Cation Effect on the Electronic Excited States of Guanine

Here, we examine first the short G-quadruplex d(TG4T)4 formed by four .... 0.33*TMP + 0.67*dGMP, 29590, 8050, 1.4 ..... Finally, cation tuning of the ...
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Cation Effect on the Electronic Excited States of Guanine Nanostructures Studied by Time-Resolved Fluorescence Spectroscopy Ying Hua,† Pascale Changenet-Barret,† Roberto Improta,‡ Ignacio Vayá,† Thomas Gustavsson,† Alexander B. Kotlyar,§ Dragoslav Zikich,§ Primož Šket,∥ Janez Plavec,∥ and Dimitra Markovitsi*,† †

CNRS, IRAMIS, SPAM, Laboratoire Francis Perrin, URA 2453, 91191 Gif-sur-Yvette, France Istituto Biostrutture e Bioimmagini-CNR, Via Mezzocannone 16, I-80134 Napoli, Italy § Department of Biochemistry, Georges S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978 Israel ∥ Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia ‡

S Supporting Information *

ABSTRACT: The effect of metal ions on the excited states of guanine nanostructures, short d(TG4T)4 quadruplexes and long G4-wires, are studied by fluorescence spectroscopy. The steadystate emission spectra show that both systems exhibit a strong cation effect. Fluorescence decays and fluorescence anisotropy decays, recorded from the femtosecond to the nanosecond timescale, reveal the following picture. In the presence of K+, emission arises mainly from delocalized ππ* states (excitons), whose decay spans several decades of times. In contrast, the fluorescence in the presence of Na+ is dominated by emission from charge transfer excited states decaying essentially on the subnanosecond time-scale. Such a difference is not due to the initially populated (Franck−Condon) states. The interproton distances derived from two-dimensional NMR measurements on the ground state of d(TG4T)4 show that the geometrical arrangement of guanines, governing the electronic coupling, is the same for both cations, in line with the UV absorption spectra. The observed cation effect is correlated with the excited state relaxation: the increased mobility of Na+ ions within the quadruplex favors trapping of ππ* excitons by charge transfer excited states, whereas such a process is hindered for the larger K+ ions. This is rationalized by quantum calculations on two stacked guanine tetrads.

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INTRODUCTION Guanine rich DNA strands have the ability to self-associate and give rise to four-stranded nanostructures called G-quadruplexes. Their repetitive unit is the guanine quartet (tetrad) where each base is connected with two others via four hydrogen bonds (Figure 1). G-quadruplexes can be formed by biologically relevant sequences, encountered, for example, in telomeres of

the eukaryotic chromosomes, and are targets for anticancer therapy.1 In addition, synthetic G-quadruplexes are considered as promising systems for applications in the field of molecular electronics and optoelectronics.2 To this end, the characterization of the electronic excited states of G-quadruplexes constitutes an important step. The knowledge of the key factors that affect their behavior will allow the design of novel photoactive four-stranded nanostructures. Moreover, such knowledge will contribute to understanding the formation of UV-induced lesions in telomeres.3 Although many studies use UV absorption, circular dichroism, or fluorescence spectra to monitor the formation of self-associated guanine structures or to determine their topology,4−6 the excited states responsible for the observed changes have been explicitly discussed only in very few articles.7−10 Such studies benefit from the knowledge accumulated during the past decade about the excited states of DNA monomers and multimers, in particular via time-

Figure 1. Schematic representation of (a) the guanine-quartet (tetrad) and (b) the d(TG4T)4 quadruplex topology with residue numbering; the four DNA strands are represented by different colors. © 2012 American Chemical Society

