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# In situ monitoring of the influence of water on DNA radiation damage by near-ambient pressure X-ray photoelectron spectroscopy

## Abstract

Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.

## Introduction

The damage to biomolecules caused by ionizing radiation is the reason behind treating of cancer via radiation therapy1. Hereby, DNA damage is of key interest due to its central role in reproduction and mutation. In isolated DNA molecules, the damage can occur at its different building blocks, the sugar–phosphate backbone, and the nucleobases. The most important types of damage, which can lead to genetic instability, are single strand breaks (SSB) and double strand breaks (DSB) at the sugar–phosphate backbone and the loss or chemical modifications of the nucleobases. To improve therapeutic outcome and to develop more effective radiosensitizers, a better understanding of the underlying damage mechanisms is necessary1,2,3. Due to the high amount of water in biological tissue, most of the inelastic scattering processes between the incoming high-energy radiation (γ) and tissue occur with the solvent. Photons used in radiation therapy have energies in the MeV range. At these energies, secondary particles are produced by inelastic scattering processes such as the photo-electric effect, Compton scattering, Auger effect, or pair production4. The former scattering events produce additional electrons, while pair production results in the creation of an electron–positron pair. For x-ray photons with 1.4 keV kinetic energy, as used in this study, ionization is the dominant inelastic scattering process4. The ionization of water molecules produces secondary particles as described by the net-ionization reaction5,6

$$\gamma +2\ {\mathrm{H}}_{2}\mathrm{O}\to {\mathrm{H}}_{2}{\mathrm{O}}^{+}+{e}^{-}+{\mathrm{H}}_{2}\mathrm{O}\to {\mathrm{H}}_{3}{\mathrm{O}}^{+}+\mathrm{O}{\mathrm{H}}^{\bullet }+{\mathrm{e}}^{-}$$
(1)

## Results

Here, we present for the first time simultaneous induction and probing of ionizing radiation damage to DNA by near-ambient pressure (NAP) XPS under H2O and N2 atmospheres, as well as standard UHV conditions (Fig. 1).

