Eli G. HvastkovsLafave, Elizabeth Rose2024-08-292024-07July 2024July 2024http://hdl.handle.net/10342/13674Electrochemical methods coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were developed and applied to rapidly analyze DNA structure, sequence variations, and damage in biological systems. Square wave voltammetry (SWV) at pyrolytic graphite electrodes with immobilized DNA allowed detection of guanine oxidation peaks catalyzed by Ru(bpy)32+. Peak current differences compared to wild-type (WT) or unaltered samples indicated DNA structural changes, which were validated through LC-MS/MS base compositional analysis of hydrolyzed DNA samples. Breast cancer 1, early onset (BRCA1) gene codes for the DNA repair enzyme Brca1 and impacts skeletal muscle function when mutated. To study Brca1's role, inducible skeletal muscle-specific BRCA1 knockout (KO) mice were generated. Initial phenotypic assays indicated increased mitochondrial DNA (mtDNA) mutation frequency in KO vs wildtype (WT). Applying the electrochemical approach, mtDNA from Brca1 KO skeletal muscle displayed statistically significantly decreased oxidative currents vs. WT, consistent with guanine depletion due to mtDNA mutations. Conversely, nuclear DNA (nDNA) showed increased currents in KO samples, suggesting accumulation of oxidatively damaged guanine (p < 0.05). Subsequent LC-MS/MS composition analysis validated these hypotheses, allowing us to quantify suspected guanine variations, as well as detect the remaining DNA bases (adenine, thymine and cytosine) and commonly damaged bases (8-oxoguanine, fapyadenine). The electrochemical – LC-MS/MS approach was further utilized to study the role of overexpressed Brca1 in various muscle locations, detect and identify DNA variations in Ni2+- treated Caenorhabditis elegans, study genetic changes in additional mouse models (i.e. Col5a1 for Ehlers-Danlos, MDX for muscular-dystrophy), and monitor DNA change in Hydrogenophaga taeniospiralis, a benzene-exposed bacteria. Combining electrochemical and mass spectrometry techniques allowed for validation of DNA structural changes observed in phenotypic studies, while also providing a versatile approach to comprehensively investigate a wide range of biological questions, as well as chemical analyses of historical artifacts. Finally, further utilization of analytical methodology, predominantly LC-MS/MS, for historical medicines provided insight into often unlisted ingredients and potential contaminants, addressing lingering historical biases towards alternative treatments during the 19th century.application/pdfEnglishChemistry, AnalyticalHistory of ScienceDoctoral Dissertation2024-08-27