Received: April 16, 2012 Revised: June 6, 2012 Published: June 11, 2012 14682

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resolved experiments.11−17 One important outcome of the latter studies, associated with theoretical calculations,18−25 is that the electronic coupling between dipolar transitions and/or due to orbital overlap is capable of inducing delocalization of the excitation over several bases. As a result, photon absorption may lead to the population of exciton states built on the ππ* states, charge transfer (CT) excited states involving neighboring bases, and combinations among them. The spatial extent of these excited states is reduced by conformational disorder.26 Thus, as G-quadruplexes are particularly rigid structures, the collective behavior of their excited states is more pronounced compared to that of DNA duplexes.10 Metal ions represent a crucial factor for the stability of Gquadruplexes,27 which have been proposed to behave as ion channels.28 These charged species interact directly with guanines through inner sphere coordination. They have been shown to enhance excitation energy transfer toward a modified fluorescent guanine incorporated within G-quadruplexes.29 Consequently, it would not be surprising if they interfere with the electronic excited states of the G-quadruplex and, in particular, with CT states which are very sensitive to environmental factors. Investigating this interference is precisely the subject of the present work focusing on two types of biologically relevant cations, K+ and Na+. Here, we examine first the short G-quadruplex d(TG4T)4 formed by four parallel DNA strands (Figure 1b). A femtosecond study of its photophysical properties in the presence of Na+, carried out by fluorescence upconversion (FU), was published previously.9 In the present work, we compare the effect of Na+ and K+ on its fluorescence behavior over six decades of time by both FU and time-correlated single photon counting (TCSPC) using the same excitation laser source (267 nm, 120 fs). This study, associated with the steadystate optical spectra and information from two-dimensional NMR measurements, allows us to disentangle processes governed by the ground state structure of these systems from those taking place during the excited state relaxation. In a second step, we examine the effect of Na+ and K+ on the fluorescence of long guanine wires (G4-wires), formed by folding a poly (dG) strand containing a few thousand bases.30,31 As G4-wires cannot be prepared in large quantities required for certain measurements, only fewer comparison experiments are performed with them. However, the data obtained on long wires confirm the picture emerging from the study of the much shorter d(TG4T)4 quadruplexes. The most important outcome of the experimental observations is that the excited state dynamics, and in particular relaxation toward CT states, are remarkably influenced by the type of the metal cation. In order to get better insight on this issue, we perform quantum chemistry calculations for two stacked G-quartets of 9-methyl guanines (9Me-G) in aqueous solution including Na+ or K+ ions. Our computations, carried out by the TD-DFT method including solvent effect via the polarizable continuum model (PCM), focus on the position of the metal cation in the ground and the CT excited states of the G-quadruplex.

