Production, Isolation,\nand Characterization of Stable\nIsotope-Labeled Standards for Mass Spectrometric Measurements of Oxidatively-Damaged\nNucleosides in RNA

RNA undergoes oxidatively induced damage in living organisms\nanalogous\nto DNA. RNA is even more vulnerable to damage than DNA due to its\ngreater abundance, single-strandedness, lack of repair and chromatin\nproteins shield, and instability, among other effects. RNA damage\ncan adversely affect gene expression, leading to protein synthesis\nalterations, cell death, and other detrimental biological consequences.\nGrowing indications suggest the involvement of oxidatively induced\nRNA damage in the pathogenesis of various human diseases, aging, and\nage-related diseases. Oxidatively induced damage can cause modifications\nto all four heterocyclic bases in RNA. Precise measurement of such\nmodifications in RNA is essential for understanding the biological\neffects of oxidatively induced RNA damage. In the past, mass spectrometry\nhas been used for this purpose. In mass spectrometric measurements,\nthe use of stable isotope-labeled analogues of analytes as internal\nstandards is essential for accurate quantifications. Past work utilized\na stable isotope-labeled analogue of 8-hydroxyguanosine only as an\ninternal standard. Thus, far, no stable isotope-labeled analogues\nof other oxidatively modified RNA nucleosides were available. In the\npresent work, we report on the preparation, isolation, and characterization\nof the <sup>13</sup>C- and <sup>15</sup>N-labeled analogues of a variety\nof modified pyrimidine- and purine-derived RNA nucleosides. We also\nshow the application of these internal standards for the measurement\nof oxidatively induced RNA damage in several commercially available\nRNA samples and in DNA along with DNA damage.