Fig. 3: Absolute quantification of oxidative lesions and abasic sites in HOCl-treated synthetic oligoribonucleotides.
From: Mapping of HOCl-oxidized RNA identifies abasic sites as major damage and oxidation product of oxo8G
a–c Binary oligonucleotides composed of U and either C, A or G were subjected to HOCl oxidation (0 to 500 µM Cl2). Quantification of parent [U (gray), C (green), A (blue), G (pink)] and oxidized nucleosides [Cl5C (dark green), Cl5U (dark gray), ho5C (light green), ho5U (light gray), Cl8A (dark blue), oxo8A (light blue), Cl8G, (dark pink), oxo8G (light pink)] was performed using LC-MS/MS and external standards for calibration purposes. Absolute amounts are indicated in µM concentrations on the left y-axis relative to the initial reaction volume. The results of the CU combination are depicted in a, the AU combination in (b) and the GU combination in (c). d–f Absolute amounts of oxidation products presented in nM concentrations of the CU combination (d), the AU combination (e) and the GU combination (f). g, h LC-MS/MS quantification of a binary AG composition expressed in µM for both parent nucleosides (g) and in nM for the corresponding oxidation products (h). i Proposed progression of multiple oxidation events preferentially occurring at guanosines, ultimately yielding abasic sites denoted as 5’-ribose phosphates (5’-RP). Chemical structures are drawn with ChemDraw. j Absolute quantification of 5’-RP (light purple) and oxo8GMP (light pink) of an unmodified 38mer oxidized in the range of 0 to 500 µM Cl2 using LC-MS/MS and external calibration. The amount is normalized to the injected amount of RNA and the number of guanosines present in the sequence. The 5’-RP shows steadily increasing amounts whereas oxo8GMP is present at maximum concentrations at 50 µM Cl2. k Absolute quantification of 5’-RP (light purple) and oxo8GMP (light pink) of an oxo8G-modified 38mer at position 16 oxidized in the range of 0 to 500 µM Cl2 using LC-MS/MS and external calibration as in j. The amount is normalized to the injected amount of RNA and the number of guanosines present in the sequence. The decreasing oxo8GMP signal at 0, 10, and 25 µM Cl2 originates from the oxo8G site at position 16, whereas oxo8GMP signals at higher applied oxidant concentrations presumably correspond to guanosines other than G16. LC-MS data of n = 3 biological replicates are shown as mean values ± SD. Source data are provided as a Source Data file.
