Power-dependent single-molecule dynamics of dark quencher blinking in QSY9/Cy3B: Diffusion-binding experiment and theory

Originally developed for bioanalytical assays such as quantitative polymerase chain reaction, dark quenchers have been adapted in other areas, from gene sequencing to ultra-sensitive chemical detection. Most applications require that the dark quenchers' presumed properties be ideal; however, it is not obvious if such assumptions are always valid. One such issue is photoblinking, whereby a quencher intermittently enters a non-absorbing state for a short period of time. Here, we investigated the role of quencher photoblinking, if detectable, by interrogating a freely diffusing QSY9-tagged single-stranded DNA (ssDNA) that stochastically binds to an immobilized complementary ssDNA tagged with Cy3B. The resulting single-molecule trajectories exhibited two-state emission levels arising from molecular binding/unbinding events, quencher photoblinking, or both. The dwell-time distributions were found to be of the exponential type and dependent on excitation power, suggesting time-independent rate parameters for the quencher. In contrast, population-level time traces resembled those arising from power-law blinking reported for other systems. To reconcile these observations, we developed an analytical theory model based on the premises that both photoblinking and DNA binding can be expressed in terms of elementary chemical steps and that the experimental observations resulted from a temporal overlap of these random and independent events. This model was able to quantitatively explain the experimental results. QSY9 photoblinking dynamics were dependent on photoexcitation power and followed first-order chemical kinetics-not power law-with a thermal recovery rate of ∼3.3×10-2 s-1 at room temperature. In addition to advancing dark quencher photophysics, this study demonstrates a quantitative understanding based on simple underlying dynamical processes for an apparent power-law photoblinking observation.