Electronically excited states of DNA oligonucleotides with disordered basesequences studied by fluorescence spectroscopy†‡
Ignacio Vayá, Johanna Brazard, Thomas Gustavsson and Dimitra Markovitsi*
Received 9th June 2012, Accepted 18th September 2012DOI: 10.1039/c2pp25180a
DNA double-stranded oligomers are studied by steady-state and time-resolved fluorescence spectroscopyfrom the femtosecond to the nanosecond time-scale, following excitation at 267 nm. It is shown thatemission arises from three types of excited states. (i) Bright ππ* states emitting around 330 nm anddecaying on the sub-picosecond time-scale with an average lifetime of ca. 0.4 ps and a quantum yieldlower than 4 × 10−6. (ii) Excimers/exciplexes emitting around 430 nm and decaying on the sub-nanosecond time-scale. (iii) Excited states emitting mainly at short wavelengths (λ < 330 nm) anddecaying on the nanosecond time-scale, possibly correlated to GC pairs. The properties of the examinedduplexes, exhibiting significant disorder with respect to the nearest neighbour base sequence, are radicallydifferent than those of the much longer and disordered calf thymus DNA. Such behaviour suggests thatlong range and/or sequence effects play a key role in the fate of excitation energy.
1. Introduction
Considerable effort has been dedicated to the characterization ofthe electronic excited states of DNA and their relaxation usingfemtosecond lasers since the beginning of the 21st century.1–11
Such studies aim to shed light on the first steps of a cascade ofevents, triggered by the absorption of UV radiation and leadingto carcinogenic mutations.12 An important outcome of the workcarried out so far is that the lifetimes of the ππ* singlet excitedstates of mono-nucleotides do not exceed 1 ps.13,14 Going fromthe mono-nucleotides to single or double strands, much longer-lived components appear in the decays as evidenced by bothtransient absorption and time-resolved fluorescence experi-ments.13,15 These were correlated to the existence of electroniccoupling (dipolar and orbital overlap) which may inducedelocalization of the excitation on several bases giving rise toexciton states, charge transfer states and combinations amongthem.5,16–23
After systematic examination of model duplexes with a repeti-tive base sequence, composed of, for example, AT or GC pairs inhomopolymeric or alternating sequences, attention was turned todisordered systems. As a matter of fact, the question arises towhat extent the cooperative effects detected for ordered systems(with respect to the base sequence) persist in the presence ofsequence disorder.
In this context, we have been interested in isolated genomicDNA. We have shown that the fluorescence spectrum of calfthymus DNA (CT-DNA) in vitro is quite similar to that of itsmonomeric constituents.24 In particular, the low energy emissionband reported in earlier publications and attributed to excimers/exciplexes25,26 is absent from the spectrum of ultrapurifiedCT-DNA when the necessary precautions are taken in order toavoid photodamage during the measurements. The astonishingfact is that, despite the “monomer-like” emission spectrum, thefluorescence decays span over several decades of time. This con-trasts with the behaviour of “ordered” model helices, for whichlong-lived fluorescence components are correlated with emissionbands clearly different from those of the mono-nucleotides.15 Weinterpreted our observations in terms of delayed fluorescenceresulting from trapping of the excitation by dark states and re-population of ππ* states.27
In parallel, a transient absorption study with UV–visibledetection, performed by Kohler and co-workers, focused on anaturally occurring 11-mer.28 The examined duplex (abbreviatedhere as ds-11) contains codons 60–62 of the human n-ras proto-oncogene. It is composed of a purine-rich single strandd(CGGACAAGAAG) (abbreviated here as ss-11a) and its comp-lementary pyrimidine-rich strand d(CTTCTTGTCCG) (abbre-viated here as ss-11b). Both single strands and the duplexpresent significant disorder regarding the nearest neighbour basesequence. It was found that, despite this disorder, long-livedcomponents are still detected in the transient signals. In theduplex, some of the longest-lived excited states are quenchedand a substantial number of the excitations decay on a sub-picosecond time-scale.
