Dye-mediated Photodamage During Single-molecule DNA Imaging (2012)

Undergraduates: Keia Faison, Catherine F. Dial, Keia Faison, Whitney Melvin Michael A. Tycon

Faculty Advisor: Christopher Fecko
Department: Biology

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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 damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single strand breaks (SSB), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSB on DNA molecules by the dye YOYO-1 using complimentary 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 (DSB). The gel assay, using supercoiled plasmid DNA, was sensitive to both SSB and DSB. This enabled the quantification of SSB 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 ?-mercaptoethanol. These results enable the estimation of the number of SSB that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.