Investigation of dissociative electron attachment to 2'-deoxycytidine-3'-monophosphate using DFT method and time dependent wave packet approach

Effect of electron correlation on single strand breaks (SSBs) induced by low energy electron (LEE) has been investigated in a fragment excised from a DNA, viz., 2'-deoxycytidine-3'-monophosphate [3'-dCMPH] molecule in gas phase at DFT-B3LYP/6-31+G(d) accuracy level and using local complex potential based time dependent wave packet (LCP-TDWP) approach. The results obtained, in conjunction with our earlier investigation, show the possibility of SSB at very low energy (0.15 eV) where the LEE transfers from π? to σ? resonance state which resembles a S(N)2 type mechanism. In addition, for the first time, an indication of quantum mechanical tunneling in strand breaking is seen from the highest anionic bound vibrational state (χ(5)), which may have a substantial role during DNA damage.     

Single,double, and multiple double strand breaks induced in DNA by 3-100 eV electrons

Nonthermal secondary electrons with initial kinetic energies below 100 eV are an abundant transient species created in irradiated cells and thermalize within picoseconds through successive multiple energy loss events. Here we show that below 15 eV such low-energy electrons induce single (SSB) and double (DSB) strand breaks in plasmid DNA exclusively via formation and decay of molecular resonances involving DNA components (base, sugar, hydration water, etc.). Furthermore, the strand break quantum yields (per incident electron) due to resonances occur with intensities similar to those that appear between 25 and 100 eV electron energy, where nonresonant mechanisms related to excitation/ionizations/dissociations are shown to dominate the yields, although with some contribution from multiple scattering electron energy loss events. We also present the first measurements of the electron energy dependence of multiple double strand breaks (MDSB) induced in DNA by electrons with energies below 100 eV. Unlike the SSB and DSB yields, which remain relatively constant above 25 eV, the MDSB yields show a strong monotonic increase above 30 eV, however with intensities at least 1 order of magnitude smaller than the combined SSB and DSB yields. The observation of MDSB above 30 eV is attributed to strand break clusters (nano-tracks) involving multiple successive interactions of one single electron at sites that are distant in primary sequence along the DNA double strand, but are in close contact; such regions exist in supercoiled DNA (as well as cellular DNA) where the double helix crosses itself or is in close proximity to another part of the same DNA molecule.     

Influence of organic ions on DNA damage induced by 1 eV to 60 keV electrons

We report the results of a study on the influence of organic salts on the induction of single strand breaks (SSBs) and double strand breaks (DSBs) in DNA by electrons of 1 eV to 60 keV. Plasmid DNA films are prepared with two different concentrations of organic salts, by varying the amount of the TE buffer (Tris-HCl and EDTA) in the films with ratio of 1:1 and 6:1 Tris ions to DNA nucleotide. The films are bombarded with electrons of 1, 10, 100, and 60?000 eV under vacuum. The damage to the 3197 base-pair plasmid is analyzed ex vacuo by agarose gel electrophoresis. The highest yields are reached at 100 eV and the lowest ones at 60 keV. The ratios of SSB to DSB are surprisingly low at 10 eV (～4.3) at both salt concentrations, and comparable to the ratios measured with 100 eV electrons. At all characteristic electron energies, the yields of SSB and DSB are found to be higher for the DNA having the lowest salt concentration. However, the organic salts are more efficient at protecting DNA against the damage induced by 1 and 10 eV electrons. DNA damage and protection by organic ions are discussed in terms of mechanisms operative at each electron energy. It is suggested that these ions create additional electric fields within the groove of DNA, which modify the resonance parameter of 1 and 10 eV electrons, namely, by reducing the electron capture cross-section of basic DNA units and the lifetime of corresponding transient anions. An interstrand electron transfer mechanism is proposed to explain the low ratios for the yields of SSB to those of DSB produced by 10 eV electrons.     

