Letter pubs.acs.org/JPCL
Stabilization of Mixed Frenkel-Charge Transfer Excitons Extended Across Both Strands of Guanine−Cytosine DNA Duplexes Miquel Huix-Rotllant,†,§ Johanna Brazard,† Roberto Improta,*,‡ Irene Burghardt,*,§ and Dimitra Markovitsi*,† †
CNRS, IRAMIS, LIDYL, Laboratoire Francis Perrin, URA 2453, F-91191 Gif-sur-Yvette, France Institut für Physikalische u. Theoretische Chemie, Goethe-Universität, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany ‡ Istituto Biostrutture e Bioimmagini- Consiglio Nazionale delle Ricerche, Via Mezzocannone 16, I-80136 Napoli, Italy §
S Supporting Information *
ABSTRACT: The photoreactive pathways that may lead to DNA damage depend crucially upon the nature of the excited electronic states. The study of alternating guanine−cytosine duplexes by fluorescence spectroscopy and quantum mechanical calculations identifies a novel type of excited states that can be populated following UVB excitation. These states, denoted High-energy Emitting Long-lived Mixed (HELM) states, extend across both strands and arise from mixing between cytosine Frenkel excitons and guanine-to-cytosine charge transfer states. They emit at energies higher than ππ* states localized on single bases, survive for several nanoseconds, are sensitive to the ionic strength of the solution, and are strongly affected by the structural transition from the B form to the Z form. Their impact on the formation of lesions of the genetic code needs to be assessed.
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Our experimental results (Figure 1) were obtained for a duplex containing ca. 1000 base pairs (GC1000) upon UVB (285 nm) excitation. The fluorescence spectra of the B-form studied in phosphate buffer are dominated by a band peaking at 307 nm. They are blue-shifted and narrower with respect to the spectrum of an equimolar mixture of dGMP and dCMP, peaking at 330 nm with a width of 0.88 eV (full width at halfmaximum (fwhm)). A 10-fold increase in the ionic strength of the phosphate buffer (from 0.065 to 0.65 mol L−1) maintains the B-form while leading to spectral narrowing from 0.63 ± 0.01 to 0.45 ± 0.01 eV (fwhm); at the same time, the fluorescence quantum yield ϕ decreases from 7 × 10−4 to 2.7 × 10−4. The spectra of the B-form in Figure 1a resemble the spectrum reported for GC1000 following UVC excitation (ϕ = 1.3 × 10−4).17 The spectral narrowing observed for the fluorescence band of the B-form with respect to noninteracting chromophores suggests emission from collective states:18,19 in the case of high ionic strength, this narrowing amounts to 0.45 eV for UVB excitation (Supporting Information (SI) Figure SI2) and 0.33 eV for UVC excitation.17 A further increase of the ionic strength (4 mol L−1 NaCl) leads to formation of the Zform,18 which is accompanied by a broadening of the spectrum to 0.61 ± 0.05 eV, a weak shift to lower energy and a further reduction of ϕ to ∼1 × 10−4. We observed that the B → Z conversion involves a rapid quantitative change followed by much slower conversion kinetics. Thus, the spectral properties observed for 4 mol L−1 NaCl solutions do not allow for a
he evolution of electronic excitations created in DNA by absorption of UV radiation is essential for photochemical reactions ultimately leading to skin cancer. During the past decade, excited states delocalized over two stacked bases, of either Frenkel exciton (ππ*) and or charge transfer (CT) type, emerged as key actors for DNA photoreactivity.1,2 However, excited states localized on single bases, or Watson−Crick CT states have also been proposed as important players in the excited state relaxation, which became one of the most debated topics in photochemistry.3−10 The degree of delocalization of Frenkel and CT states is a highly controversial issue. While most theoretical models found that fluorescent states extend at most over two stacked bases, even when a larger number of bases are considered,11−15 there are indications of more extensive delocalization from the experimental side.6,10,16 Here, we show that mixed Frenkel exciton/CT states, delocalized over four bases on both strands and characterized by an energy higher than that of the emitting ππ* states, can be stabilized during the excited state relaxation in duplex DNA. This unexpected relaxation path, determined for alternating guanine−cytosine (GC) sequences using fluorescence spectroscopy and characterized by quantum mechanical calculations, is found to be more efficient for the B-form compared to the Z-form. The high energy long-lived excited states can be populated upon UVB excitation, present in the solar light reaching the surface of the Earth, and are sensitive to changes of the ionic strength which is a biologically relevant parameter. Stabilization up to the nanosecond time-scale of such blueshifted, collective excited states of mixed Frenkel/CT character is novel, not only for DNA, but also for multichromophoric systems in general. © XXXX American Chemical Society
Received: April 20, 2015 Accepted: May 14, 2015
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DOI: 10.1021/acs.jpclett.5b00813 J. Phys. Chem. Lett. 2015, 6, 2247−2251
Letter
The Journal of Physical Chemistry Letters
Figure 1. Fluorescence spectra (a), normalized fluorescence decays (b) and fluorescence anisotropies (c) recorded at 305 nm for GC1000: B-form in phosphate buffer of low (red) and high (blue) ionic strength, and Z-form (green); λexc: 285 nm. In panel a, intensities are representative of the fluorescence quantum yields, and the black line corresponds to the spectrum of an equimolar mixture of dGMP and dCMP.19,20 In panels b and c, the apparatus response function is shown in gray.
the polarizable continuum model (PCM);24 all calculations were performed with the Gaussian09 package.25 Additional reference calculations were performed at the ADC(2) level in the gas phase using the Turbomole package.26 For the above systems, we carried out calculations for both the B- and the Zform, starting from the idealized structures of each form according to the x3DNA software.27 The observed effects are governed by the symmetry of the duplex as well as by the relative energetics and the strength of the interaction between CT states and Frenkel excitons (Figure 2). The extent of mixing at the Franck−Condon geometry is
precise characterization of the Z-form behavior. However, they indicate a clear trend showing that the high energy long-lived fluorescence is quenched in the Z-form. Details related to the sample preparation and the fluorescence spectra are given in SI. The differences among the three fluorescence spectra in Figure 1a are reflected in the time-resolved fluorescence signals recorded at 305 nm by time-correlated single photon counting (Figure 1b). All three decays are composed of a fast component that cannot be resolved by this technique (
