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

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.

Neuroprotective Properties of Quinone Reductase 2 Inhibitor M-11, a 2-Mercaptobenzimidazole Derivative

“…In addition, the contribution of NQO2 to cellular damage may also be associated with the oxidation of the co-substrate NRH to NR+ with subsequent formation of 4-pyridone-3-carboxamide riboside in the presence of O 2 •− , which activates cell autophagy in vitro [ 90 ]. Therefore, to study ROS production in the presence of adrenochrome and BNAH, the DNA-binding CellROX Green Reagent [ 91 ] and CM-H 2 DCFDA [ 92 ] dyes were chosen. CellROX Green Reagent is oxidized by O 2 •− and hydroxyl radical (OH • ), unlike H 2 O 2 , and is used in ROS generation models with menadione (vitamin K3)-exogenous para-quinone with NQO2 substrate properties [ 93 , 94 ].…”

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR

“…We tested the Tm values of primers (ASPro4se-FP, ASPro4as-RP) and their perfectly complementary strands (ASPro4se-FP-C, ASPro4as-RP-C) in the presence or absence of ammonium bismuth citrate or bismuth subcarbonate and compared with those with addition of additives. In the excitation, reactive oxygen species (ROS) are produced, which are very active and will attack the double bond, causing the internal double bond of the Cy5 dye to break, resulting in a decrease of the fluorescence signal [36]. So, we took the fluorescence signal of the oligonucleotide chain labelled with the Cy5 dye as a control, We also tested the Tm values of primers (APOE-FP, APOE-RP) and their perfectly complementary strands (APOE-FP-C, APOE-RP-C) in the presence or absence of ammonium bismuth citrate or bismuth subcarbonate and compared with those with addition of additives.…”