1.3. Photoluminescence of ds-DNA molecule

For a DNA molecule the optical transitions correspond to the electron transition between energy levels of the various single bases (Fig. 3), i.e. intra-base excitations. The band gap respects the energy difference between the top of the HOMO band and the bottom of the LUMO band, which are corresponding to different bases [4].

Now we can analyze the photoluminescence spectra of DNA polymerized molecules in the layer and determine HOMO - LUMO transition [5]. Using this value we predict the types of IV characteristics of the structures with ds-DNA molecules as metal, semiconductor, and insulator [3].

1.4. Charge transport in DNA

One way to detect DNA damage and its configuration involves the investigation of DNA film conductivity in wet and dry samples. Since direct investigation of UV-induced electronic process in DNA in cells requires complicated equipment [2], mostly research is made using DNA extracted from cells or synthesized DNA molecules with fixed length and known base sequences [4, 6-8].

The choice of conductivity measurements as a sensitive method for the detecting could be based on a models of ds-DNA as a "one-dimensional electronic material" [3, 9, 10]. The n-electron overlap between the base pairs within the double helix implies that the stacked base pairs might be a one-dimensional pathway for electronic charge transport. In classical DNA structure, the bases within the double helix are oriented perpendicular to the axis of the helix and parallel to each other with separation distance of 0.34 nm along the axis of the helix [6].

The observation of 1D conductivity in ds-DNA requires aligned DNA helixes and sufficient ^-electron overlap. Oriented films of a ds-DNA-surfactant complex satisfy both these criteria. The aligned double helixes separated by 4.1 nm with face to face base pair stacking and base separated by 0.34 nm [6].

The film hydration drives the base pairs into planar stacking with relatively strong n-electron overlap along the axis (wet DNA). There is relatively little information in the literature concerned specifically with the direct observation of base-pair stacking dynamics driven by water content in film [11]. The results of X-ray diffraction study of base-pair stacking in aligned films of DNA- surfactant complex are obtained [6]. The base-pair spacing within the DNA helix are 0.41 nm when the sample is dried in air at 50% relative humidity and 0.34 nm in aqueous environment at room temperature. Dehydration of DNA (dry DNA) causes the bases to rotate from planar to edge stacking with correspondingly poor ^-electron overlap.

When DNA film is not aligned (for example, DNA polymerized molecules form network [12]) the conductivity is defined by morphology of absorbed DNA molecules on a substrate (single- or multilayer films), DNA molecule conformation (wet or dry DNA) and nucleoside bases set in synthesized DNA. For such DNA layers it's necessary to account additional pathway of charge transport, for example, transport with including of barriers between adjacent molecules [3, 13]. Then contribution of charge transport on one-dimensional pathway through base stacking in single molecules could be only the part of general current film.

Besides carrying out of these wet DNA film conductivity measurements it's important to take in account the electrolysis of a buffer solution and a deposition of ions on metal contacts [14].

From 1.1, 1.2, 1.3 analyses we can assume that UV induced bases excitations and cyclobutane- pyrimidine dimer and (4-6) adducts formation in DNA molecules can leads to the changes in rc-orbitals overlapping and therefore ones can be observed in changes of DNA conductivity.

On the base of analyzes in 1.1 -1.4, with the aim to reveal UV induced damage in DNA molecules, in contrast to earlier listed investigations, we studied absorbed wet ds-DNA polymerized molecules layer from networks on mica surface, using DNA polymerized molecules in pure buffer solution, and dry layer from ordered networks with added SiO2 spheres (4.5 nm in size) [15]. These layers were dried in air (70% relative humidity) under visible light.

This study by optical spectroscopy, photoluminescence, conductivity gives the evidence that UV induced changes in ds(ss)-DNA molecules in buffer solutions and wet, dry layers are photochemical reactions with cyclobutane- pyrimidine dimer and (4-6) adducts formation and repair in applied electrical field.

2. Experimental

In experiments we used 2 mM plant ds-DNA polymerized molecules (Eris, France [11]) that were dissolved in 3 mM NaOH buffer and 2 mM ss-DNA with 15 oligonucleotides bases: 3' -CCA CCG CTG CTG AGG - 5', length 5.4 nm, wide 1 nm (Jena Bioscience, Germany) were dissolved in 100 mM carbonate/bicarbonate buffer [7] .We prepared ds-DNA polymerized molecules water solutions dissolving 0.1 ml of DNA in buffer solution in 1, 40, 50 ml of water showed a pH of 7.4 (the volume ratios are 1:1, 1:40, 1:50, correspondingly) and ss-DNA water solution dissolving 1 ml of DNA in buffer solution in 5 ml of water showed pH of 7.4. The solutions in quartz cuvvettes were irradiated by UV (X = 200-400 nm) during 5-90 min with the light power 1020 photon/(cm2 s).