K2HPO4, and 0.48 M KCl) or Tris−HCl buffer (pH 7.2; 0.57 M KCl) for d(TG4T)4/K+. G-quadruplex formation was induced by heating the sample at 96 °C for 5 min followed by slow cooling down to 20 °C. The solution was incubated for 3 weeks at 4 °C. Formation of d(TG4T)4/Na+ was confirmed by following its melting at 295 nm.32 Formation of d(TG4T)4/K+ was monitored by recording successive fluorescence spectra; no spectral change was observed after the incubation period. Long monomolecular G4-wires were prepared as previously described,30 following a procedure consisting of two main steps: (i) enzymatic synthesis and purification of continuous 2800 base dG strands and (ii) folding of the purified dG strands into G4-wires. The structures were characterized by AFM, UV spectroscopy, circular dichroism, as well as by the sensitivity to enzymes.30,33,34 G4-wires were dissolved in the same buffer solutions as d(TG4T). Ultrapure water was produced by a MILLIPORE (Milli-Q Synthesis) system. Spectroscopic Measurements. Steady-state absorption and fluorescence spectra were recorded with a Perkin-Elmer Lambda 900 spectrophotometer and a SPEX Fluorolog-3 fluoremeter, respectively, using 1 cm optical path quartz cells. Fluorescence spectra were recorded in two steps: between 280 and 450 nm without optical filter on the emission side and then between 400 and 700 nm using a Schott GG 385 filter to eliminate contribution from the second order of the scattered excitation beam, Raman scattering, and the UV part of the quadruplex fluorescence. Fluorescence spectra were corrected for the instrumental response after subtraction of the signal arising from the pure solvent. TMP in water (ϕ = 1.54 × 10−4)35 was used as a reference for the determination of the fluorescence quantum yields. The excitation source for the time-resolved measurements was the third harmonic (267 nm, 120 fs) of a Ti-sapphire laser (Coherent MIRA 900). The repetition rate was 76 and 4.75 MHz for FU and TCSPC operation, respectively. The FU setup is described in detail elsewhere.36 The apparatus function was ca. 330 fs (fwhm). The TCSPC setup used a Becker & Hickl GmbH PC card. A Schott WG 295 filter was placed in front of a SPEX monochromator. The detector was a microchannel plate (R1564 U Hamamatsu) providing an instrumental response function of 70 ps (fwhm), determined by the Raman line of water. Temporal scans at the different emission wavelengths were made for parallel (Ipar) and perpendicular (Iperp) excitation/ detection configurations by controlling the polarization of the exciting beam with a half-wave plate (267 nm). For TCSPC experiments, a Glan Thomson prism was placed at the detection side either in vertical polarization. The excitation energies under parallel and perpendicular conditions were identical, giving a G factor of 1. For FU (peak intensity: 0.2 GWcm−2), 25 mL of solution was circulating through a flow cell, whereas, for TCSPC (peak intensity: 3 kW cm−2), ca. 3 mL of solution contained in 10 × 10 mm2 quartz cells was continuously stirred. Successive measurements gave identical decays which were eventually merged to increase the signal-to-noise ratio. Quantum Chemistry Calculations. Ground state geometry optimizations in aqueous solution were performed at the M052X/6-31G(d) level,37,38 including solvent effect by the polarizable continuum model (PCM).39 This computational approach has been already successfully employed to study oligonucleotide single and double strands.24,25 The M052X

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MATERIALS AND METHODS Materials. The oligonucleotide d(TG4T) was purchased from Eurogentec; it was purified by reverse-phase HPLC. The lyophilized powder was dissolved (1 mM in single strand) in the following: phosphate buffer (pH 6.8; 30 mM NaH2PO4, 30 mM Na2HPO4, and 0.48 M NaCl) for d(TG4T)4/Na+; phosphate buffer (pH 6.8; 30 mM KH2PO4, 30 mM 14683

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it was shown quite recently that water molecules rather than cations occupy the binding sites between G-quartet and G−C base pairs in d(G3CT4G3C)2.44 Despite the similarity of their ground state geometry, reflected in their absorption spectra, the steady-state fluorescence spectra of d(TG4T)4/K+ and d(TG4T)4/Na+ (Figure 2) are substantially different. The d(TG4T)4/K+

functional has been indeed specifically tailored to provide a reliable description of stacking interactions.37,38 In order to study the cation effect on the CT transitions within the Gquartet, we instead resorted to PCM/TD-PBE0/6-31G(d) calculations: PBE0,40,41 as all the standard density functionals, is known to overestimate the stability of CT transitions, making geometry optimizations less cumbersome.24,25 On the other hand, in order to avoid overestimation of the conformational flexibility of the tetrads arising by the absence of the backbone, and to limit the effect of possible inaccuracies of PBE0 in the determination of the stacking geometry, we constrained the 9Me-G bases to keep the same orientation and the same intertetrad distance as in the ground state minimum located by PCM/M052X/6-31G(d) calculations.

