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Hirofumi Kurita1 Tomoko Nakajima1 Kaori Sano1 Saki Miyachika1 Yoshito Uchihashi1 Natsuki Haruta1 Hachiro Yasuda1 Kazunori Takashima1 Akira Mizuno1

1, Toyohashi University of Tech, Toyohashi, , Japan

Cold atmospheric pressure plasmas have been intensively studied due to growing interest in biological and medical applications. Especially the plasma has been considered as a promising tool for cancer therapy. One of the proposed molecular mechanism is DNA damage-associated cell death. Therefore DNA is one of the most important biomolecular targets for investigating the effects of exposure to the plasma. Over the last decade, many studies have attempted to characterize DNA damage and the associated cellular responses induced by plasma irradiation. In the early stage of the investigation, most of the reports used isolated plasmid DNA molecules in liquids and the analysis was based on gel electrophoresis. For example, it has been reported that oxidative damage is induced by exposure to the plasma, resulting in single-strand breaks (SSBs) and double-strand breaks (DSBs) separated by conventional agarose gel electrophoresis [1]. In recent years, the analysis of genomic DNA in the plasma-irradiated cells were reported. For example, the single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring DNA damage. Although gel electrophoresis is relatively inexpensive and easy to perform, it requires long run times. Therefore we have developed non-electrophoretic methodologies. Our first investigation is a single-molecule-based method for evaluating strand breaks in large linear DNA molecules that allows the length of individual DNA molecules to be measured [2]. In this investigation plasma-induced DNA breakages have been kinetically analyzed. However, the single-molecule method requires the acquisition and processing of many fluorescence images for reliable analysis. We have also investigated rapid detection of DNA strand breaks induced by plasma irradiation using a molecular beacon (MB) [3]. MBs are oligonucleotides that adopt a stem-and-loop structure and carry a 5’-fluorescent moiety and a 3’-nonfluorescent quenching moiety. Scission of the stem by plasma irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the extent of DNA strand breaks. In addition, we reported that a plasma jet readily induced DNA strand breaks in synthetic models of tissue and cells, surprisingly without any significant rupture of the phospholipid membrane [4]. Furthermore, the feasibility of MB-based methodology for detecting intracellular DNA damage was investigated. Our novel methodology may allow investigations of the effects of atmospheric pressure plasma on DNA damage-associated cell death in plasma treatments.

[1] H. Yasuda, et al., Plasma Process. Polym., 5, 615-621 (2008)
[2] H. Kurita, et al., Appl. Phys. Lett., 99, 191504 (2011); H. Kurita, et al., Jpn. J. Appl. Phys., 53, 05FR01A (2014)
[3] H. Kurita, et al., Appl. Phys. Lett., 107, 263702 (2015)
[4] E. Szili, et al., J. Phys. D: Appl. Phys., 50, 274001 (2017)

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