Production, Isolation, and Characterization of Stable Isotope-Labeled Standards for Mass Spectrometric Measurements of Oxidatively-Damaged Nucleosides in RNA

RNA undergoes oxidatively induced damage in living organisms analogous to DNA. RNA is even more vulnerable to damage than DNA due to its greater abundance, single-strandedness, lack of repair and chromatin proteins shield, and instability, among other effects. RNA damage can adversely affect gene expression, leading to protein synthesis alterations, cell death, and other detrimental biological consequences. Growing indications suggest the involvement of oxidatively induced RNA damage in the pathogenesis of various human diseases, aging, and age-related diseases. Oxidatively induced damage can cause modifications to all four heterocyclic bases in RNA. Precise measurement of such modifications in RNA is essential for understanding the biological effects of oxidatively induced RNA damage. In the past, mass spectrometry has been used for this purpose. In mass spectrometric measurements, the use of stable isotope-labeled analogues of analytes as internal standards is essential for accurate quantifications. Past work utilized a stable isotope-labeled analogue of 8-hydroxyguanosine only as an internal standard. Thus, far, no stable isotope-labeled analogues of other oxidatively modified RNA nucleosides were available. In the present work, we report on the preparation, isolation, and characterization of the ¹³C- and ¹⁵N-labeled analogues of a variety of modified pyrimidine- and purine-derived RNA nucleosides. We also show the application of these internal standards for the measurement of oxidatively induced RNA damage in several commercially available RNA samples and in DNA along with DNA damage.