Methylation of DNA CpG domains in cellular DNA is a key mechanism of epigenetic regulation. Disruptions in the processes maintaining DNA methylation can lead to diseases like cancer. The radiation response of certain cancer cells may be affected by their DNA methylation levels, which may have consequences in their response to radiotherapy. In this work, we utilized DNA origami nanotechnology to examine whether DNA methylation impacts DNA response to ionizing radiation in solution before biological processes come into play. Our findings reveal that a protective effect is achieved with just a few methylated CpG adducts. Both low-LET (electron) and high-LET (carbon ion) irradiation show a reduced lesion count in methylated DNA, as indicated by qPCR results. AFM single-molecule observations using DNA origami nanoframes suggest fewer double-strand breaks in methylated DNA after carbon ion irradiation. This radioprotective effect may contribute to the differential radiation response of cellular DNA and should be considered when predicting and evaluating DNA radiation damage yields.

Department of Dynamics of Molecules and Clusters J Heyrovský Institute of Physical Chemistry of the CAS Dolejškova 3 182 23 Prague Czech Republic

Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics of the CAS Vídeňská 1083 142 20 Prague Czech Republic

Normandie Univ ENSICAEN UNICAEN CEA CNRS CIMAP Boulevard Henri Becquerel BP 5133 140 70 Caen cedex 5 France

Nuclear Physics Institute of the CAS Řež 130 250 68 Řež Czech Republic

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Moore L. D., Le T., Fan G.. DNA Methylation and Its Basic Function. Neuropsychopharmacology. 2013;38:23–38. doi: 10.1038/npp.2012.112. PubMed DOI PMC

Greenberg M. V. C., Bourc’his D.. The Diverse Roles of DNA Methylation in Mammalian Development and Disease. Nat. Rev. Mol. Cell Biol. 2019;20:590–607. doi: 10.1038/s41580-019-0159-6. PubMed DOI

Smith J., Sen S., Weeks R. J., Eccles M. R., Chatterjee A.. Promoter DNA Hypermethylation and Paradoxical Gene Activation. Trends Cancer. 2020;6:392–406. doi: 10.1016/j.trecan.2020.02.007. PubMed DOI

Sulkowski P. L., Oeck S., Dow J., Economos N. G., Mirfakhraie L., Liu Y., Noronha K., Bao X., Li J., Shuch B. M., King M. C., Bindra R. S., Glazer P. M.. Oncometabolites Suppress DNA Repair by Disrupting Local Chromatin Signalling. Nature. 2020;582:586–591. doi: 10.1038/s41586-020-2363-0. PubMed DOI PMC

Young I.-C., Brabletz T., Lindley L. E., Abreu M., Nagathihalli N., Zaika A., Briegel K. J.. Multi-Cancer Analysis Reveals Universal Association of Oncogenic LBH Expression with DNA Hypomethylation and WNT-Integrin Signaling Pathways. Cancer Gene Ther. 2023;30:1234–1248. doi: 10.1038/s41417-023-00633-y. PubMed DOI PMC

Schübeler D.. Function and Information Content of DNA Methylation. Nature. 2015;517:321–326. doi: 10.1038/nature14192. PubMed DOI

Zhu X., Wang Y., Tan L., Fu X.. The Pivotal Role of DNA Methylation in the Radio-Sensitivity of Tumor Radiotherapy. Cancer Med. 2018;7:3812–3819. doi: 10.1002/cam4.1614. PubMed DOI PMC

Jin Z., Liu Y.. DNA Methylation in Human Diseases. Genes Dis. 2018;5:1–8. doi: 10.1016/j.gendis.2018.01.002. PubMed DOI PMC

Zhu X., Gao Y., Feng Y., Zheng J., Dong Y., Zhang P., Zhu Y., Fan G.. DNA Methylation in Small Cell Lung Cancer. Clin. Transl. Discovery. 2023;3:e191. doi: 10.1002/ctd2.191. DOI

Miousse I. R., Kutanzi K. R., Koturbash I.. Effects of Ionizing Radiation on DNA Methylation: From Experimental Biology to Clinical Applications. Int. J. Radiat. Biol. 2017;93:457–469. doi: 10.1080/09553002.2017.1287454. PubMed DOI PMC

Belli M., Tabocchini M. A.. Ionizing Radiation-Induced Epigenetic Modifications and Their Relevance to Radiation Protection. Int. J. Mol. Sci. 2020;21:5993. doi: 10.3390/ijms21175993. PubMed DOI PMC

Chi H.-C., Tsai C.-Y., Tsai M.-M., Lin K.-H.. Impact of DNA and RNA Methylation on Radiobiology and Cancer Progression. Int. J. Mol. Sci. 2018;19:555. doi: 10.3390/ijms19020555. PubMed DOI PMC

Lima F., Ding D., Goetz W., Yang A. J., Baulch J. E.. High LET 56Fe Ion Irradiation Induces Tissue-Specific Changes in DNA Methylation in the Mouse. Environ. Mol. Mutagen. 2014;55:266–277. doi: 10.1002/em.21832. PubMed DOI