### Direct DNA damage

In the following, the vacuum results are compared with previous studies, before the modifications of the underlying damaging channels by the presence of N2 and H2O atmosphere, are discussed. In Fig. 2, photoelectrons ejected under vacuum conditions at C1s (left column), O1s (central column), and N1s (right column) binding energies (BE) are shown together with the deconvolution results at the beginning (top row) and end (bottom row) of the exposure. The peaks are assigned to different chemical bonds (Table 1) based on literature values19,21,26. The C1s signal is deconvoluted into four different peaks at BEs at (1) 285 eV which belongs to hydrocarbons, (2) around 286–287 eV originating from alcohol (C–OH), backbone (C–O–P), cyclic ether (C–O–C), and carbon bond to nitrogen (C–N), while (3) at 288 eV to C=O, C=N, and (4) at 289 eV to N–(C=O)–N. The O1s spectra are deconvoluted into components assigned either to double bonded oxygen at 531 eV (C=O, P=O) or single bonded oxygen in the backbone (C–O–P), sugar (C–O–C), or alcohol (C–OH) at 532 eV, as well as water at a BE of around 536 eV. The N1s spectra are deconvoluted into contributions from imines around 399 eV and from amines, amides, and urethanes around 400.5 eV. Alternative deconvolution strategies based on three peaks are discussed in the Supplementary Information (SI), Sec. 1.4. Changes in the peak intensities during the course of the irradiation (Figs. 2 and 3) occur due to damage at the different DNA subunits. An increase of a certain species can be assigned to an addition to or formation of radicals at the DNA, which are precursors of new products. Decrease of a relative peak area with time corresponds to the cleavage of an associated bond (Table 1) or the release of a fragment from the surface19. The various types of DNA damage, namely strand breaks, sugar decomposition, and base damage, are related to different molecular groups. Although additional minor effects might contribute to the chemical changes observed, they are still of highest biological relevance, since they can be the starting point of mutation and apoptosis7. Thus, changes of the N1s signals can be assigned to dehydrogenation of the amino groups or to the breaking of the N-glycosidic bond, which can lead to a base release19. Sugar decomposition can result in the loss of C–OH groups and a decrease of the C1s and O1s signals over time. The decrease of signals associated with the C–O–P bonds at the DNA backbone at C1s 286 eV and O1s 532 eV with simultaneous formation of P–O and C–C at 285 eV can be interpreted as formation of strand breaks at the sugar–phosphate backbone21. This behavior, associated with strand break induction, is the predominant trend observed during XPS measurements under vacuum conditions (Fig. 4, top row). On the same time scale, the total peak intensities and the peak areas associated with nitrogen bonds are relatively unaffected. Both trends are in excellent agreement with XPS data of dsDNA as measured by Rosenberg et al.21. Furthermore, the slight decrease of the N1s signal at 400.5 eV compared to the imine signal (Fig. 4, top row right) is similar to the results by Ptasinska et al. reported for calf-thymus dsDNA19. Recent results from XPS studies on nucleotides show similar trends of oxygen-related peaks, while differing for the imine-related signals which decreased during irradiation24. This variation might be explained by the differences in LEE localization at the nucleobases between dsDNA and single nucleotides. Especially, since the formation of a TNI at the nucleobases and subsequent ET to the backbone is one of the most frequent damage pathways for strand break induction by LEE11,27. All this damage can be attributed to direct effects originating from x-ray photons or secondary electrons, since water is mostly absent under vacuum conditions. Here, on average less than 0.3 water molecules per nucleotide were present, as determined from the O1s spectral intensities (Fig. 2, center column). Thus, the initial distribution of direct ionization events at DNA molecules correlates with the electron density at the different subgroups7,28. Upon ionization, an electron is ejected, while the hole can migrate within the DNA. The hole can lead to a SSB via reactions at the DNA backbone or localize at the bases with a preference for guanine28,29. Besides ionization events, LEE with energies below 20 eV can form TNI and damage DNA by resonant processes such as DEA or shape resonances. The associated LEE damage yields depend on the initial electron capture probability of the DNA bases to form TNIs. Strand breaks are predominantly produced by cleavage of the C–O bond in the backbone, while base releases occur after cleavage of the N-glycosidic bond30. For LEE between 4 and 16 eV, the yield of strand breaks is approximately twice as high as the yield of base release31. Thus, taking into account the high abundance of LEE and their preferential cleavage of the DNA backbone (Fig. 2), we can confirm previous studies21,24,25, and conclude that LEE cause the majority of the strand breaks observed under vacuum conditions.

### The influence of nitrogen

The measurements we presented so far were performed in an UHV-XPS system which provides the charge compensation needed in vacuum. They were used to compare the behavior of our DNA samples with the literature data. In contrast, during NAP-XPS measurements, the charge compensation is achieved by the presence of gases. Since vacuum and NAP measurements were performed in devices which differ in a variety of properties (e.g., x-ray fluence, incident angle, spot geometry, transmission function, charge compensation), results can only be compared qualitatively. Thus, to enable a direct comparison between hydrated and nonhydrated DNA, measurements under N2 and H2O atmosphere were performed within the same NAP-XPS setup (compare SI, Fig. S2). The qualitative evolution of the XPS data between vacuum (Fig. 4, top row) and N2 (Fig. 4, center row) are similar, even though the overall damage observed over time is slightly higher for N2. Here, modifications of various damaging channel under the presence of N2 might occur. Thus, x-ray interactions with N2 molecules have to be considered, since their dissociation products are also potential damaging agents. However, in a previous study it was concluded that irradiated pure N2 produces much less damage by indirect effects than other gases, such as O2 or NO2, which agrees with our results32. There, it appeared that N2 molecules mostly damage DNA in combination with ROS, which are not present under the anaerobic conditions applied here. Furthermore, at NAP conditions, the gas molecules have a mean free path of less than 1 μm. Thus, only x-ray interaction near the surface can produce reactive species which are able to reach the DNA32. To gain deeper insight into the inelastic scattering processes, and herewith to direct damage effects, the production of reactive species and the energy deposit in DNA, particle scattering simulations were performed. Hereby, the impact of x-ray photons passing through the respective vacuum, nitrogen, or water atmosphere, an additional surface layer of absorbed gases, and the DNA itself was simulated. Since XPS approximately probes the DNA until a depth of 10 nm, only scattering events in this region were evaluated. The results show that each photon deposits on average 1.9 eV in the DNA layer, whereby less than 0.2 % of them ionize a DNA molecule and produce an electron–hole pair. Approximately, the same number of electrons thermalizes in the DNA region, and therefore are able to form TNIs. All these results vary less than 5% between vacuum, N2, and H2O atmosphere (compare SI, Table S3). Thus, the changes observed in DNA damage induction under different atmospheric conditions (Fig. 4) are not caused by direct effects. From these considerations, we can conclude that experiments under N2 atmosphere allow us to study direct damage effects caused by high-energy radiation and LEE, as suggested by Alizadeh et al.32. Thus, comparison with results from water atmosphere allows in situ monitoring of chemical changes induced by indirect effects.

