Abstract
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 dA8, dT8, dG8, dC8, and dA2dT2dG2dC2 as chemical building blocks of DNA with ARTP for 1–4 min, identified the mononucleotide products using 31P- and 1H-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 31P-and 1H-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 dT8 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., dT8 > dC8 > dA8 > dG8 > dA2dT2dG2dC2. 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.
Highlights
Center, Wuxi Research Institute of Applied Technologies, Tsinghua University, Wuxi 214072, People’s Republic of China. 3TmaxTree Biotechnology Co
The γ-phosphate group of dATP disappeared after 4-min treatment with ARTP, and the α- and β -phosphate groups were cleaved when treated for an additional time (Fig. 2a,b)
We found that various chemical structural changes to the artificial mononucleotides and oligonucleotides that may account mutagenesis for the wide and successful applications reported for ARTP-induced mutation breeding of diverse microbes and plants as well animals caused by ARTP treatment
Summary
Based on APGD, we developed an “atmospheric and room-temperature plasma (ARTP)” driven by a radio-frequency power supply and equipped with water-cooled bare-metallic copper electrodes (Fig. 1) We have employed this system to mutate the genomes of various microbes and plants as well as animals, including bacteria, microalgae, fungi, and yeast[1,4, 19,20,21,22,23,24]. The mechanism(s) behind ARTP mutagenesis is (are) presumably related to the actions of reactive oxygen species (ROS)/reactive nitrogen species (RNS) generated by the reaction of plasma with the water environment of ARTP-treated cellular samples These reactive species damage the cellular DNA and, induce the activation of repair systems such as the bacterial SOS repair s ystem[4,25]. The molecular dynamic simulation was performed using Discovery Studio v2.5 (DS) to estimate the stability of the synthesized oligonucleotides
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