Direct and Indirect Photochemical Reactions in Viral RNA Measured with RT-qPCR and Mass Spectrometry

RNA carries the genetic instructions for many viruses to replicate in their host cells. The photochemical reactions that take place in RNA and affect viral infectivity in natural and engineered environments, however, remain poorly understood. We exposed RNA oligomer segments from the genome of bacteriophage MS2 to UV₂₅₄, simulated sunlight, and singlet oxygen (¹O₂) and analyzed the oligomer reaction kinetics with reverse transcription quantitative PCR (RT-qPCR) and quantitative matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Following UV₂₅₄ exposure, quantitative MALDI-TOF-MS detected significantly more RNA modifications than did RT-qPCR, suggesting that certain chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In contrast, MALDI-TOF-MS tracked as much ¹O₂-induced RNA damage as RT-qPCR. After 5 h of simulated sunlight exposure (5100 J/m² UVB and 1.2 × 10⁵ J/m² UVA), neither MALDI-TOF-MS nor RT-qPCR detected significant decreases in the oligomer concentrations. High-resolution electrospray ionization (ESI)–Orbitrap MS analyses identified pyrimidine photohydrates as the major UV₂₅₄ products, which likely contributed to the discrepancy between the MS- and RT-qPCR-based results. Reactions between RNA oligomers and ¹O₂ resulted in an unidentified major product with a mass change of +6 Da. These results shed light on the photochemical reactions that take place in RNA and suggest that the analytical techniques used to detect RNA reactivity could bias the observed reaction kinetics.