Development of An Spr Method To Monitor the Chemical Interaction of Dna With A Small Reactive Molecule: Peroxynitrite As An Example
Date of Award
Master of Science In Chemistry Degree
Dr. Valentin Gogonea
Dr. Christopher W. Boyd
Peroxynitrite is an anion with the formula ONOO-. It is an unstable structural isomer of nitrate, NO3-. Although its conjugate acid (ONOOH) is highly reactive, it is quite relatively stable in basic solutions. Generation of peroxynitrite in-vivo occurs through the diffusion-controlled reaction between superoxide anion and nitric oxide free radical. It is a strong oxidizing and nitrating agent, and its formation has been correlated with many pathological conditions. Because of its oxidizing properties, peroxynitrite can damage a wide array of molecules in cells, including DNA and proteins. Reactions of peroxynitrite with DNA, proteins, and lipids, trigger a wide array of cellular responses ranging from subtle modifications of cell signaling to oxidative injury committing cells to necrosis and apoptosis. Peroxynitrite-induced DNA modifications include formation of 8-nitroguanine and 8-oxoguanine as well as DNA single strand breakage and base modifications when exposed to different concentrations of peroxynitrite. However, the accurate measurement of peroxynitrite concentration has been a challenge since this analyte reacts with many cellular targets. Development of analytical techniques capable of rapid and sensitive detection of this fast reacting and damaging agent is an important research goal not only to monitor its dynamic concentration but also to correlate its amount with observed chemical damage that this oxidant generates. iii Surface Plasmon Resonance (SPR) is an analytical platform that can detect interactions of immobilized ligand with running analytes. The change detected is related to a change in the refractive index as a result of analyte binding to immobilized ligands. Peroxynitrite reaction with DNA is expected to result in significant structural changes in the tethered DNA double helix of attached probes. We hypothesize that although peroxynitrite is a small molecule and is not expected to bind or cause significant changes in refractive index on SPR chips, the expected effect of its reactivity with DNA bases will result in collective structural configuration of immobilized DNA probes with can be monitored using SPR chips. To address this hypothesis, we propose the following 4 specific aims: Specific Aimi: Optimization of methods of immobilization of oligonucleotides on nanogold SPR interfaces. Specific Aim2: Characterization of the DNA-functionalized SPR gold chip. Specific Aim3: Study ofthe interaction ofPON with immobilized DNA structures on the SPR chips. Specific Aim 4: Compare and contrast SPR results with electrochemical methods using the same DNA sequences immobilized on electrode surfaces.
Sharma, Divya, "Development of An Spr Method To Monitor the Chemical Interaction of Dna With A Small Reactive Molecule: Peroxynitrite As An Example" (2019). ETD Archive. 1260.