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

Area of Science:

• Photophysics and Spectroscopy
• Biophysical Chemistry
• Molecular Biology

Background:

• Dark quenchers are crucial in bioanalytical assays like quantitative polymerase chain reaction (qPCR).
• Their presumed ideal properties are often assumed, but photoblinking, a temporary loss of absorption, can affect performance.
• Investigating quencher photoblinking is essential for optimizing sensitive detection methods.

Purpose of the Study:

• To investigate the photoblinking dynamics of QSY9 dark quenchers in a single-molecule DNA binding assay.
• To determine if photoblinking follows power-law kinetics or other models.
• To develop a theoretical model reconciling experimental observations with underlying photophysical processes.

Main Methods:

• Utilized single-molecule fluorescence spectroscopy with QSY9-tagged single-stranded DNA (ssDNA) binding to immobilized complementary ssDNA tagged with Cy3B.
• Analyzed single-molecule trajectories and dwell-time distributions under varying excitation power.
• Developed an analytical theory model based on chemical kinetics to interpret photoblinking and binding events.

Main Results:

• Single-molecule trajectories showed two-state emission levels due to binding/unbinding and/or photoblinking.
• Dwell-time distributions were exponential and excitation power-dependent, indicating first-order kinetics for the quencher.
• The developed model quantitatively explained experimental results, showing QSY9 photoblinking follows first-order kinetics, not power law, with a specific thermal recovery rate.

Conclusions:

• QSY9 photoblinking dynamics are governed by first-order chemical kinetics, not power-law blinking.
• This study provides a quantitative understanding of dark quencher photophysics and apparent power-law observations.
• The findings are crucial for accurate interpretation and optimization of assays employing dark quenchers.