Fig. 2: HOCl induces concentration-dependent RNA damage and formation of distinct nucleoside oxidation products in vitro and in vivo.
From: Mapping of HOCl-oxidized RNA identifies abasic sites as major damage and oxidation product of oxo8G
a, b Denaturing PAGE of a 32P-labeled synthetic oligonucleotide treated in an in vitro HOCl oxidation series at concentrations ranging from 0 to 500 µM Cl2. Lanes 1 and 11 contain unlabeled ladders indicating 50, 100, and 200 nucleotides (nt). Lane 2 contains an untreated control, while lanes 3 to 10 contain samples oxidized from 0 to 500 µM Cl2. 100 ng was loaded for each sample. RNA was visualized using GelRed™ staining (a) and a 32P-phosphor scan (b). c Corresponding absorbance spectra of oxidized (0 - 500 µM Cl2) RNA samples after purification measured on a Nanodrop™ spectrophotometer. d Structures of HOCl oxidation products. Main nucleosides: uridine (U in gray), cytidine (C in green), adenosine (A in blue), guanosine (G in pink) and their corresponding oxidation products: 5-hydroxyuridine (ho5U in light gray), 5-chlorouridine (Cl5U in dark gray), 5-hydroxycytidine (ho5C in light green), 5-chlorocytidine (Cl5C in dark green), 8-oxoadenosine (oxo8A in light blue), 8-chloroadenosine (Cl8A in dark blue), 8-oxoguanosine (oxo8G in light pink) and 8-chloroguanosine (Cl8G in dark pink). Chemical structures are drawn with ChemDraw. e Absolute quantification of nucleosides derived from the oxidized oligonucleotide via LC-MS/MS using external standards for calibration. The synthetic, unmodified RNA oligonucleotides were subjected to HOCl treatment at the indicated concentrations. Nucleoside concentrations are indicated in µM relative to the volume of the oxidation reaction. The right y-axis is normalized to the total nucleoside content of the untreated control. Note that the apparent increase in pyrimidines at high concentrations of Cl2 results from a UV normalization bias caused by purine chromophore oxidation. f Low-abundant oxidation products are presented in nM concentrations. g Absolute quantification of a HOCl concentration series performed in vivo on S. cerevisiae cells. Cl2 concentrations are indicated on the x-axis and range from 0 to 20 mM Cl2. Total RNA was extracted and subjected to nucleoside analysis. The left y-axis indicates nucleoside concentrations in pmol per OD, and the right y-axis shows relative proportions normalized to the total nucleoside concentration in the untreated control. h Absolute amounts of the oxidized nucleosides displayed in fmol per OD on the left y‑axis. The right y-axis is normalized to the total nucleoside concentration in the untreated control. Data are presented as the mean of three biological replicates ± SD, n = 3. Source data are provided as a Source Data file. g, h Created in BioRender. Weber, M. (2025) https://BioRender.com/ap8gmkc.
