An oxygen-scavenging system without impact on DNA mechanical properties in single-molecule fluorescence experiments

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Oxygen scavenging systems (OSSs) are essential for maintaining dye stability in single-molecule fluorescence (SMF) experiments.
• The conventional protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) OSS can negatively impact DNA mechanical properties, restricting its use in force spectroscopy applications.

Purpose of the Study:

• To evaluate the efficacy of bilirubin oxidase (BOD) and pyranose oxidase and catalase (POC) OSSs as alternatives to PCD OSS for SMF experiments.
• To assess the impact of these OSSs on DNA mechanics and dye photobleaching lifetimes.

Main Methods:

• Investigated BOD and POC OSSs in single-molecule fluorescence experiments.
• Assessed the effects of OSSs on DNA mechanical properties using buffer-dependent analyses.
• Measured the photobleaching lifetimes of Cy3 and Cy5 dyes under different OSS conditions.
• Monitored pH changes induced by the OSSs.

Main Results:

• BOD OSS demonstrated no significant alteration of DNA mechanical properties across various buffer conditions.
• POC OSS exhibited buffer-dependent effects on DNA mechanics.
• BOD OSS substantially prolonged the photobleaching lifetimes of Cy3 and Cy5 dyes.
• BOD OSS induced minimal pH variations compared to PCD OSS.

Conclusions:

• BOD OSS is a highly effective oxygen scavenger for SMF, preserving DNA integrity and enhancing dye stability.
• BOD OSS presents a significant advantage over PCD OSS, particularly for integrated single-molecule fluorescence and force spectroscopy (SMFS) studies.
• The findings support the broad applicability of BOD OSS in advanced single-molecule biophysical techniques.