title: Quantification of dye-mediated photodamage during single-molecule DNA imaging.
abstract: 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.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tycon
authors.forename: Catherine F
authors.initials: CF
authors.lastname: Dial
authors.forename: Keia
authors.initials: K
authors.lastname: Faison
authors.forename: Whitney
authors.initials: W
authors.lastname: Melvin
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fecko
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry; D001583:Benzoxazoles / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D005456:Fluorescent Dyes / Q000737:chemistry*; D016166:Free Radical Scavengers / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry*; D007700:Kinetics; D007834:Lasers; D008856:Microscopy, Fluorescence; D011806:Quinolinium Compounds / Q000737:chemistry
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D007364:Intercalating Agents; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D001205:Ascorbic Acid