Charge separation and charge delocalization identified in long-living states of photoexcited DNA

Base stacking in DNA is related to long-living excited states whose molecular nature is still under debate. To elucidate the molecular background we study well-defined oligonucleotides with natural bases, which allow selective UV excitation of one single base in the strand. IR probing in the picosecond regime enables us to dissect the contribution of different single bases to the excited state. All investigated oligonucleotides show long-living states on the 100-ps time scale, which are not observable in a mixture of single bases. The fraction of these states is well correlated with the stacking probabilities and reaches values up to 0.4. The long-living states show characteristic absorbance bands that can be assigned to charge-transfer states by comparing them to marker bands of radical cation and anion spectra. The charge separation is directed by the redox potential of the involved bases and thus controlled by the sequence. The spatial dimension of this charge separation was investigated in longer oligonucleotides, where bridging sequences separate the excited base from a sensor base with a characteristic marker band. After excitation we observe a bleach of all involved bases. The contribution of the sensor base is observable even if the bridge is composed of several bases. This result can be explained by a charge delocalization along a well-stacked domain in the strand. The presence of charged radicals in DNA strands after light absorption may cause reactions—oxidative or reductive damage—currently not considered in DNA photochemistry.DNA photophysics is crucial for the understanding of light-induced damage of the genetic code (1). The excited state of single DNA bases is known to decay extremely fast on the subpicosecond time scale, predominantly via internal conversion (2, 3). This ultrafast decay is assumed to suppress destructive decay channels, thereby protecting the DNA from photodamage and avoiding disintegration of the genetic information. In contrast to this ultrafast deactivation of single nucleobases, the biological relevant DNA strands show further long-living states (4, 5). Several explanations for these long-living states and the size of their spatial extent have been discussed in the literature (5–9). Delocalized excitons (9); excitons that decay to charge-separated states or neutral excimer states (10, 11); exciplexes located on two neighboring bases (5, 8, 12, 13); or even excited single bases, where steric interactions in the DNA strand impedes the ultrafast decay (14), have been proposed. Further computations suggest a decay of an initially populated delocalized exciton to localized neutral or charged excimer states (15–17). However, to our knowledge, a final understanding of the nature of these long-living states has not been reached. Related experiments were performed in the last decade to investigate charge transport processes in DNA, motivated by DNA electronics and oxidative damage (18, 19). Charge transport was initiated by photoexcitation of modified DNA bases or chromophores and followed by transient absorption (20–23). The transport mechanism was described by charge-hopping, superexchange, or transfer of charge along delocalized domains in DNA (18).Until now, most experimental investigations of the long-living state were performed with transient absorption spectroscopy in the UV-visible (UV/Vis) regime (5, 9, 12) or with time-resolved fluorescence (10, 24, 25). Due to the broad, featureless, and overlapping absorption bands of the different DNA bases in this spectral region, it is difficult to investigate the molecular origin of the long-living states using these methods. A further drawback is the unselective and simultaneous excitation of several bases used in most experiments. To circumvent these problems, we used for the present study well-defined oligonucleotides, which enable selective excitation of one single base. Observation of the long-living excited states was performed via time-resolved IR spectroscopy, which can profit from the many “fingerprint” vibrational bands (26, 27). IR spectroscopy is able to distinguish between different DNA bases and their molecular states. It can also reveal changes in the electronic structure and identify charge-separated states.In this study we used single-stranded DNA, in which π stacking between neighboring bases leads to structured domains, similar to the structure in a double helix (28). This interaction is known to be crucial for the long-living states (5). The investigation of single-stranded DNA enables us to construct special sequences, where only one base can be selectively excited. We used the natural bases 2′-deoxyuridine (U), 2′-deoxyadenosine (A), 5-methyl-2′-deoxycytidine (mC), and 2′-deoxyguanosine (G). The nucleobase U occurs naturally in RNA and is similar to the DNA base thymine but shows a blue-shifted absorbance spectrum. mC occurs with a frequency of 4–5% in mammalian DNA (29) and plays an important role as an epigenetic marker (30). The UV/Vis absorbance of mC and G are red-shifted in comparison with A and U, which allows selective excitation at 295 nm in oligonucleotides consisting of mC, A, and U (Fig. 1 A and B) or G and A. This selectivity can only be obtained in single-stranded DNA because G and its complementary base mC have overlapping absorbance bands in the UV range (Fig. S1). Selectivity in probing is based on the significant differences in the IR-absorption spectra of these bases, which display distinct marker bands for each base (Fig. 1 A and E).Open in a separate windowFig. 1.Selective excitation of mC in mCUA and probing of characteristic A, U, and mC marker bands in the IR. (A, B, and E) Picosecond UV light pulses at 295 nm allow selective excitation of mC (shown in bold) in mixed DNA sequences consisting of mC, U, and A. (B) Absorbance spectra of 2′-deoxyadenosine monophosphate (A), 2′-deoxy-5-methylcytidine (mC), and uridine monophosphate (U). (C) Time-resolved absorption difference (color-coded) plotted vs. wavenumber and delay time for mCUA and (D) for a mixture of the corresponding monomers. (E) Probing the individual contribution of each base is possible in the IR at 1,625 cm⁻¹ (A), 1,655 cm⁻¹ (U), and 1,667 cm⁻¹ (mC) (marked by dashed lines). (F) Transients at 1,667 cm⁻¹ for mCUA and the mixture of monomers.With the combination of selective excitation and selective probing we are able to elucidate the nature of the long-living states in DNA strands. Investigation of dinucleotides clearly shows that light absorption in DNA leads to charge separation between stacked neighboring bases, which recombine on the 100-ps time scale. In longer oligonucleotides we observe simultaneous bleach of several bases, which points to a delocalization of the charges along the strand. Our results show that charge transfer in DNA is a natural process, induced by UV-light absorption of DNA.     

Structure and Oligonucleotide Binding Efficiency of Differently Prepared Click Chemistry-Type DNA Microarray Slides Based on 3-Azidopropyltrimethoxysilane

The structural characterization of glass slides surface-modified with 3-azidopropyltrimethoxysilane and used for anchoring nucleic acids, resulting in the so-called DNA microarrays, is presented. Depending on the silanization conditions, the slides were found to show different oligonucleotide binding efficiency, thus, an attempt was made to correlate this efficiency with the structural characteristics of the silane layers. Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and X-ray reflectometry (XRR) measurements provided information on the surface topography, chemical composition and thickness of the silane films, respectively. The surface for which the best oligonucleotides binding efficiency is observed, has been found to consist of a densely-packed silane layer, decorated with a high-number of additional clusters that are believed to host exposed azide groups.