Single wavelength optical experiments may, however, containcontributions from many overlapping transitions. Fluorescencemeasurements, associating dynamics with information on energy
†This article is published as part of a themed issue in honour ofJean-Pierre Desvergne on the occasion of his 65th birthday.‡Electronic supplementary information (ESI) available. See DOI:10.1039/c2pp25180a
CNRS, IRAMIS, SPAM, Laboratoire Francis Perrin, URA 2453 F-91191Gif-sur-Yvette, France. E-mail: [email protected];Fax: +33 1 69 08 76 39; Tel: +33 1 69 08 46 44
and polarization of the electronic transitions, can contribute todisentangle various intricate processes. For this reason, we haveundertaken an investigation of the duplex ds-11 and its parentsingle strands ss-11a and ss-11b, using steady-state and time-resolved fluorescence spectroscopy with 267 nm excitation.Moreover, knowing that a significant size effect is observed inthe fluorescence of “ordered duplexes”,29–31 we also comparethe behaviour of ds-11 with that of ds-22, e.g. a duplex contain-ing two consecutive repeats of the examined disorderedsequence. The objective of the work is twofold. On the onehand, we aim at completing the picture regarding the excitedstate relaxation of these systems, for which transient absorptiondata, obtained following excitation at 266 nm, are available. Onthe other hand, we want to compare the fluorescence propertiesof small sized “disordered” duplexes with our previous findingson the equally disordered but very long CT-DNA, both studiedunder the same experimental conditions.
Here we present fluorescence and fluorescence anisotropydecays from the femtosecond to the nanosecond time-scale,obtained by two different detection techniques, fluorescenceupconversion (FU) and time-correlated single photon counting(TCSCP) using the same femtosecond laser as an excitationsource. The former technique detects fluorescence from brightexcited states whereas the latter detects photons from all types ofexcited states. We also report time-resolved fluorescence spectra.A key point in these experiments is the application of specificprotocols developed for the measurements of DNA systemswhose fluorescence quantum yields are of the order of 10−4.32 Inthis way, detection of photons from helices which are damagedduring the measurements is avoided.
2. Experimental section
Single and double stranded oligonucleotides, purified byreversed phase HPLC, were obtained from Eurogentec. Theywere dissolved in phosphate buffer (0.1 mol L−1 NaH2PO4,0.1 mol L−1 Na2HPO4 and 0.25 mol L−1 NaCl). Water wasdelivered by a MILLIPORE (Milli-Q Synthesis) system. Themelting curves of ss-11a, ss-11b and ds-11, shown in the insetsof Fig. 1, are the same as those reported in reference.28 Themelting curves of ds-11 and ds-22 are compared in the ESI(Fig. ESI-1‡).
Steady-state absorption and fluorescence spectra wererecorded with a Perkin-Elmer Lambda 900 spectrophotometer
and a SPEX Fluorolog-3 fluorometer, respectively, using 1 cmoptical path quartz cells. The fluorometer was equipped with twograting monochromators centred at 330 and 500 nm, respecti-vely. Fluorescence spectra were recorded simultaneously from280 to 700 nm on both detectors using a Schott WG385 filter toeliminate contributions from the second order of the scatteredexcitation beam, Raman scattering and the UV part of the oligo-mer fluorescence on the grating centred at 500 nm. Fluorescencespectra were corrected for the instrumental response after sub-traction of the signal arising from the pure solvent. TMP in water(ϕ = 1.54 × 10−4)3 was used as a reference for the determinationof the fluorescence quantum yields.
The excitation source for the time-resolved measurements wasthe third harmonic (267 nm) of a mode-locked Ti-Sapphirelaser (Coherent MIRA 900) pumped by a cw solid state laser(Coherent VERDI V10, 10 W), delivering ≈ 120 fs pulses. Forthe third harmonic generation, a home built frequency triplingunit based on two 0.5 mm BBO type I crystals (Fujian Institute)and 10 mm spherical mirrors was used. The repetition rate was76 and 4.75 MHz for FU and TCSPC experiments, respectively.The average excitation power was measured by a Melles Griotbroadband powermeter.