Resonant Formation of Strand Breaks in Sensitized Oligonucleotides Induced by Low‐Energy Electrons (0.5–9 eV)

Halogenated nucleobases are used as radiosensitizers in cancer radiation therapy, enhancing the reactivity of DNA to secondary low‐energy electrons (LEEs). LEEs induce DNA strand breaks at specific energies (resonances) by dissociative electron attachment (DEA). Although halogenated nucleobases show intense DEA resonances at various electron energies in the gas phase, it is inherently difficult to investigate the influence of halogenated nucleobases on the actual DNA strand breakage over the broad range of electron energies at which DEA can take place (<12 eV). By using DNA origami nanostructures, we determined the energy dependence of the strand break cross‐section for oligonucleotides modified with 8‐bromoadenine (^8BrA). These results were evaluated against DEA measurements with isolated ^8BrA in the gas phase. Contrary to expectations, the major contribution to strand breaks is from resonances at around 7 eV while resonances at very low energy (<2 eV) have little influence on strand breaks.     

SINGLE-STRAND BREAKS INDUCED IN DNA BY VACUUM-ULTRAVIOLET RADIATION

Abstract— The efficiency of vacuum u.v. for producing single-strand breaks in DNA was determined for wavelengths between 58 and 254 nm (corresponding to photon energies of 21·2 and 4·9 eV, respectively) by using the supertwisted RF-DNA of bacteriophage φX174. The cross-section for production of single-strand breaks increases continuously by about 5 orders of magnitude between 5 and 10 eV photon energy, whereas from 11 to 21 eV the number of strand breaks produced per unit of incident radiation energy is approximately constant. Thus, absorption of a 10-eV photon causes DNA strand breaks with maximum efficiency. In addition, the number of electrons liberated from DNA by photons below 10 eV is one or two orders of magnitude higher than the frequency of strand breaks, demonstrating that in this energy range only a small fraction of the ionizations leads to strand breakage in DNA.     

Vacuum-UV and Low-Energy Electron-Induced DNA Strand Breaks – Influence of the DNA Sequence and Substrate

DNA is effectively damaged by radiation, which can on the one hand lead to cancer and is on the other hand directly exploited in the treatment of tumor tissue. DNA strand breaks are already induced by photons having an energy below the ionization energy of DNA. At high photon energies, most of the DNA strand breaks are induced by low-energy secondary electrons. In the present study we quantified photon and electron induced DNA strand breaks in four different 12mer oligonucleotides. They are irradiated directly with 8.44 eV vacuum ultraviolet (VUV) photons and 8.8 eV low energy electrons (LEE). By using Si instead of VUV transparent CaF₂ as a substrate the VUV exposure leads to an additional release of LEEs, which have a maximum energy of 3.6 eV and can significantly enhance strand break cross sections. Atomic force microscopy is used to visualize strand breaks on DNA origami platforms and to determine absolute values for the strand break cross sections. Upon irradiation with 8.44 eV photons all the investigated sequences show very similar strand break cross sections in the range of 1.7–2.3×10⁻¹⁶ cm². The strand break cross sections for LEE irradiation at 8.8 eV are one to two orders of magnitude larger than the ones for VUV photons, and a slight sequence dependence is observed. The sequence dependence is even more pronounced for LEEs with energies <3.6 eV. The present results help to assess DNA damage by photons and electrons close to the ionization threshold.     

Dissociative electron attachment to furan, tetrahydrofuran, and fructose

We study dissociative electron attachment to furan (FN) (C(4)H(4)O), tetrahydrofuran (THF) (C(4)H(8)O), and fructose (FRU) (C(6)H(12)O(6)) using crossed electron/molecular beams experiments with mass spectrometric detection of the anions. We find that FN and THF are weak electron scavengers and subjected to dissociative electron attachment essentially in the energy range above 5.5 eV via core excited resonances. In striking contrast to that, FRU is very sensitive towards low energy electrons generating a variety of fragment ions via a pronounced low energy feature close to 0 eV. These reactions are associated with the degradation of the ring structure and demonstrate that THF cannot be used as surrogate to model deoxyribose in DNA with respect to the attack of electrons at subexcitation energies (<3 eV). The results support the picture that in DNA the sugar moiety itself is an active part in the initial molecular processes leading to single strand breaks.     

FORMATION OF SINGLE AND DOUBLE STRAND BREAKS IN DNA ULTRAVIOLET IRRADIATED AT HIGH INTENSITY

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.     