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RESULTS AND DISCUSSION d(TG4T)4. The absorption spectra of d(TG4T)4, in the presence of K+ or Na+, abbreviated as d(TG4T)4/K+ and d(TG4T)4/Na+, respectively, are practically identical; their maximum molar absorption coefficients differ by less than 4%. Thus, it appears that the metal ion does not affect the Franck− Condon (FC) excited states, which are expected to be delocalized over several bases.9 Indeed, a theoretical study performed for d(GGG)4/Na+ in the frame of the exciton theory, combining data from quantum chemistry calculations and molecular dynamics simulations, reported that the FC states are extended, on average, over 57% of the guanines of the system.10 The driving force for such delocalization of the excitation is the dipolar coupling between ππ* electronic transitions of the guanines (S0 → S1 and S0 → S2), which depends on their geometrical arrangement within the Gquadruplex. In order to examine if the local structure of the d(TG4T)4 ground state is affected by the type of the metal ion, we evaluated proton−proton distances derived from NOESY NMR spectra published recently.27 Only resolved cross-peaks were considered for distance calculations. Specifically, we analyzed interproton distances that reflect helical rise and potential G-quartet opening as well as structural variations of thymine residues on outer G-quartets. Comparison of distances among imino−imino and imino−aromatic protons belonging to guanine residues in different G-quartets indicates almost no changes as a function of cation nature (see the Supporting Information, Table SI-1). Proton−proton distances between imino and H8 protons of guanines within the same G-quartet are very similar as well for both d(TG4T)4/K+ and d(TG4T)4/ Na+. These demonstrate that the opening of G-quartets is not affected by the nature of the cation, which results in the same size of G-quadruplex cores. Furthermore, similar distances derived from cross-peaks in aromatic−aromatic, methyl− aromatic, and imino−methyl regions of NOESY spectra show that thymine residues are with regards to outer G-quartets at similar positions, irrespective of the cation type. We stress that, according to NMR studies, cations are dehydrated before entering into the G-quadruplex core.42 In fact, the preferred coordination of K+ over Na+ is driven by the greater energetic cost of Na+ dehydration with respect to K+ dehydration.43 Several later studies clearly confirmed that only bare cations without water molecules are present inside the central G-quadruplex cation channel. Thus, in the case of Gquadruplex structures with all binding sites occupied by cations, most likely no water molecules, which would affect ion mobility, are involved in the middle of the structure. Moreover,

Figure 2. Fluorescence spectra of d(TG4T)4 (solid lines; a, b, c, and d) and G4-wires (dashed lines; c and d) in the presence of Na+ (red) and K+ (blue). The spectra in part a are representative of the quantum yields, while the others are normalized. The spectra in black correspond to noninteracting mononucleotides (0.33TMP + 0.67dGMP). Excitation wavelength: 267 nm.

spectrum peaks at 330 nm, suggesting emission from ππ* states, as in the case for noninteracting nucleotides, thymidine monophosphate (TMP) and 2′-deoxygunanosine monophosphate (dGMP). The band around 330 nm is much weaker for d(TG4T)4/Na+, whose spectrum displays a shoulder at ca. 400 nm, indicating the presence of a second band. The fluorescence quantum yield of d(TG4T)4/Na+ (3 × 10−4) is only slightly lower than that of d(TG4T) 4/K+ (4 × 10−4). When concentrated buffered solutions of d(TG4T)4/Na+ or d(TG4T)4/K+ are diluted in ultrapure water up to a factor of 100, their fluorescence spectra remain the same. This means that the spectral properties depend only on the “internal” metal ions, interacting directly with guanines, while those interacting with the backbone play a minor role. A more quantitative comparison of the spectra is made after their conversion on an energy scale (Figure 2b and Table 1). The energy difference between the maxima of these spectra is 2500 cm−1. The presence of Na+ gives rise to a broader emission band compared to K+: 8940 and 5870 cm−1 (fwhm), respectively. The d(TG4T)4/K+ spectrum is even narrower by 2180 cm−1 than that of the noninteracting monomers, although their maxima coincide. Figure 3 shows the fluorescence decays and fluorescence anisotropy decays recorded for d(TG4T)4/K+ at various wavelengths, ranging from 310 to 450 nm, by FU. Upon increasing wavelength, the decays slow down. The signal at 310 nm is significantly faster than the others; its amplitude drops to 14684

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photons emitted per decade of time (Figure 4). The most striking feature is that, in the presence of Na+, the decay at 450

