Insights into the molecular-level effects of atmospheric and room-temperature plasma on mononucleotides and single-stranded homo- and hetero-oligonucleotides

Atmospheric and room-temperature plasma (ARTP) has been successfully developed as a useful mutation tool for mutation breeding of various microbes and plants as well animals by genetic alterations. However, understanding of the molecular mechanisms underlying the biological responses to ARTP irradiation is still limited. Therefore, to gain a molecular understanding of how irradiation with ARTP damages DNA, we irradiated the artificially synthesized mononucleotides of dATP, dTTP, dGTP, and dCTP, and the oligonucleotides of dA(8), dT(8), dG(8), dC(8), and dA(2)dT(2)dG(2)dC(2) as chemical building blocks of DNA with ARTP for 1–4 min, identified the mononucleotide products using (31)P- and (1)H-nuclear magnetic resonance spectroscopy (NMR), and identified the oligonucleotide products using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) during ARTP treatment. The observed (31)P-and (1)H-NMR spectrum signals for the plasma-treated and untreated mononucleotides indicated that dATP was less stable to plasma irradiation than the other mononucleotides. The oligonucleotides after treatment with ARTP were found to have been broken into small fragments as shown by mass spectrometry, with the cleaved bonds and produced fragments identified according to their expected spectral m/z values or molecular weights derived from their m/z values. The stabilities of the oligonucleotides differed to ARTP irradiation, with dT(8) being the most stable and was more beneficial to stabilizing single-stranded oligonucleotide structures compared to the other base groups (A, G, and C). This was consistent with the average potential energy level obtained by the molecular dynamic simulation of the oligonucleotides, i.e., dT(8) > dC(8) > dA(8) > dG(8) > dA(2)dT(2)dG(2)dC(2). In summary, we found that ARTP treatment caused various structural changes to the oligonucleotides that may account for the wide and successful applications reported for ARTP-induced mutation breeding of various organisms.