title: Designing a Single-Molecule Biophysics Tool for Characterising DNA Damage for Techniques that Kill Infectious Pathogens Through DNA Damage Effects.
abstract: Antibiotics such as the quinolones and fluoroquinolones kill bacterial pathogens ultimately through DNA damage. They target the essential type IIA topoisomerases in bacteria by stabilising the normally transient double-strand break state which is created to modify the supercoiling state of the DNA. Here we discuss the development of these antibiotics and their method of action. Existing methods for DNA damage visualisation, such as the comet assay and immunofluorescence imaging can often only be analysed qualitatively and this analysis is subjective. We describe a putative single-molecule fluorescence technique for quantifying DNA damage via the total fluorescence intensity of a DNA origami tile fully saturated with an intercalating dye, along with the optical requirements for how to implement these into a light microscopy imaging system capable of single-molecule millisecond timescale imaging. This system promises significant improvements in reproducibility of the quantification of DNA damage over traditional techniques.
authors.affiliation: Departments of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK. hlm531@york.ac.uk.
authors.forename: Helen
authors.initials: H
authors.lastname: Miller
authors.affiliation: Departments of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK.
authors.forename: Adam J M
authors.initials: AJ
authors.lastname: Wollman
authors.affiliation: Departments of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Leake
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D055808:Drug Discovery; D054884:Host-Pathogen Interactions; D006801:Humans; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D013329:Structure-Activity Relationship; D059005:Topoisomerase II Inhibitors / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II
keywords: DNA damage; DNA gyrase; DNA origami; Quinolone; Single molecule; YOYO