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.

The Effect of Gold Nanoparticle Concentration and Laser Fluence on the Laser-Induced Water Decomposition

“…are formed during the ionization of pure water . Hydrogen peroxide, hydroxyl radical, hydroperoxide radical, superoxide anion radical, etc., are often called reactive oxygen species. , In the current paper, we have shown the process of the formation of all three stable products (Figures and ) and also the process of the generation of a hydroxyl radical (Figure ). It is established that the generation rates of various products depend significantly on the concentration of nanoparticles.…”

Single-molecule visualization of ROS-induced DNA damage in large DNA molecules

“…18 A number of studies followed to develop several methods for detecting ROS-induced DNA breaks, such as alkaline elution, 19 comet assay, 20,21 32 p-postlabeling methods, 22,23 and AFM. 24 Chemical modifications of DNA damage have also been identified via chromatography-coupled mass spectrometry. 25,26 Specifically, 8-oxo-dG is used as an oxidative damage marker because it can be detected in the femtomolar range by electrochemical HPLC.…”

AFM-based force spectroscopy for bioimaging and biosensing

“…Via calculating the distribution of single strand DNA, the effects of different strength of ROS could be quanticationally evaluated. 111 DNA has highly specic self-assembly ability to form periotic patterns, and that is why it becomes an extraordinarily ideal potential bottom-up materials for nanoelectronics, biosensors, and programmable molecular machines. With the high resolution and sensitivity, AFM is used as one of the most important instruments to characterize the unique self-assembly DNA structure, especially the articial DNA.…”