Absorption spectra of ds (ss)-DNA solutions were recorded with a Jasco V-570 double beam spectrometer in quartz cuvettes (1 and 0.5 sm wide, respectively) in the range of 200-300 nm at T=293 K.

Photoluminescence and absorption spectra of ds-DNA polymerized molecules in tris HCl buffer (0.5 M Tris base, pH 7.6); before and after UV irradiation at 60 min (337 nm) and 90 min (200-400 nm), respectively were recorded. The solutions in quartz cuvettes were irradiated for 60 min by impulse N+ laser (X = 337 nm) and 365 nm wavelength separated from mercurial lamp illumination by a filter. The laser provided impulses with a frequency at 100 Hz, long time of impulse 10 ns, power 1.3 kW, and a light-illuminated square 3 mm in diameter. Emission spectra of ds-DNA solutions were recorded with KSV-23 spectrometer with vacuum photo detector FEU- 100(300-700 nm) having the resolution 2A/mm at T=293 K. The time of recording of every spectrum was 2 min.

To obtain absorbed ds-DNA layers the prepared solution (1:50) was dropped on the insulator surface and dried in air ( 70% relative humidity) under visible light for 10 min. A dry layer was formed from ds-DNA with added SiO2 spheres (4.5 nm in size) [15] with aim to absorb bonded water molecules from ds- DNA. SiO2 spheres with a surface modified by CH chains with NH2 end groups were used.

AFM images were studied to the characterize the morphology of the absorbed ds-DNA as a wet and dry layer on mica surface. AFM images were obtained by Digital Instruments Nanoscope Ilia. The measurements were performed at ambient condition in tapping mode to increase sensitive and resolution and to decrease the contact force between sample surface and AFM tips during surface topography measurements. The scanning was taken by a 100 ^m G-scamier using an etched silicon tip with a nominal radius of curvature 10 nm [16].

All contacts fYMA

Conductivity measurements were taken for the absorbed ds-DNA layer between and on two golden strips on the insulator as shown in Fig. 4. For the wet-absorbed ds-DNA layer the measurements were carried out after 10 min. of layer preparation, and for the dry layer - after 24 h drying in the air in dark at room temperature. A typical value of the current through the layer was nanoamperes.

The current through the Au-DNA-Au structure as a function of applied voltage during 30 s was measured before and during UV irradiation. The dependence of current through the structure under fixed voltage 1 V (conductivity under fixed voltage) on the time for the periodical switched UV irradiation was investigated. Measurements were carried out on automatic complex equipment in our Lab [3].

3. Results and discussions

3.1. Recognition of UV induced changes in ds(ss)-DNA absorption and photoluminescence

For the comparison of UV irradiation influence on different types of DNA sequences the experiments used ds(ss)-DNA molecules in water solutions (Figs 5,7).

Figure 4. a- Scheme of measurements of current and voltage in Au strip-DNA-Au strip structure; b- Configuration and sizes of Au strips.

Figure 4. a- Scheme of measurements of current and voltage in Au strip-DNA-Au strip structure; b- Configuration and sizes of Au strips.


3.1.1 Absorption spectra of different concentration of ds-DNA molecules in water solution. Influence of UV irradiation on ds-DNA molecule absorption

Two absorption bands with maxima at 208, 204, 196 and only at 256 nm based on re-re* transition of nucleotide bases (Fig. 3a) are observed for different concentrations of ds-DNA molecules in water solutions. Volume ratios were ds-DNA:H2O 1:1, 1:40, 1:50, correspondingly. A weak shift of the absorption maximum was seen at 208 nm (8 nm) with the ds-DNA molecules and decreasing concentration in water. This was caused by ds-DNA hydration in water [3]. The absorption intensity at these maxima and at others wavelengths (190-300 nm) was reduced with the decreased concentration of ds-DNA molecules in water.

Absorption spectrum of ds-DNA molecules in tris-HCl buffer (the volume ratios 1:10) in Fig. 5a has two wide absorption bands with maxima at 218 and 252 nm. Then this experiment confirms the influence of ds-DNA environment in solution on absorption spectrum, especially at band with maximum 218 nm that is sensitive to ds-DNA molecules hydration in water.

To minimize the ds-DNA molecules hydration in water we chose a tris-HCl buffer: the intensities of both maxima are comparable and the wavelength distance between them is 34 nm in comparison with water solutions 48, 52, 54 nm (1:1, 1:40, 1:50). To search for UV-induced changes in ds-DNA in water we selected ds-DNA molecules in tris-HCl buffer solution.