Aypar U., Morgan W. F., Baulch J. E.. Radiation-Induced Epigenetic Alterations after Low and High LET Irradiations. Mutat. Res./Fundam. Mol. Mech. Mutagen. 2011;707:24–33. doi: 10.1016/j.mrfmmm.2010.12.003. PubMed DOI

Li T., Mao C., Wang X., Shi Y., Tao Y.. Epigenetic Crosstalk Between Hypoxia and Tumor Driven by HIF Regulation. J. Exp. Clin. Cancer Res. 2020;39:224. doi: 10.1186/s13046-020-01733-5. PubMed DOI PMC

Solov’yov A. V., Verkhovtsev A. V., Mason N. J.. et al. Condensed Matter Systems Exposed to Radiation: Multiscale Theory, Simulations, and Experiment. Chem. Rev. 2024;124:8014–8129. doi: 10.1021/acs.chemrev.3c00902. PubMed DOI PMC

Schürmann R., Vogel S., Ebel K., Bald I.. The Physico-Chemical Basis of DNA Radiosensitization: Implications for Cancer Radiation Therapy. Chem. - Eur. J. 2018;24:10271–10279. doi: 10.1002/chem.201800804. PubMed DOI

Ameixa J., Bald I.. Unraveling the Complexity of DNA Radiation Damage Using DNA Nanotechnology. Acc. Chem. Res. 2024;57:1608–1619. doi: 10.1021/acs.accounts.4c00121. PubMed DOI PMC

Leung W. Y., Murray V.. The Influence of DNA Methylation on the Sequence Specificity of UVB- and UVC-Induced DNA Damage. J. Photochem. Photobiol., B. 2021;221:112225. doi: 10.1016/j.jphotobiol.2021.112225. PubMed DOI

Kogikoski S., Ameixa J., Mostafa A., Bald I.. Lab-on-a-DNA Origami: Nanoengineered Single-Molecule Platforms. Chem. Commun. 2023;59:4726–4741. doi: 10.1039/D3CC00718A. PubMed DOI PMC

Rajendran A., Endo M., Sugiyama H.. Single-Molecule Analysis Using DNA Origami. Angew. Chem., Int. Ed. 2012;51:874–890. doi: 10.1002/anie.201102113. PubMed DOI

Endo M., Katsuda Y., Hidaka K., Sugiyama H.. Regulation of DNA Methylation Using Different Tensions of Double Strands Constructed in a Defined DNA Nanostructure. J. Am. Chem. Soc. 2010;132:1592–1597. doi: 10.1021/ja907649w. PubMed DOI

Sala L., Lyshchuk H., Sáchová J., Chvátil D., Kočišek J.. Different Mechanisms of DNA Radiosensitization by 8-Bromoadenosine and 2’-Deoxy-2’-fluorocytidine Observed on DNA Origami Nanoframe Supports. J. Phys. Chem. Lett. 2022;13:3922–3928. doi: 10.1021/acs.jpclett.2c00584. PubMed DOI PMC

Sala L., Zerolová A., Rodriguez A., Reimitz D., Davídková M., Ebel K., Bald I., Kočišek J.. Folding DNA into Origami Nanostructures Enhances Resistance to Ionizing Radiation. Nanoscale. 2021;13:11197–11203. doi: 10.1039/D1NR02013G. PubMed DOI PMC

Ameixa J., Sala L., Kocišek J., Bald I.. Radiation and DNA Origami Nanotechnology: Probing Structural Integrity at the Nanoscale. ChemPhysChem. 2025;26:e202400863. doi: 10.1002/cphc.202400863. PubMed DOI PMC

Králík M., Šolc J., Chvátil D., Krist P., Turek K., Granja C.. Microtron MT 25 as a Source of Neutrons. Rev. Sci. Instrum. 2012;83:083502. doi: 10.1063/1.4739404. PubMed DOI

Rothard H.. CIRIL: Interdisciplinary Research at GANIL. Nucl. Phys. News. 2022;32:29–33. doi: 10.1080/10619127.2021.1990680. DOI

Sala L., Zerolová A., Vizcaino V., Mery A., Domaracka A., Rothard H., Boduch P., Pinkas D., Kocišek J.. Ion Beam Processing of DNA Origami Nanostructures. Beilstein J. Nanotechnol. 2024;15:207–214. doi: 10.3762/bjnano.15.20. PubMed DOI PMC

Brabcová K. P., Sihver L., Ukraintsev E., Štěpán V., Davídková M.. How Detection of Plasmid DNA Fragmentation Affects Radiation Strand Break Yields. Radiat. Prot. Dosim. 2019;183:89–92. doi: 10.1093/rpd/ncy222. PubMed DOI

Beaudier P., Zein S. A., Chatzipapas K., Tran H. N., Devès G., Plawinski L., Liénard R., Dupuy D., Barberet P., Incerti S., Gobet F., Seznec H.. Quantitative Analysis of Dose Dependent DNA Fragmentation in Dry pBR322 Plasmid using Long Read Sequencing and Monte Carlo Simulations. Sci. Rep. 2024;14:18650. doi: 10.1038/s41598-024-69406-3. PubMed DOI PMC

