Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Anal Biochem. 2012 Jul 1;426(1):13-21. doi: 10.1016/j.ab.2012.03.021. Epub 2012 Apr 4.
Single-molecule fluorescence imaging of DNA-binding proteins has enabled detailed investigations of their interactions. However, the intercalating dyes used to visually locate DNA molecules have the undesirable effect of photochemically damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single-strand breaks (SSBs), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSBs on DNA molecules by the dye YOYO-1 using complementary single-molecule imaging and gel electrophoresis-based damage assays. The single-molecule assay imaged hydrodynamically elongated lambda DNA, enabling the real-time detection of double-strand breaks (DSBs). The gel assay, which used supercoiled plasmid DNA, was sensitive to both SSBs and DSBs. This enabled the quantification of SSBs that precede DSB formation. Using the parameters determined from the gel damage assay, we applied a model of stochastic DNA damage to the time-resolved DNA breakage data, extracting the rates of single-strand breakage at two dye staining ratios and measuring the damage reduction from the radical scavengers ascorbic acid and β-mercaptoethanol. These results enable the estimation of the number of SSBs that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.
单分子荧光成像技术已被用于研究 DNA 结合蛋白的相互作用。然而，用于可视化定位 DNA 分子的嵌入染料会通过自由基中间体对 DNA 造成不可取的光化学损伤。不幸的是，这种损伤以单链断裂（SSB）的形式发生，SSB 是不可见的，但会严重影响蛋白质的行为。我们使用互补的单分子成像和基于凝胶电泳的损伤检测方法，研究了染料 YOYO-1 对 DNA 分子造成的 SSB 的形成。单分子检测法对水力延伸的 λ DNA 进行成像，能够实时检测双链断裂（DSB）。该凝胶检测法使用超螺旋质粒 DNA，对 SSB 和 DSB 均敏感。这使我们能够定量 SSB 的形成。使用凝胶损伤检测法确定的参数，我们将随机 DNA 损伤模型应用于时间分辨的 DNA 断裂数据，从两个染料染色比中提取单链断裂率，并从自由基清除剂抗坏血酸和β-巯基乙醇测量损伤减少。这些结果使我们能够估计在成像过程中发生的 SSB 的数量，并且可以在荧光显微镜中使用的广泛激光强度范围内进行扩展。
