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.

中文翻译：

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在单分子荧光实验中对 DNA 机械性能无影响的除氧系统


除氧系统 （OSS） 对于单分子荧光 （SMF） 实验中的染料稳定性至关重要。然而，常用的原儿茶酸 （PCA）/原儿茶酸-3,4-双加氧酶 （PCD） OSS 会改变 DNA 的机械特性，从而限制其适用性。为了解决这一限制，我们检查了以前未在单分子实验中使用的胆红素氧化酶 （BOD） OSS，以及吡喃糖氧化酶和过氧化氢酶 （POC） OSS。我们的结果表明，POC OSS 以缓冲液依赖性方式影响 DNA 力学，而 BOD OSS 在所有测试的缓冲液条件下都没有明显的影响。此外，与 PCD OSS 相比，BOD OSS 显著延长了 Cy3 和 Cy5 染料的光漂白寿命，并引起了最小的 pH 值变化。总的来说，这些发现突出了 BOD OSS 的卓越性能，表明其具有广泛应用的潜力，特别是在将 SMF 与单分子力谱 （SMFS） 测量相结合的实验中。