Designing a single-molecule biophysics tool for characterizing DNA damage for techniques that kill infectious pathogens through DNA damage effects

TL;DR

This paper introduces a novel single-molecule fluorescence method using DNA origami tiles to quantitatively assess DNA damage, enhancing reproducibility over traditional qualitative techniques in biophysics and microbiology.

Contribution

It develops a new fluorescence-based single-molecule technique for quantifying DNA damage with improved reproducibility, applicable to studying antibiotics' effects.

Findings

Proposed a fluorescence method for DNA damage quantification

Designed a DNA origami tile saturated with intercalating dye

Outlined optical setup for single-molecule imaging

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.