Table 1. Characteristics of the Fluorescence Spectra: Maximum (νmax), Width (Δv; fwhm), Quantum Yield (ϕ) (Excitation Wavelength: 267 nm) system +

d(TG4T)4/K d(TG4T)4/Na+ G-wires/K+ G-wires/Na+ dGMP TMP 0.33*TMP + 0.67*dGMP

νmax (cm−1)

Δv (cm−1)

ϕ × 104

29670 27170 30130 27300 29500 29850 29590

5870 8940 5050 8300 8950 6290 8050

4.0 3.0 9.2 9.5 1.3 1.5 1.4

Figure 4. Percentage of emitted photons per decade of time. They are reconstructed after a fitting/deconvolution procedure of fluorescence decays recorded by TCSPC for d(TG4T)4 and G4-wires in the presence of Na+ or K+.

Figure 3. Fluorescence decays (a and c) and fluorescence anisotropy decays (b and d) recorded by fluorescence upconversion; (a and b) d(TG4T)4/K+; (c and d) comparison of the d(TG4T)4/K+ (blue) and the d(TG4T)4/Na+ (red) signals at 360 nm. Dashes correspond to TMP (upper line in part b), dGMP (lower line in part b), and their stoichiometric mixture (d).35 The apparatus function is shown in gray.

nm is dominated by photons emitted between 0.1 and 1 ns. In contrast, in the presence of K + , photon emission is predominant in the 0.01−0.10 ns range and it lasts for more than 1 ns. Not only the fluorescence decays but also the fluorescence anisotropies recorded by TCSPC (Figure 5) exhibit a quite different behavior compared to those obtained by FU. When comparing r(t) signals determined by TCSPC and FU, we should keep in mind that the former technique detects

ca. 20% within 8 ps (Figure 3a). A more rapid decrease is observed for the fluorescence anisotropy which dwindles down to ca. 0.05 within 2 ps (Figure 3b). No clear wavelength dependence is noticed in the case of the anisotropy with the exception of the signal at 310 nm, whose initial value is higher than that observed for longer emission wavelengths. At all wavelengths, the fluorescence decays are slower in the presence of K+ compared to that of Na+.9 However, the fluorescence anisotropy decays are not sensitive to the nature of the cation. An example is shown in Figure 3c and d, where the cation effect on the signals recorded at 360 nm is presented. For both Na+ and K+, the fluorescence anisotropy r(t) exhibits the same fast decrease; then it remains constant from 2 ps to at least 8 ps. The plateau value (0.06 ± 0.02) is lower than the corresponding values of TMP (0.36) and dGMP (0.13), whose fluorescence lifetimes are ca. 0.5 ps.35,45 The low r(t) values, compared to those detected for noninteracting chromophores on the femtosecond time-scale, suggest that energy transfer takes place within d(TG4T)4 in the presence of either cation. The fluorescence decays recorded by TCSPC present a more complex behavior. After a fitting/deconvolution procedure (see the Supporting Information), we determined the percentage of

Figure 5. Time dependence of the fluorescence anisotropy determined at 310 nm (violet), 330 nm (blue), 360 nm (green), and 450 nm (red) for d(TG4T)4 in the presence of Na+ (a) and K+ (b). 14685

