**5. Electron attachment to short single‐stranded and plasmid DNA**

Cellular DNA consists of a double‐stranded helical structure, composed of two long polynu‐ cleotide chains [75]. Thus, as already mentioned in the Introduction section, in order to systematically understand LEE damage mechanisms and their role in radiation DNA damage, molecular targets of increasing complexity were studied, from simple molecules containing just two of the basic subunits (e.g., a phosphate group coupled with a sugar or a nucleoside having a DNA base + sugar), via synthetic, single‐ and double‐stranded oligonucleotides, containing multiple nucleotides to plasmid and other cellular DNA with many thousands of base pairs.

Even though most simple DNA components may be easily vaporized for experimental investigation in the gas phase, the larger units such as nucleosides and nucleotides usually decompose during evaporation [12]. In any case, the condensed phase is certainly the more appropriate environment to study problems relevant to radiation damage in biomolecular systems. The experimental methods and techniques, used in the condensed phase, differ from those in the gas phase. Most condensed phase experiments are achieved by bombarding thin films (2–10 nm) of oligonucleotides or plasmid DNA with an energy‐selected beam of LEEs from an electron gun or an electron monochromator. To prevent excessive charging, these thin‐ film biological samples are deposited onto a metal substrate by spin‐coating, lyophilisation (freeze‐drying), or molecular self‐assembly, as in the case of thiolated DNA on gold substrates [10] and 1,3‐diaminopropane layer plasmid on graphite [76]. The LEE‐induced damage to plasmid and linear DNA films has then been investigated by (1) measuring electron‐stimulated desorption (ESD) of anions, (2) imaging the breaks by atomic force and scanning tunneling microscopies, and (3) analyzing, after bombardment, the change of DNA topology by gel electrophoresis or the molecular content by high‐ performance liquid chromatography (HPLC) and mass spectroscopy [35, 77].

chemistry induced by DEA was completely suppressed by methylation in the electron energy

In recent years, platinum‐based drugs were also investigated regarding their decomposition by LEEs. It was suggested that in concomitant treatment in which chemotherapeutic drugs and radiotherapy are combined, one possible mechanism responsible for the observed synergy between treatments is the enhancement in the number of secondary species induced by primary radiation in the vicinity of the binding site of the platinum compounds in DNA (see Section 8). The gas‐phase DEA studies of PtBr<sup>2</sup> in the electron energy range between 0 and 10 eV showed the formation of the Br anion via two possible channels. The most dominant channels were assigned to the Br‾ + PtBr dissociation limit reached at ∼1 eV and the higher

The observation that all these radiosenisitizers exhibit DEA with high effciency, even close to 0 eV, may have significant implications for the development and use of these drugs in tumor radiation therapy. Considering their use as radiosensitizers, their fragmentation and the resulting generation of radicals at very low electron energies may be a key in understanding

Cellular DNA consists of a double‐stranded helical structure, composed of two long polynu‐ cleotide chains [75]. Thus, as already mentioned in the Introduction section, in order to systematically understand LEE damage mechanisms and their role in radiation DNA damage, molecular targets of increasing complexity were studied, from simple molecules containing just two of the basic subunits (e.g., a phosphate group coupled with a sugar or a nucleoside having a DNA base + sugar), via synthetic, single‐ and double‐stranded oligonucleotides, containing multiple nucleotides to plasmid and other cellular DNA with many thousands of

Even though most simple DNA components may be easily vaporized for experimental investigation in the gas phase, the larger units such as nucleosides and nucleotides usually decompose during evaporation [12]. In any case, the condensed phase is certainly the more appropriate environment to study problems relevant to radiation damage in biomolecular systems. The experimental methods and techniques, used in the condensed phase, differ from those in the gas phase. Most condensed phase experiments are achieved by bombarding thin films (2–10 nm) of oligonucleotides or plasmid DNA with an energy‐selected beam of LEEs from an electron gun or an electron monochromator. To prevent excessive charging, these thin‐ film biological samples are deposited onto a metal substrate by spin‐coating, lyophilisation (freeze‐drying), or molecular self‐assembly, as in the case of thiolated DNA on gold substrates [10] and 1,3‐diaminopropane layer plasmid on graphite [76]. The LEE‐induced damage to plasmid and linear DNA films has then been investigated by (1) measuring electron‐stimulated desorption (ESD) of anions, (2) imaging the breaks by atomic force and scanning tunneling microscopies, and (3) analyzing, after bombardment, the change of DNA topology by gel

their action and the molecular mechanisms necessary to improve radiotherapy.

**5. Electron attachment to short single‐stranded and plasmid DNA**

range below 2 eV.

192 Radiation Effects in Materials

base pairs.

energy channel to Br‾ + Pt + Br [74].

Oligomers of single‐stranded DNA containing the four bases (e.g., G, C, A, and T), which are among the simplest forms of DNA, have made the analysis of degradation products much simpler than would be the case for longer single‐ and double‐stranded configurations. Short oligomers deposited onto metal surfaces (e.g., tantalum, platinum, and gold) as films of different thicknesses (1–5 ML) were bombarded with LEEs and produced fragments analyzed by HPLC [77]. The results for the GCAT oligonucleotide indicated that strand breaks occur preferentially by cleavage of the C–O bond rather than the P–O bond, with two maxima at electron energies of 6 and 10 eV [78, 79].