Low-energy electron diffraction and induced damage in hydrated DNA

Elastic scattering of 5-30 eV electrons within the B-DNA 5'-CCGGCGCCGG-3' and A-DNA 5'-CGCGAATTCGCG-3' DNA sequences is calculated using the separable representation of a free-space electron propagator and a curved wave multiple scattering formalism. The disorder brought about by the surrounding water and helical base stacking leads to a featureless amplitude buildup of elastically scattered electrons on the sugar and phosphate groups for all energies between 5 and 30 eV. However, some constructive interference features arising from diffraction are revealed when examining the structural waters within the major groove. These appear at 5-10, 12-18, and 22-28 eV for the B-DNA target and at 7-11, 12-18, and 18-25 eV for the A-DNA target. Although the diffraction depends on the base-pair sequence, the energy dependent elastic scattering features are primarily associated with the structural water molecules localized within 8-10 A spheres surrounding the bases and/or the sugar-phosphate backbone. The electron density buildup occurs in energy regimes associated with dissociative electron attachment resonances, direct electronic excitation, and dissociative ionization. Since diffraction intensity can be localized on structural water, compound H2O:DNA states may contribute to energy dependent low-energy electron induced single and double strand breaks.     

The soluble eumelanin precursor 5,6-dihydroxyindole-2-carboxylic acid enhances oxidative damage in human keratinocyte DNA after UVA irradiation.

Soluble melanin precursors are present in serum and may act as skin chromophores contributing to UVR-induced oxidative damage. Our study aimed to determine whether the soluble eumelanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) photosensitizes DNA damage in human keratinocytes exposed to UVA irradiation. The HaCaT keratinocytes were incubated with and without DHICA, before irradiation with broadband UVA (320-400 nm). The DNA photodamage was assessed using the comet assay that detects frank single-strand breaks (SSB) and specific oxidative lesions with the addition of endonuclease III. Without DHICA incubation, there was no significant increase in SSB, compared to unirradiated cells, for doses up to 48.5 J/cm2 (< 1 minimum erythemal dose). Preincubation with 0.5 microM DHICA caused an increase in SSB at every UVA dose (significant from 12.1 to 48.5 J/cm2), while varying the DHICA concentration (0.125-2 microM) showed this effect to be concentration dependent such that SSB increased and endonuclease III-sensitive sites decreased with increasing DHICA concentration. The irradiation of cells in the presence of antioxidants (catalase, mannitol and histidine) suggests that DHICA-induced photosensitization is mediated via singlet oxygen and, to a lesser extent, hydroxyl radicals. These results indicate that DHICA can induce strand breaks with UVA at clinically relevant doses via a mechanism involving reactive oxygen species.     

Low energy electron induced damage to plasmid DNA pQE30

Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5-22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and ～18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.     

Comparison between X-ray photon and secondary electron damage to DNA in vacuum

Both monolayer and thick (20 microm) films of dry pGEM-3Zf(-) plasmid DNA deposited on tantalum foil were exposed to Al Kalpha X-rays (1.5 keV) for various times in an ultrahigh vacuum chamber. For monolayer DNA, the damage was induced mainly by low energy secondary electrons (SEs) emitted from the tantalum. For the thick films, DNA damage was induced chiefly by X-ray photons. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis. The exposure curves for the formation of nicked circular (single strand break, SSB), linear (double strand break, DSB), and interduplex cross-link forms 1 and 2 were obtained for both monolayer and thick films of DNA, respectively. The lower limits of G values for SSB and DSB induced by SEs were derived to be 86 +/- 2 and 8 +/- 2 nmol J(-1), respectively. These values are 1.5 and 1.6 times larger than those obtained with 1.5 keV photons. The projected X-ray energy dependence of the low energy electron (LEE) enhancement factor for the SSB and DSB in monolayer DNA is also discussed. This new method of investigation of the SE-induced damage to large biomolecules allows direct comparison of the yield of products induced by high energy photons and LEEs under identical experimental conditions.     