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fluorescence from all types of excited states, whereas the latter probes fluorescence only from bright states. Moreover, the r(t) signal recorded by TCSPC up to about 100 ps is distorted by the instrumental response function, representing an average anisotropy value. In the presence of Na+, the r(t) values recorded by TCSPC exhibit very strong wavelength dependence. At 0.2 ns, when the signals are no more significantly distorted by the instrumental response function, r(t) ranges from 0.12 to −0.04, the negative values being associated with the lower energy photons. In the presence of K+, after an initial decrease, a constant value, equal to 0.08 ± 0.03, is found at 330 and 360 nm. At 450 nm, the anisotropy decreases, reaching a minimum at about 0.16 ns, and subsequently it increases again, overlapping the signals recorded at shorter wavelengths (inset in Figure 5b). The ensemble of the data obtained for d(TG4T)4 indicates that fluorescence arises from excited states with different polarization, in particular in the presence of Na+. In the presence of K+, the excited states associated with lower fluorescence anisotropy are not those having the longest lifetime. NMR experiments have shown that part of the thymines adopt a quartet structure.27 As the lowest in energy absorption bands of TMP and dGMP largely overlap, a contribution of thymines to the FC exciton states is probable. However, thymines, located at the end of the strands, undergo large conformational motions which tend to localize the excitation. In view of the above considerations, excitation at 267 nm will populate a large variety of excited states, mainly of ππ* character, whose properties depend on the precise conformation of the system: exciton states built on the excited states of only guanines or with some participation from thymines but also ππ* states localized on guanines (S1 and S2) or thymines (S1).18 Subsequently, relaxation of the ππ* states will follow complex competing routes: nonradiative decay to the ground states, intraband scattering (internal conversion among exciton states), localization of the excitation on single bases, or trapping by charge transfer excited states. G4-Wires. The fluorescence spectra of G4-wires in the presence of Na+ and K+ are compared with the corresponding spectra of d(TG4T)4 in Figure 2c and d. As in the case of short G-quadruplexes, the presence of Na+ renders the spectra of G4wires broader, reinforcing the longer wavelength part of the spectrum. The spectral features, determined after conversion on a wavenumber scale, are given in Table 1. The difference in the emission maxima (2830 cm−1) as well as in their width (3250 cm−1; fwhm), due to the cation effect, is larger than that observed for d(TG4T)4. The latter contains an important fraction of thymine residues which emit at shorter wavelengths than the guanines. This explains the slight blue shift of the d(TG4T)4 spectra compared with the corresponding spectra of the G4-wires, containing only guanines. The fluorescence quantum yield of the G4-wires, similar for both types of cations, is 2−3 times higher than that of d(TG4T)4 (Table 1). The fluorescence decays recorded for the G4-wires by TCSPC are much longer than those of d(TG4T)4. In particular, the fraction of photons emitted after 1 ns increases with the size of the system (Figure 4). However, the effect of Na+ cations on the fluorescence decay is independent of the size of the system: it enhances photon emission mainly between 0.1 and 1 ns. Nature of the Emitting Excited States. In order to correlate the observed fluorescence properties to the various relaxation pathways, we first focus on the G-quadruplexes in the

presence of K+, whose steady-state spectra closely resemble those of the noninteracting mononucleotides (Figure 2). One could think that their emission arises from excited states localized on guanines whose nonradiative decay rate is simply slowed down as they are embedded within a rigid system. Such a possibility is ruled out because the dGMP fluorescence spectra are known to shift to lower energies and broaden with time.45 However, just the opposite is observed for Gquadruplexes: photon emission from d(TG4T)4, and even more from G4-wires, lasts much longer compared to dGMP, but their spectra are narrower than that of the mononucleotide. Although intratetrad hydrogen bonding could affect the emission line-shape, the large dependence of the spectral narrowing on the size of the system suggests that, at least partly, emission arises from exciton states. Depending on the precise conformation of the G-quadruplex,10 the emitting exciton may correspond to an electronic transition with strong oscillator strength and detected by both FU and TCSPC or to a weak transition and probed only by TCSPC. Their properties will be reflected in both the fluorescence decays and the fluorescence anisotropy decays. Fluorescence anisotropy provides information about the electronic excited states if rotational diffusion is slow compared to the probed time-scales. This is indeed the case for d(TG4T)4, whose time constant for rotational diffusion, calculated from its hydrodynamic radius including the first hydration layer (33.1 Å),27 is 4.7 ns. The fluorescence anisotropy determined for d(TG4T)4/K+ at 330 nm by TCSPC (Figure 5) is nearly constant (0.08 ± 0.03) and close to the value 0.1, corresponding to in-plane depolarization.46 This means that the transition dipoles associated with photon absorption and photon emission are randomly distributed within the plane, the plane of the G-quartets in the examined system. It is possible that that weak coupling of ππ* states with CT states leads to a small deviation of the transition vectors from the tetrad plane, lowering slightly the fluorescence anisotropy below to what is expected from inplane depolarization. Emission from excited states localized on individual bases is probable at times shorter than 2 ps. In particular, the signals at 310 nm contain the fingerprint of excited states localized on thymines (Figure 3a and b). Indeed, the fluorescence spectrum of TMP is slightly blue-shifted with respect to that of dGMP and its fluorescence anisotropy is higher than 0.35.35 The astonishing similarity between the r(t) signals determined by FU for d(TG4T)4/Na+ and d(TG4T)4/K+ shows that, in both cases, the same type of ππ* states emit at short times. However, their dynamics are different, the presence of Na+ rendering the fluorescence decays faster. This partial quenching is accompanied by the appearance, at lower energy, of a new fluorescence band (Figure 2). It is correlated with charge transfer excited states involving guanines located on different quartets. The corresponding electronic transition is expected to be polarized perpendicular to the quartet plane, while ππ* transitions are polarized within this plane. The fluorescence anisotropy determined for d(TG4T)4/Na+ by TCSPC (Figure 5) strongly supports this attribution: upon increasing the emission wavelength, r(t) decreases, in line with increased contribution from CT states. The r(t) signal recorded for d(TG4T)4/K+ at 450 nm reaches a minimum at ca. 0.16 ns and goes up again. Such a behavior indicates that the nanosecond component of the fluorescence is rather due to ππ* states and not to CT states. 14686

