title: AFM-based quantification of conformational changes in DNA caused by reactive oxygen species.
abstract: Radical induced modification of DNA plays an important role in many pathological pathways like cancer development, aging, etc. In this work, we quantify radical-induced DNA damage that causes transitions from double to single stranded DNA using atomic force microscopy (AFM). The plasmid pBR322 is attacked by free hydroxyl radicals that are produced by Fenton's reaction; the strength of the radical attack is controlled via the ratio of hydroxyl radical molecules to DNA base pairs. The extent of DNA modification is assessed by AFM tapping mode (TM) imaging of the plasmids (after adsorption onto PAH-functionalized mica) in air. As single stranded DNA chains (height ∼2 Å) are much smaller than intact DNA strands (∼5 Å), their fraction can be quantified based on the height distribution, which allows a simplified data analysis in comparison to similar AFM-based approaches. It is found that the amount of damaged DNA strands increases with increasing strength of radical attack, and decreases if ROS scavengers like sodium acetate are added. Competition curves are calculated for the interaction of hydroxyl radicals with DNA and sodium acetate, which finally allows calculation of relative rate constants for the respective reactions.
authors.affiliation: Institut für Physik, Ernst-Moritz-Arndt Universität , Felix-Hausdorff-Strasse 6, D-17487 Greifswald, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Berg
authors.forename: Janine
authors.initials: J
authors.lastname: Wilken
authors.forename: Christiane A
authors.initials: CA
authors.lastname: Helm
authors.forename: Stephan
authors.initials: S
authors.lastname: Block
mesh_terms: D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D010957:Plasmids; D017382:Reactive Oxygen Species / Q000737:chemistry*
chemical_list: D017382:Reactive Oxygen Species; D004247:DNA