## Discussion

In summary, we have quantified radiation induced damage at fully hydrated DNA molecules directly by XPS. The comparison between irradiations under N2 and H2O atmosphere revealed a strong increase in the overall damage upon hydration. Contributions from direct and indirect processes were separated by combining NAP-XPS experiments with Monte Carlo particle scattering simulations. In dry DNA, high-energy photons and LEE showed a preference for the induction of DNA strand breaks at the backbone. In contrast, deoxyribose and nucleobases were affected much less by direct damage. This behavior changed dramatically when water was present. Here, excited water molecules and hydroxyl radicals initiated indirect damage processes which lead to base modification and base release. It was revealed that base modifications become predominant here, even when the total amount of strand breaks increased further as well.

## Methods

### Sample preparation

Herring sperm DNA in ultrapure water was obtained from Sigma-Aldrich. The DNA was dropcasted on carefully cleaned microscopy slides and dried with Ar gas (Linde). All three DNA samples were stored on dry ice until they were placed in the XPS chambers.

### XPS measurements

All XPS measurements were performed with Al Kα radiation (E = 1486.6 eV). The UHV-XPS measurements were done with an AXIS Ultra DLD photoelectron spectrometer (Kratos Analytical, Manchester, UK). Here, the pressure was below 1 × 10−8 mbar. Laboratory NAP XPS measurements were done with an EnviroESCA (SPECS GmbH, Berlin, Germany)47,48. During the NAP-XPS measurements, the pressure was kept in the NAP regime between 4 and 14 mbar. Details on experimental procedures (SI Secs. 1.1 and 1.2) and data analysis (SI Sec. 1.3) are provided in the SI. XPS fitting results are summarized in SI in Tables S1–S3.

### Particle scattering simulations

Particle scattering simulations were performed with the Geant4 10.5 framework and the Topas 3.3 interface, the Livermore scattering cross sections and 1 nm particle cut length49,50. Simulation details are given in SI Sec. 2 and results are summarized in SI Table S4.

## Data availability

The data that support the findings of this study are available from M.B.H. upon request.

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## Acknowledgements

The authors thank Dr. Tihomir Solomun for useful discussions and critical reading of the manuscript, L. Cordsmeier for assistance with ChemDraw and K. Fast for preparing sketches of the experimental setup. M.B.H. acknowledges funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) 442240902/HA 8528/2-1.

## Funding

Open Access funding enabled and organized by Projekt DEAL.

## Author information

Authors

### Contributions

M.B.H. conceived the study, prepared the DNA samples, and discussed results and wrote the paper with contributions from the other authors. J.R. performed the vacuum measurement and measurement of uncertainty analysis and contributed to the interpretation of the data and the discussion. P.M.D. performed NAP-XPS measurements and contributed to the interpretation of the data and the discussion.

### Corresponding author

Correspondence to Marc Benjamin Hahn.

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### Competing interests

The authors declare no competing interests.

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Hahn, M.B., Dietrich, P.M. & Radnik, J. In situ monitoring of the influence of water on DNA radiation damage by near-ambient pressure X-ray photoelectron spectroscopy. Commun Chem 4, 50 (2021). https://doi.org/10.1038/s42004-021-00487-1

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