Sensitizing DNA Towards Low‐Energy Electrons with 2‐Fluoroadenine

2‐Fluoroadenine (^2FA) is a therapeutic agent, which is suggested for application in cancer radiotherapy. The molecular mechanism of DNA radiation damage can be ascribed to a significant extent to the action of low‐energy (<20 eV) electrons (LEEs), which damage DNA by dissociative electron attachment. LEE induced reactions in ^2FA are characterized both isolated in the gas phase and in the condensed phase when it is incorporated into DNA. Information about negative ion resonances and anion‐mediated fragmentation reactions is combined with an absolute quantification of DNA strand breaks in ^2FA‐containing oligonucleotides upon irradiation with LEEs. The incorporation of ^2FA into DNA results in an enhanced strand breakage. The strand‐break cross sections are clearly energy dependent, whereas the strand‐break enhancements by ^2FA at 5.5, 10, and 15 eV are very similar. Thus, ^2FA can be considered an effective radiosensitizer operative at a wide range of electron energies.     

On the mechanism of anion desorption from DNA induced by low energy electrons

Our knowledge of the mechanisms of radiation damage to DNA induced by secondary electrons is still very limited, mainly due to the large sizes of the system involved and the complexity of the interactions. To reduce the problem to its simplest form, we investigated specific electron interactions with one of the most simple model system of DNA, an oligonucleotide tetrameter compound of the four bases. We report anion desorption yields from a thin solid film of the oligonucleotide GCAT induced by the impact of 3-15 eV electrons. All observed anions (H-, O-, OH-, CN-, and OCN-) are produced by dissociative electron attachment to the molecule, which results in desorption peaks between 6 and 12 eV. Above 14 eV nonresonant dipolar dissociation dominates the desorption yields. By comparing the shapes and relative intensities of the anion yield functions from GCAT physisorbed on a tantalum substrate with those obtained from isolated DNA basic subunits (i.e., bases, deoxyribose, and phosphate groups) from either the gas phase or condensed phase experiments, it is possible to obtain more details on the mechanisms involved in low energy electron damage to DNA, particularly on those producing single strand breaks.     

Gel electrophoresis method for quantitation of gamma ray induced single- and double-strand breaks in DNA irradiated in vitro

We describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single- and double-strand breaks formed by gamma-rays for DNA irradiated in vitro. For single-strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double-strand breaks doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single-strand breaks is linear while the dose response function for double-strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.     

Low energy electron-induced reactions in gas phase 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose: a model system for the behavior of sugar in DNA

Dissociative electron attachment to 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (TAR) is studied in a crossed electron-molecular beam experiment with mass spectrometric detection of the observed fragment ions. Since in TAR acetyl groups are coupled at the relevant positions to the five membered ribose ring, it may serve as an appropriate model compound to study the response of the sugar unit in DNA towards low energy electrons. Intense resonances close to 0 eV are observed similar to the pure gas phase sugars (2-deoxyribose, ribose, and fructose). Further strong resonances appear in the range of 1.6-1.8 eV (not present in the pure sugars). Based on calculations on transient anions adopting the stabilization method, this feature is assigned to a series of closely spaced shape resonances of pi* character with the extra electron localized on the acetyl groups outside the ribose ring system. Further but weaker resonant contributions are observed in the range of 7-11 eV, representing core excited resonances and/or sigma* shape resonances. The decomposition processes involve single bond ruptures but also more complex reactions associated with substantial rearrangement. The authors hence propose that the sugar unit in DNA plays an active role in the molecular mechanism towards single strand breaks induced by low energy electrons.     