In the FU experiments, fluorescence was collected by para-bolic mirrors (Janos), passed through a 1 mm WG320 orWG305 glass filter (Schott) in order to suppress residual exci-tation light and focused into a 0.5 mm thick BBO type I crystal(Fujian Institute) together with the residual beam of the funda-mental radiation (800 nm) in order to generate the sum-frequency(SF) radiation. The resulting SF radiation (215–250 nm) wasfocused onto the entrance slit of a double grating monochroma-tor (SPEX 1680) and detected by a photomultiplier (HamamatsuR1527P) connected to a lock-in photon counter (StanfordSR400). The spectral resolution was set to 4 nm. The instrumen-tal response function was determined by measuring the rise-time in the blue wing (320–340 nm) of the fluorescence froma dimethylquaterphenyl (Lambda Physics) solution in cyclo-hexane. It was found to be around 330 fs (fwhm).
The TCSPC setup used a Becker & Hickl GmbH SPC630card. A Schott WG 295 filter was placed in front of a SPEXmonochromator in order to suppress residual excitation light.The detector was a microchannel plate (Hamamatsu R1564 U)providing an instrumental response function of around 70 ps(fwhm) as given by the Raman line of water at 295 nm.
For both FU and TCSPC measurements, temporal scans at thedifferent emission wavelengths were made either at the magicangle or for parallel (Ipar) and perpendicular (Iperp) excitation/detection configurations by controlling the polarization of theexciting beam with a half-wave plate (267 nm). The excitationenergies under parallel and perpendicular conditions were identi-cal, giving a G factor of 1. We checked that the total fluorescencedecays, calculated as F(t) = Ipar(t) + 2Iper(t), were identical tothose recorded directly at the magic angle.
For FU (excitation peak intensity at 267 nm: 0.2 GW cm−2),25 mL of solution were circulating through a 0.4 mm quartzflow cell whereas for TCSPC (excitation peak intensity at267 nm: 3 kW cm−2), 3.5 mL of solution were continuouslystirred in a 10 × 10 mm quartz cell. The number of absorbedphotons per pulse was approximately 10−4 and 10−9 times lowerthan the number of bases in the excited volume for FU and
Fig. 1 Normalised UV absorption spectra and melting curves at260 nm (insets) of (A) ss-11a (blue), (B) ss-11b (green) and (C) ds-11(red). Black lines correspond to stoichiometric mixtures of mono-nucleo-tides, determined experimentally. The grey line in C represents theaverage spectrum of ss-11a and ss-11b; it was calculated as a linear com-bination of spectra recorded independently.
TCSPC, respectively. In both experiments, successive measure-ments gave identical decays, showing that no significant photo-degradation occurred during the measurements. The data setswere finally merged to increase the signal-to-noise ratio.
3. Results and discussion
3.1 Steady-state spectra
Fig. 1 compares the UV absorption spectra of ss-11a, ss-11b andds-11 with those of the stoichiometric mixtures of the corres-ponding mono-nucleotides. For all three oligonucleotides, theabsorption maximum is blue-shifted with respect to that of themonomers (Table 1). The same effect was detected previouslyfor model oligomers29,30,33 and attributed to the existence ofFranck–Condon exciton states.20,34 Although the fingerprint ofthe electronic coupling is present in the spectra of the singlestrands, the profile of the duplex spectrum differs from that cor-responding to the sum of ss-11a and ss-11b (Fig. 1C), deter-mined using the molar absorption coefficients given by Tataurovet al.:35 the former spectrum is narrower and slightly shifted toshorter wavelengths. This observation, together with the moreimportant hypochromism exhibited by the duplex (insets inFig. 1), proves that base pairing increases the strength of elec-tronic interactions in the ground state conformation. The normal-ized spectra of ds-11 and ds-22 practically overlap (Fig. ESI-2‡);yet, the hypochromism of the longer duplex is about 25% higherthan that of the shorter one (Fig. ESI-1‡).
The steady-state fluorescence spectra of ss-11a, ss-11b andds-11, together with those of the corresponding monomermixtures, are shown in Fig. 2. In addition to the peak located
close to the maximum of the ππ* emission of monomers, theyall present a low energy band. The latter is more pronouncedin the case of the purine rich single strand (Fig. 2A) but appearsas a shoulder for the pyrimidine rich single strand (Fig. 2B).Low energy emission of DNA has been attributed to excimers/exciplexes,36 associated with charge transfer states, as recentlyrationalized by quantum chemical calculations.37–39
Upon base-pairing, the intensity of the low energy banddiminishes as derived from comparison of the spectra obtainedfor ds-11 and that corresponding to an equimolar mixture of thetwo parent single strands. A further decrease in the intensity ofthe low energy band is observed upon increasing the size of theduplex (Fig. 3A). In the duplex spectra, the maximum of thehigh energy band is located at somewhat shorter wavelengthscompared to the mono-nucleotide mixture, the difference beingmore important for the longer duplex (325 nm vs. 330 nm;Table 2).