The influence of UV irradiation on ds-DNA absorption spectra from Fig. 5a, curves a-f revealed that the shapes of absorption curves b-e at range 235-280 nm changed. The absorption band had a maximum at 252 nm and the absorption intensity of left shoulder at X < 252 nm reduced more essentially than for the right shoulder. The position of absorption maxima shifts from 252 to 256 nm). Changes of band shape with absorption maximum at 218 nm were caused by changes in absorption intensity of right shoulder. The saturation of changes in the absorption spectra - curve e and f are identical. This result is illustrated by dependence of ds-DNA absorption intensity (for wavelengths 252 nm) on UV-radiation time (Fig. 6 curve a). The absorption intensity doesn't change after 60, 80 min.

200 220 240 260 280 300 Wavelength, nm Figure 5. Absorption spectra of ds-DNA molecules in water solution before (a) and after UV irradiation: b, c, d, e, f during 15, 30, 45, 60, 90 min correspondingly

Figure 6. Absorption intensity of: a - ds-DNA molecules at 252 nm; b - ss-DNA molecules at 256 nm versus UV irradiation time.

200 220 240 260 280 300 Wavelength, nm Figure 5. Absorption spectra of ds-DNA molecules in water solution before (a) and after UV irradiation: b, c, d, e, f during 15, 30, 45, 60, 90 min correspondingly

Figure 6. Absorption intensity of: a - ds-DNA molecules at 252 nm; b - ss-DNA molecules at 256 nm versus UV irradiation time.

Comparison of observed UV-induced shifts and intensity of ds- DNA absorption maxima with positions and intensity of nucleotide bases absorption maxima is shown in Fig. 3b. The changes of absorption intensity in the range of 235 < X < 252 nm corresponds to absorption on thymine base. Decreasing thymine's contribution to ds-DNA absorption spectrum with a long-wave shift (maximum at 252 nm nearest to the maximum) corresponded to the adenine base. We assume that these shifts and decreasing of absorption intensity was caused by reducing nucleotide bases (T and C) number in ds-DNA sequences with the formation of cyclobutane-pyrimidine dimers and/or (4-6) adduct formation. Absorption intensity doesn't change after 60, 90 min (Fig. 6/ We assume that this represent the time of UV- induced reactions .

On the other hand, the band in short wavelengths (X < 235 nm) with a maximum at 218 nm is not sensitive to UV irradiation. We assume that this confirms that UV irradiation influences only the nucleotide bases.

We compared the absorption (Fig. 5a) and photoluminescence spectra (Fig. 9) of ds-DNA molecules in tris-HCl buffer solution before and after UV irradiation of 60 min (200-400 nm) and 60 min (337 nm), respectively. We observed that the absorption and photoluminescence spectra are well separated (Fig. 10). The positions of the sub-bands in absorption spectra are at 253 (4.9), 265 (4.7), 274 (4.5), 285 nm (4.3 eV) and 251 (4.9), 269 (4.6), 274 nm (4.5 eV) in Fig. 7 a b, correspondingly. Comparison of spectra before and after UV irradiation revealed: a main electronic transition at 253 nm (4.9 eV) that determines the maximum absorption of DNA molecules before irradiation remains, but the value of absorption decreases over 1.5 a.u.. The transition at 265 nm (4.7 eV) shifts to 269 nm (4.6 eV), and the transition at 274 nm (4.5 eV) doesn't change. The transition at 285 nm (4.3 eV) did not appear after irradiation. The transition shift at 265 nm (4.7 eV) to 269 nm (4.6 eV) is caused by the thymine bases (Fig. 2b) that took part in photochemical reaction (Fig. 1, 2). The absence of transition at 285 nm (4.3 eV) after irradiation also confirms our results.

Photochemical reactions in the nucleotide base pairs with the formation of dimers and/or (4-6) adducts led to the change of electronic levels of the DNA molecule. This was revealed in absorption spectra (Fig. 5):

- absorption maximum at 265 nm (4.7 eV) shifts to 269 nm (4.6 eV) - this caused by additional absorption on formed dimers;

- maximum at 285 nm (4.3 eV) is absent - absorption on cytosine base becomes minimal: we assume that cytosine was used in dimer formation.

3.1.2. Absorption spectra of ss-DNA molecules in water solution. Influence of UV irradiation on ss-DNA molecule absorption

In ss-DNA absorption spectra two bands are seen with maxima are at 200 and 256 nm (Fig. 7). Differences between these absorption spectra and ds-DNA molecules spectra (Fig. 5) appear in another shape of the band with maxima over 252, 256 nm: the shoulder with maxima at 269 nm. This corresponds to the absorption of guanine, thymine bases, which number more then adenine, cytosine base number in ss-DNA (Fig. 3), is good seen.

The intensity of absorption spectra of the band with maximum at 256 nm decreases with the time of UV irradiation, but the shape of curves doesn't change. However, the weak long wave shift of left shoulder at X < 256 nm is observed. Differences between these absorption

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