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

Oxygen scavenging systems (OSSs) are critical for dye stability in single-molecule fluorescence (SMF) experiments. However, the commonly used protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) OSS alters DNA mechanical properties, limiting its applicability. To address this limitation, we examine the bilirubin oxidase (BOD) OSS, which had not been previously used in single-molecule experiments, alongside the pyranose oxidase and catalase (POC) OSS. Our results revealed that POC OSS affected DNA mechanics in a buffer-dependent manner, while BOD OSS had no discernible effect across all tested buffer conditions. Furthermore, BOD OSS significantly extended the photobleaching lifetimes of Cy3 and Cy5 dyes and caused minimal pH changes compared to PCD OSS. Collectively, these findings highlight the superior performance of BOD OSS, suggesting its potential for widespread application, particularly in experiments combining SMF with single-molecule force spectroscopy (SMFS) measurements.