In order to quantify the intensity of the high and low energyemission bands of the oligonucleotides, we have fitted theirspectra with the sum of two Gaussians (Fig. ESI-3‡). This isonly a rough estimation because in the case of multichromopho-ric systems, containing at least three different types of chromo-phores and existing in a multitude of conformations, theobserved bands are expected to be the envelop of a large numberof electronic transitions.40 The emission maxima and the totalfluorescence quantum yields ϕTOT are shown in Table 2, wherewe have also added the quantum yields of the high (ϕH) and low(ϕL) energy bands, derived from the fits with the Gaussians.
The ϕH values show only weak variation, ranging from 0.34 ×10−4 to 0.55 × 10−4, and are smaller than the quantum yield of
Table 1 Maxima of the absorption spectra of the studiedoligonucleotides (νO) and the corresponding stoichiometric mixture ofmono-nucleotides (νM) and energy difference between them (Δν)
Fig. 2 Normalised fluorescence spectra of (A) ss-11a (blue), (B) ss-11b (green) and (C) ds-11 (red). Black lines correspond to measurementswith stoichiometric mixtures of mono-nucleotides. The grey line in Crepresents the spectrum of an equimolar mixture of ss-11a and ss-11b; itwas calculated as a linear combination of spectra recorded indepen-dently, taking into account their respective absorbances and quantumyields.
Fig. 3 Effect of the duplex size on the fluorescence properties: ds-11(red) and ds-22 (dark red). (A) Steady-state emission spectra whose rela-tive intensity is representative of the quantum yields. (B) Normalised FUdecays at 330 nm and (C) normalised TCSPC decays at 420 nm. Theinstrumental response function is shown in grey.
Table 2 Maxima of the steady-state emission spectra of the studiedoligomers (λO) and fluorescence quantum yields corresponding to theirhigh (ϕH) and low energy (ϕL) bands and total emission (ϕTOT); λM andϕM denote the fluorescence maximum and quantum yield ofstoichiometric mixtures of mono-nucleotides, respectively
the mono-nucleotides. In the case of duplexes, ϕH is aboutone order of magnitude lower compared to that of CT-DNA(3 × 10−4) which does not exhibit any low energy emissionband.24 In contrast to ϕH, ϕL undergoes larger variations, decreas-ing by a factor of six going from ss-11a to ds-22.
3.2 Time-resolved properties
The fluorescence decay of ds-11 recorded by FU at 330 nm isnot very different from those of the corresponding mono-nucleotide mixture and the purine rich single strand (Fig. 4A). Asomewhat slower decay is observed for the pyrimidine richstrand. The fluorescence spectrum of the duplex ds-11 recordedby FU at 0.3 ps (Fig. 4B) is located at the same position as thatof the mono-nucleotide mixture but it is slightly narrower.
In order to obtain a quantitative description of the decays, wefollowed a fitting/deconvolution procedure using a bi-exponen-tial model function: α × exp(−t/τ1) + (1 − α) × exp(−t/τ2). Wedetermined an average lifetime <τ>, defined as α × τ1 + (1 − α)× τ2, whose values fall in the range 0.3–0.6 ps (Table 3), that ismuch the same as those reported for the mono-nucleotides.3
They are also quite close to the shortest time-constants deter-mined for these systems by transient absorption (0.43 ps).28 Weremark that the <τ> value of ds-11 is lower than the averagevalue found for the parent single strands as well as that of ds-22.In other terms, base pairing or a decrease in the duplex sizeaccelerates the deactivation of the bright ππ* states.
Schwalb and Temps reported that the introduction of GC pairsin AT sequences quenches the fluorescence of the bright ππ*states.7 Comparison of the FU decay obtained for ds-22 withthose of model duplexes of about the same size, studied underthe same experimental conditions, is in qualitative agreementwith their conclusion. As a matter of fact, the <τ> value of ds-22is larger than that of alternating GC (0.21 ps)41 and smaller thanthose of alternating AT (1.1 ps)29 and homopolymeric AT(2.5 ps)30 duplexes.