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This is corroborated by the histograms in Figure 4. Replacement of K+ by Na+ increases the fraction of photons emitted between 0.1 and 1 ns at 450 nm, where emission from CT states is dominant. In contrast, a weak yet detectable fraction of photons is emitted at 330 and 360 nm after 1 ns in the case of d(TG4T)4/K+ (Figure 4), showing that ππ* excitons may persist on the nanosecond time-scale. This effect is more pronounced for G4-wires, suggesting emission from more extended exciton states. Photo-Induced Geometrical Changes. NMR experiments performed for the ground states of d(TG4T)4 have demonstrated that Na+ ions are more mobile compared to K+ ions. Hence, Na+ ions could change more easily their position during the excited state relaxation. Photoinduced motion of counterions correlated with important changes in the dipole moment has been reported for other types of chromophores.47 Moreover, metal ions are known to affect charge transport in DNA.48 In order to get a hint if the excited state relaxation of G-quadruplexes could be modulated by the metal ion, we studied by quantum chemistry calculations two stacked Gquartets composed of 9Me-guanines in their ground and CT excited states. As a first step, we optimized the ground state geometry at the PCM/M052X/6-31G(d) level. Although the absence of the backbone does not allow detailed comparison with experimental results, our model is able to reproduce the different location of K+ and Na+ ions within the G-quadruplexes. A similar intertetrad distance is found for Na+ (3.35 Å) and K+ (3.36 Å), in agreement with NMR measurements;49 Na+ is located closer to one of the G-quartets: the distances Na1−X1 and Na2−X2, where X1 and X2 are the centers of G1 and G2 tetrads, are both ca. 0.5 Å. In contrast, K1+ is sandwiched between the G-quartets, being almost equidistant from the two planes, located at 1.4 and 1.8 Å from them. The K2−X2 distance (1.6 Å) is slightly larger than the Na2−X2 one. In this respect, it is important to stress that our computational model, including only two tetrads, does not provide a perfectly symmetric arrangement of the K+ ions. In other words, due to the repulsion with the other K+ ion, the “internal” K+ will be closer to one of the tetrads. Subsequently, we partially optimized, at the PCM/TDPBE0/6-31G(d) level, the geometry corresponding to the lowest CT transition involving guanines at different tetrads. As expected, independently of the type of cation, the two 9Me-G involved in the electronic transition adopt the structure of a 9Me-G+ cation and a 9Me-G− anion. The position of all the ions changes with respect to that found in the ground state minimum. The metal ions lose the symmetric arrangement with respect to the G-quadruplex axis; they move farther from the 9Me-G+ cation and get closer to the 9Me-G− anion. In the ground state, the Na+ ions are equidistant from the oxygen atoms of the four 9Me-G carbonyl groups (2.46 Å) of the coordinating G-quartet. In the CT excited state, its distance from the positively charged 9Me-G+ increases noticeably and that from the negatively charged 9Me-G− decreases (Figure 6). A similar picture is obtained in the case of K+. However, on the balance, the smaller Na+ exhibits a larger mobility because it also changes its position with respect to the axis of the Gquadruplex. The angle NaXiO deviates from the 90° value found in the ground state by more than 5°. In the case of K+, the geometrical changes are mainly due to movement of the 9Me-G involved in the CT transition from the average tetrad plane than to the shift of the metal cations. For example, the

