Work

In Situ Sensing of Reactive Oxygen Species on Dye-Stained Single DNA Molecules under Illumination

Year: 2019

Type: article

Abstract: Reactive oxygen species (ROS) are a necessary evil in many biological systems and have been measured with fluorescent probes at the ensemble levels both in vitro and in vivo. Measuring ROS generated from a single molecule is important for mechanistic studies, yet measuring ROS near a dye-labeled single-molecule under illumination has been challenging. Here, we use CellROX, a group of ROS probes, to sense ROS near dye-stained DNA that has been flow-stretched and immobilized on a surface. ROS is responsible for the photodamage of DNA molecules under this circumstance. In this report, we confirmed the ROS sensing reaction in bulk solutions and optimized the conditions for single-molecule experiments including the selection of substrates, dye concentrations, probes in the CellROX series, excitation lasers, and emission filter-sets. We observed a correlation between ROS and the dye-labeled DNA and localized the ROS-activated CellROX probe molecules at both the ensemble level and the single-molecule level.

Source: Langmuir

DOI: 10.1021/acs.langmuir.9b01822

Authors: Joseph R. Pyle, Kurt Waldo E. Sy Piecco, Juvinch R. Vicente, Jixin Chen

Institutions: Ohio University, University of the Philippines Visayas

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Institutes of Health, National Human Genome Research Institute

Award: Super-Resolution Optical Mapping for DNA Analysis Using Triplex-Forming Oligonucleotides as Stochastic Molecular Probes