity to a large macromolecule such as a protein. So far, the green-absorbing symmetric cyanine known as Cy3 has been the probe of choice in this field because the magnitude of the increase observed upon protein binding (usually 2–4 –fold) is large enough to allow for the analysis of protein dynamics on the inherently noisy single-molecule signals. Here, we report the characterization of the photophysical properties of the red-absorbing hemicyanine dye Dy-630 in the context of its potential application as a single-molecule PIFE probe. The behavior of Dy-630 in solution is similar to that of Cy3; the fluorescence quantum yield and lifetime of Dy-630 increase with increasing viscosity, and decrease with increasing temperature indicating the existence of an activated nonradiative process that depopulates the singlet state of the dye. As in the case of Cy3, the results of transient spectroscopy experiments are consistent with the formation of a photoisomer that reverts to the ground state thermally in the microsecond timescale. Unfortunately, experiments with DNA samples paint a more complex scenario. As in the case of Cy3, the fluorescence quantum yield of Dy-630 increases significantly when the dye interacts with the DNA bases, but in the case of Dy-630 attachment to DNA results in an already long fluorescence lifetime that does not provide a significant window for the protein-induced enhancement observed with Cy3. Although we show that Dy-630 may not be well-suited for PIFE, our results shed light on the optimal design principles for probes for PIFE applications.

Submillisecond Conformational Transitions of Short Single-Stranded DNA Lattices by Photon Correlation Single-Molecule Förster Resonance Energy Transfer

“…We fit the first 20 μs of the TCFs to a model monoexponential decay, and the optimized time constants for each of the ss-dsDNA constructs are listed in Table S2 of the Supporting Information. Relaxations on a time scale of ∼3 μs have been observed previously in photophysical studies of the Cy3 chromophore and are known to be associated with excited-state intersystem crossing and forward photoisomerization, which undergo the reverse step of ground-state recovery within ∼10 μs. − This photoisomerization leads to quenching of the Cy3 donor chromophore excited state, which leads to a change in the relative intensities of Cy3 donor and Cy5 acceptor fluorescence in our ss-dsDNA constructs and thus to a change in the FRET efficiency. We therefore assigned the ∼3 μs decay in our current studies to photoisomerization of the Cy3 chromophore, and we assumed that these relatively fast processes contributed to the “time-averaged” background of the signal fluctuations that we observed on tens-of-microseconds and longer time scales, which we interpreted to be due to structural changes of the oligo­(dT) n templates.…”

Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

“…We fit the first 20 microseconds of the TCFs to a model mono-exponential decay, and the optimized time constants for each of the ss-dsDNA constructs are listed in Table S2 of the SI. Relaxations on the time scale of ~3 µs have been observed previously in photophysical studies of the Cy3 chromophore, and are known to be associated with excited state intersystem crossing and forward photoisomerization, which undergo the reverse step of ground state recovery within ~10 µs (38)(39)(40)(41)(42). We therefore assign the ~ 3 µs decay in our current studies to photoisomerization of the Cy3 chromophore, and we assume that these relatively fast processes contribute to the 'time-averaged' background of the signal fluctuations that we observe on tens-of-microseconds and longer time scales, and which we interpret to be due to structural changes of the oligo(dT)n templates.…”

Impact of Cyanine Conformational Restraint in the Near-Infrared Range

“… For example, while the fluorescence lifetime of trimethine indocyanine is ca. 18-fold greater in glycerol than in water, the fluorescence lifetime of 2 is shorter in glycerol (τ F = 0.83 ns) than in all measured solvents except water …”

“…18-fold greater in glycerol than in water, the fluorescence lifetime of 2 is shorter in glycerol (τ F = 0.83 ns) than in all measured solvents except water. 24 To analyze the role of solvent in greater detail, the rate constant for nonradiative deactivation (k nr ) was calculated from Φ F and τ F as k nr = (1 − Φ F )/τ F (Figure S4 and Table S1). Experiments with various trimethine and penthamethine cyanines show an excellent correlation between k nr and solvent viscosity.…”