PHOTOSENSITIZATION OF SUPERCOILED DNA DAMAGE BY 5,6-DIHYDROXYINDOLE-2-CARBOXYLIC ACID, A PRECURSOR OF EUMELANIN

The photosensitizing or photoprotecting action of 5,6-dihydroxyindole-2-carboxylic acid (DICA), an intermediate in the biosynthesis of eumelanins, was investigated. Under irradiation at 313 nm, aqueous buffered solutions of DICA (22.5 μW) photosensitized the cleavage of phage φX174 DNA. The number of single strand breaks (SSB) depended on the dose of irradiation and was more important in the absence than in the presence of oxygen. In the presence of oxygen, the quantum yield of SSB was around 6′10 ⁷ (Φ_SSB) The influence of specific scavengers, such as mannitol, sodium azide or superoxide dismutase, indicated that hydroxyl radicals, superoxide anions and perhaps singlet oxygen were involved in these processes. The increase in SSB in D₂O was also indicative of the participation of singlet oxygen. Comparative experiments performed with indole-2-carboxylic acid (IC), a dehydrox-ylated analog of DICA, showed that this compound, although lacking a phenol group, also photosensitized DNA cleavage via a mechanism involving hydroxyl radicals. Various sources of these radicals were envisioned. Furthermore, under our conditions, DICA was not found to photoinduce the formation of DNA dimers: No increase in SSB was observed in DNA irradiated in the presence of DICA, after treatment by phage T4 endonuclease V (an enzyme that selectively cuts DNA at dimer sites), whereas, in contrast, a significant increase in SSB was detected after treatment of DNA irradiated alone. So it appears that DICA may both photosensitize DNA cleavage and reduce UV-induced DNA dimer formation.     

Shape and core‐excited resonances of thionucleobases

Thionucleobases can be used in chemoradiation therapy of cancer. Shape resonances (SRs) and core‐excited resonances (CERs) can lead to fragmentation and eventually result in strand breaks of DNA. In particular, the more energetic CERs are believed to cause double‐strand breaks that can hardly be repaired. In this work, both the SRs and CERs of exemplary 2‐thiouracil, 4‐thiouracil, 2‐thiothymine, 4‐thiothymine, and 6‐aza‐2‐thiothymine are investigated using stabilization method in conjunction with long range corrected time‐dependent density functional theory. Results indicate that the energies of (1) π*₁ and π*₂ SRs, (2) n‐π* CERs, and (3) mixed resonances of π‐π* CERs with π* SRs can be significantly stabilized due to thionation of uracil or thymine. It is noteworthy that the resonant cases of (2) and (3) can be accessed by electrons even at energies below 4 eV. Consequently, the increased decay of temporary anions can enhance strand breaks of DNA.     

Molecular Characterization of DNA Double Strand Breaks with Tip‐Enhanced Raman Scattering

DNA double strand breaks (DSBs) are deadly lesions that can lead to genetic defects and cell apoptosis. Techniques that directly detect DNA DSBs include scanning electron microscopy, atomic force microscopy (AFM), and fluorescence based approaches. While these techniques can be used to identify DSBs they provide no information on the molecular events occurring at the break. Tip‐enhanced Raman scattering (TERS) can provide molecular information from DNA at the nanoscale and in combination with AFM provides a new way to visualize and characterize the molecular structure of DSBs. DSBs result from cleavage at the 3’‐ and 5’‐bonds of deoxyribose upon exposure to UVC radiation based on the observation of P? O? H and methyl/methylene deformation modes enhanced in the TERS spectra. It is hypothesized that strand fragments are hydrogen‐terminated at the lesion, indicating the action of free radicals during photon exposure.     

UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine

Reactive oxygen species (ROS) are involved in the oxidative damage of the cyanobacterium Anabaena sp. caused by UV-B (280-315 nm) radiation. UV-B-induced overproduction of ROS as well as the oxidative stress was detected in vivo by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Thiobarbituric acid reactive substances (TBARS) and fluorometric analysis of DNA unwinding (FADU) methods were adapted to measure lipid peroxidation and DNA strand breaks in Anabaena sp. Moderate UV-B radiation causes an increase of ROS production, enhanced lipid peroxidation and DNA strand breaks, yielding a significantly decreased survival. In contrast, the supplementation of UV-A in our work only showed a significant increase in total ROS levels and DNA strand breaks while no significant effect on lipid peroxidation, chlorophyll bleaching or survival was observed. The presence of ascorbic acid and N-acetyl-L-cysteine (NAC) reversed the oxidative stress and protected the organisms from chlorophyll bleaching and the damage of photosynthetic apparatus induced by UV-B significantly, resulting in a considerably higher survival rate. Ascorbic acid also exhibited a significant protective effect on lipid peroxidation and DNA strand breaks while NAC did not show a substantial effect. These results suggest that ascorbic acid exhibited significantly higher protective efficiency with respect to DNA strand breaks and survival than NAC while NAC appears to be especially effective in defending the photosynthetic apparatus from oxidative damage.