Recently, Bald and co‐workers demonstrated the visualization of LEE‐induced bond cleavage in DNA origami‐based DNA nanoarrays on the single‐molecule level using atomic force microscopy (AFM) [80–82]. This novel method has a number of advantages: (1) only miniscule amounts of material are required to create submonolayer surface coverage, because of the facility to detect the DNA strand breaks at a single‐molecule level; (2) within a single experi‐ ment, more than one oligonucleotide sequence with various arrangements can be irradiated to efficiently compare a number of different DNA structures; (3) the method represents a simple way to obtain absolute strand break cross sections, thus providing benchmark values for further experimental and theoretical studies, and finally (4) this technique is not limited to single strands, but can be extended to quantify DSBs and to investigate higher order DNA structures.

Applying this technique, Bald and coworkers compared the absolute strand break cross sections of different 13‐mer oligonucleotide sequences (i.e., 5'‐TT(*X*T*X*)3TT, with *X* = A, C, or G) to evaluate the role of the different DNA nucleobases in DNA strand breakage. They also studied the sensitizing effect of incorporation of 5‐bromouracil (BrU) by comparing the absolute strand break cross sections for the sequences 5'‐TT(*X*BrU*X*)3TT, with *X* = A, C, or G. The observed trend in the absolute strand break cross sections agrees qualitatively with the previous HPLC studies investigating the fragmentation of oligonucleotide trimers of the sequence T*X*T, with *X* = A, C, G, irradiated with 10 eV electrons [83]. Additionally, the cross sections measured with this method are comparable in magnitude with the cross sections for strand breaks in different plasmid DNA molecules induced by 1–10 eV electrons, as deter‐ mined by agarose gel electrophoresis [84, 85]. The DNA nanoarray technique thus bridges the gap between very large genomic double‐stranded DNA and very short oligonucleotides, and enables the detailed investigation of sequence‐dependent processes in DNA radiation damage. Further experimental and theoretical studies are carried out covering a broad range of electron energies and DNA sequences to elucidate the most relevant damage mechanisms [86].

In order to increase the complexity of targeted biomolecules, several studies have investigated the damage induced by LEEs in double‐stranded plasmid DNA. Due to the supercoiled arrangement of plasmid DNA, a single‐bond rupture in a DNA with a few thousand base pairs can produce a conformational change in the topology of the entire molecule. These changes include base alterations, abasic sites, intra‐ and inter‐strand base cross‐links, DNA adducts, and SSBs or DSBs; hence, these can be detected efficiently by techniques such as gel electro‐ phoresis. This technique can identify supercoiled (SC), nicked circular (C), full‐length linear (L), cross‐linked (CL), and short linear forms of DNA, which can be assigned to undamaged DNA, SSBs, DSBs, several types of cross‐linked DNA, and multiple double‐strand breaks (MDSBs), respectively [87].

Though it has been established that most of the strand breaks induced by ionizing radiation have been repaired by a DNA ligation step, a DSB represents a particularly detrimental lesion that poses a serious threat to the cell, since it usually cannot be easily repaired [88]. Indeed, even a single DSB can lead to cell death if left unrepaired or, more worryingly, it can cause mutagenesis and cancer if repaired improperly [89].

The results obtained for LEE‐irradiated supercoiled plasmid DNA in several investigations are well described in the literature and summarized in authoritative review articles [10, 11, 35]. These studies have shown that SSBs can occur as a result of DEA at electron energies well below electronic excitation and ionization thresholds (0.8–10 eV) [83, 90]. The results of Martin et al. [90] reveal two resonant peaks at 0.8 and 2.2 eV in the SSB yield function (i.e., the number of strand breaks versus the incident electron energy) via the formation of TMAs. These findings are consistent with theoretical calculations indicating that SSBs induced by near‐zero energy electrons are thermodynamically feasible [91–93]. Theoretical simulations of electron scatter‐ ing and electron capture via "shape" resonances support the role of LEEs in DNA strand breaks [94]. Theoretical calculations on scattering and attachment of LEEs to DNA components up to supercoiled plasmid DNA have been intensively reviewed in recent years [95, 96].

Another spatially resolved technique that exploits the use of graphene‐coated Au thin films and surface‐enhanced Raman spectroscopy (SERS) has recently emerged. Utilizing this technique, the sequence dependence of DNA damage at excitation energies < 5 eV can be studied [97]. Currently, Ptasińska and coworkers are performing a quantitative and qualitative investigation of the various types of damages to dry and hydrated DNA induced by exposure to helium and nitrogen atmospheric pressure plasma jets (APPJs). Since an APPJ contains multiple reactive species, including LEEs, also found in radiation chemistry, exposure to these plasma jets provides information on both the direct and indirect pathways to damaging DNA. Ptasińska and coworkers have employed nitrogen APPJ to induce DNA damage in SCC‐25 oral cancer cells, and have thus provided new insight into radiation damage to a cellular system [98].