Georgakilas A. G., O’Neill P., Stewart R. D.. Induction and Repair of Clustered DNA Lesions: What Do We Know So Far? Radiat. Res. 2013;180:100–109. doi: 10.1667/RR3041.1. PubMed DOI

Aten J. A., Stap J., Krawczyk P. M., van Oven C. H., Hoebe R. A., Essers J., Kanaar R.. Dynamics of DNA Double-Strand Breaks Revealed by Clustering of Damaged Chromosome Domains. Science. 2004;303:92–95. doi: 10.1126/science.1088845. PubMed DOI

Bertolet A., Ramos-Méndez J., McNamara A., Yoo D., Ingram S., Henthorn N., Warmenhoven J.-W., Faddegon B., Merchant M., McMahon S. J., Paganetti H., Schuemann J.. Impact of DNA Geometry and Scoring on Monte Carlo Track-Structure Simulations of Initial Radiation-Induced Damage. Radiat. Res. 2022;198:207–220. doi: 10.1667/RADE-21-00179.1. PubMed DOI PMC

Psonka K., Gudowska-Nowak E., Brons S., Elsässer T., Heiss M., Taucher-Scholz G.. Ionizing Radiation-Induced Fragmentation of Plasmid DNA – Atomic Force Microscopy and Biophysical Modeling. Adv. Space Res. 2007;39:1043–1049. doi: 10.1016/j.asr.2007.02.089. DOI

Pachnerová Brabcová K., Sihver L., Yasuda N., Matuo Y., Štěpán V., Davídková M.. Clustered DNA Damage on Subcellular Level: Effect of Scavengers. Radiat. Environ. Biophys. 2014;53:705–712. doi: 10.1007/s00411-014-0557-2. PubMed DOI

Vyšín L., Brabcová K. P., Štěpán V.. et al. Proton-induced direct and indirect damage of plasmid DNA. Radiat. Environ. Biophys. 2015;54:343–352. doi: 10.1007/s00411-015-0605-6. PubMed DOI

Roush A. E., Riaz M., Misra S. K., Weinberger S. R., Sharp J. S.. Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris­(hydroxymethyl)­aminomethane. J. Am. Soc. Mass Spectrom. 2020;31:169–172. doi: 10.1021/jasms.9b00088. PubMed DOI PMC

Sala L., Rakovský J., Zerolová A., Kočišek J.. Light-Induced Damage to DNA Origami Nanostructures in the 193 nm-310 nm Range. J. Phys. B: At. Mol. Opt. Phys. 2023;56:185101. doi: 10.1088/1361-6455/acf3bd. DOI

Rezaee M., Adhikary A.. The Effects of Particle LET and Fluence on the Complexity and Frequency of Clustered DNA Damage. DNA. 2024;4:34–51. doi: 10.3390/dna4010002. PubMed DOI PMC

Vyšín L., Burian T., Ukraintsev E., Davídková M., Grisham M. E., Heinbuch S., Rocca J. J., Juha L.. Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation. Radiat. Res. 2018;189:466–476. doi: 10.1667/RR14825.1. PubMed DOI

Perstin A., Poirier Y., Sawant A., Tambasco M.. Quantifying the DNA-damaging Effects of FLASH Irradiation With Plasmid DNA. Int. J. Radiat. Oncol. Biol. Phys. 2022;113:437–447. doi: 10.1016/j.ijrobp.2022.01.049. PubMed DOI

Prise K. M., Gillies N. E., Michael B. D.. Further Evidence for Double-Strand Breaks Originating from a Paired Radical Precursor from Studies of Oxygen Fixation Processes. Radiat. Res. 1999;151:635–641. doi: 10.2307/3580201. PubMed DOI

Buglewicz D. J., Su C., Banks A. B., Stenger-Smith J., Elmegerhi S., Hirakawa H., Fujimori A., Kato T. A.. Metal Ions Modify In Vitro DNA Damage Yields with High-LET Radiation. Toxics. 2023;11:773. doi: 10.3390/toxics11090773. PubMed DOI PMC

Madugundu G. S., Cadet J., Wagner J. R.. Hydroxyl-Radical-Induced Oxidation of 5-Methylcytosine in Isolated and Cellular DNA. Nucleic Acids Res. 2014;42:7450–7460. doi: 10.1093/nar/gku334. PubMed DOI PMC

Rooman M., Pucci F.. Estimating the Vertical Ionization Potential of Single-Stranded DNA Molecules. J. Chem. Inf. Model. 2023;63:1766–1775. doi: 10.1021/acs.jcim.2c01525. PubMed DOI

Uddin I. A., Stec E., Papadantonakis G. A.. Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs. ChemPhysChem. 2024;25:e202300946. doi: 10.1002/cphc.202400391. PubMed DOI

Kumari N., Vartak S. V., Dahal S., Kumari S., Desai S. S., Gopalakrishnan V., Choudhary B., Raghavan S. C.. G-quadruplex Structures Contribute to Differential Radiosensitivity of the Human Genome. iScience. 2019;21:288–307. doi: 10.1016/j.isci.2019.10.033. PubMed DOI PMC