RNA Oxidative Damage by the Iron-Fenton Reaction is Influenced by Bicarbonate Concentration and Ligand Strength

Oxidative damage to RNA is associated with neurodegeneration, cardiovascular diseases, and cancer development. Studies that monitor RNA damage by H₂O₂ often omit the physiological buffer bicarbonate in the reaction, which fails to account for the influence of the buffer on the iron-Fenton reaction. Herein, we monitored two in vitro systems to understand how bicarbonate redirects the iron-Fenton reaction from a hydroxyl radical (HO^•) generator in the absence of bicarbonate to one that predominantly yields carbonate radical anion (CO₃^•-) in the presence of this buffer. Using the HO^•-selective fluorophore terephthalic acid, we found that the Fe(II)-ligand identity impacted the bicarbonate concentration required to transition the Fenton reaction to predominantly yield CO₃^•-. These findings were then corroborated by following the oxidation of guanosine (rG), which reports on oxidation by both radicals, and uridine (rU) oxidation, which responds to only HO^• as the oxidizing species. The studies found that as the Fe(II)-ligand complex stability increased, the bicarbonate concentration inflection point to favor CO₃^•- production and rG oxidation also increased. Regardless of the ligand strength, the crossover values obtained were below physiologically relevant bicarbonate concentrations (<20 mM). Next, <i>Escherichia coli</i> or HEK293T cells were pre-equilibrated with bicarbonate from 0 to 20 mM before a bolus addition of H₂O₂. The bicarbonate-dependent inflection points for favoring CO₃^•- over HO^• (or ferryl) for <i>E. coli</i> (7.3 mM) and HEK293T (11.3 mM) cells differed, but were below physiologically relevant concentrations, supporting the hypothesis that the cellular iron-Fenton reaction normally yields CO₃^•-. The redox-cycling compound menadione was used for continuous in-cell generation of H₂O₂ to find bicarbonate dependencies in oxidation reactions of RNA. The studies herein point toward the redirection of the iron-Fenton reaction in cells to predominantly yield CO₃^•- that selectively damages rG sites in the transcriptome.