Figure 6. Schematic drawing of the G-quadruplexes studied by PCMTD-DFT calculations. The distances between the metal ions and the carbonyl oxygen of the 9-methylguanines are depicted in red. The 9methylguanines involved in the charge transfer transitions are denoted by “+” and “−”; in black are shown the distances found in the ground state minimum. X1 and X2 are the centers of the G-quartets. PCM/ TD-PBE0/6-31G(d) geometry optimizations.

variations of the angle KXiO are within 1° with respect to the 90° value found in the ground state. From the energetic point of view, the change in the position of Na+ ions within the Gquadruplex increases the stability of the CT state. However, this statement should be considered with caution because the backbone is not taken into account in the calculations.

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CONCLUSIONS The ensemble of fluorescence measurements shows that Frenkel excitons in G-quadruplexes may be trapped by charge transfer excited states, as depicted schematically in Figure 7. This trapping is clearly favored by the presence of Na+ ions. Our calculations on two stacked tetrads of 9Me-G have shown that the change in the position between the ground and CT excited states involving guanines in different tetrads is more important for Na+ than K+ ions. The cation motion could

Figure 7. Schematic representation of the processes occurring in Gquadruplexes following excitation at 267 nm. Trapping of Frenkel excitons by charge transfer excited states is favored by Na+ ions whose mobility is larger compared to that of K+ ions. 14687

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therefore play a key role in modulating the accessibility of the decay channel involving the CT excited states. It could affect their thermodynamic stability and change their electronic coupling both with the ππ* states and with the ground electronic state. In addition, it could influence the rate of formation of CT excited states and of their deactivation to the ground state via charge recombination. The present study was carried out for four-stranded structures formed by natural guanines which absorb in the UV spectral domain, emit fluorescence with very low quantum yield, and therefore are not appropriate for optical applications. However, the demonstration that the interplay between Frenkel excitons and CT excited states of G-quadruplexes can be modulated by the nature of the metal cation could inspire the design of synthetic systems with desired properties. The rules to take into account concern not only the nature of metal ions but also that of the monomeric units. They may be chosen, for example, with respect to their capacity to prevent or enhance the formation of excited CT states at certain sites and/or modify the opening of the G-quadruplex core to change ion mobility. Finally, cation tuning of the electronic excited states could affect the first steps of a cascade of events leading to carcinogenic mutations or cell death. Therefore, it would be interesting to study the UV-induced processes in Gquadruplexes formed by the sequence TTAGGG, encountered in human telomeres, in the presence of Na+ or K+.

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ASSOCIATED CONTENT

S Supporting Information *

Proton−proton distances in d(TG4T)4 as a function of metal ions; computed details for the ground state geometry of 2(9Me-G)4; fits of the fluorescence decays. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The European Union (COST Action MP0802 and LASERLAB-EUROPE/grant agreement no. 228334), the French Agency for Research (ANR-10-BLAN-0809-01), and the Conselleria de Educacion-Generalitat Valenciana (VALi+D program to I.V., no. 2010033) are acknowledged for financial support.

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