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, Escherichia coli 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 E. coli (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.