In Fig. 5, we compare the fluorescence decays of ds-11 at330 nm with those of CT-DNA.31 The FU signals of the two“disordered” systems are similar. In contrast, the differencebetween their TCSPC signals is more striking, that of CT-DNAbeing clearly slower (Fig. 5B). But despite the large amplitudeof the ultrafast part of the ds-11 signal, coinciding with theinstrumental response function, even for this system, the majorityof the photons are emitted at times longer than 100 ps.
Weak amplitude long-lived components are present in all theTCSPC decays recorded from 305 to 420 nm for the four exam-ined oligomers (Fig. ESI-4‡). As found so far for other DNAmultimers, at least three-exponential functions are needed to fitcorrectly the fluorescence decays over four decades of time, andthe time constants derived from the fitting/deconvolution pro-cedure vary with the emission wavelength. In fact, such func-tions are not appropriate for multichromophoric systems, inwhich electronic coupling is operative. Therefore, we considerthat histograms describing the percentage of photons emitted perdecade of time, constructed using the fitted functions, provide arough but a more representative picture of the decays. The fittedparameters are given in the ESI.‡
We show in Fig. 6 the histograms at 305 and 420 nm whichcorrespond approximately to the decays of the high energy andlow energy emission bands of the fluorescence spectra (Fig. 2).It is clear that the high energy band is dominated by long-livedcomponents, the ultrafast emission from bright ππ* states rep-resenting only a small fraction of the emitted photons; the pyrimi-dine rich single strand ss-11b, for which 55% of the photons areemitted in less than 100 ps, being an exception. For theduplexes, about half of the photons at 305 nm are emitted after1 ns. Considering that the ϕH values determined for the duplexesare ca. 4 × 10−5 (Table 1) and the percentage of photons emittedat 330 nm before 10 ps is lower than 10% (ESI-4B‡), we deducethat the fluorescence quantum yield of the bright ππ* states is
Fig. 4 (A) Fluorescence decays at 330 nm and (B) fluorescence spectraat 0.3 ps recorded by FU for the ss-11a (blue), ss-11b (green), ds-11(red) and the stoichiometric mixture of mono-nucleotides correspondingto the duplex (black). The latter was determined experimentally. Theinstrumental response function is shown in grey. The symbols and solidlines in B correspond to the experimental points and the fitted Gaussianfunctions, respectively.
Fig. 5 Fluorescence decays recorded by (A) FU and (B) TCSPC at330 nm for ds-11 (red) and CT-DNA (violet). The instrumental responsefunctions are shown in grey.
Table 3 Parameters derived from the fits of the FU decays at 330 nmby bi-exponential functions: α × exp(−t/τ1) + (1 − α) × exp(−t/τ2). Theaverage lifetime is defined as <τ> = α × τ1 + (1 − α) × τ2. Mduplexdenotes the stoichiometric mixture of mono-nucleotides correspondingto the duplexes
lower than 4 × 10−6. We recall that these states, whose lifetimewithin double stranded structures does not exceed a fewps,7,8,13,15,42 emit around 330 nm. The radiative lifetime deter-mined for the duplexes using this ϕ value and the average life-times in Table 3 is larger than 100 ns. This contrasts with theradiative lifetimes of the monomeric chromophores which areonly a few ns. Such a discrepancy shows that approximating theultrafast component by a single time constant is not correct andprobably complex processes take place at very short times.
The largest percentage of photons emitted at 420 nm isencountered in the 0.1–1 ns interval. Thus, low energy excimer/exciplex emission is not associated with the longest livedspecies, which emit mostly at higher energy. This can also beseen in Fig. 3, where the duplexes ds-11 and ds-22 are com-pared. For the shorter duplex, for which the low energy band ismore pronounced, the fluorescence decay at 420 nm is slower fortimes shorter than 1 ns, but at longer times the decays of theshort and long duplexes coincide.
The time-resolved fluorescence anisotropy determined by FUfor ds-11 is compared in Fig. 7A to that of the stoichiometricmixture of monomers. Note that for all the studied modelduplexes,17,29,30,41 but also for CT-DNA,27 the fluorescence an-isotropy at time zero is lower than that of the mono-nucleotidesand decreases further on the femtosecond time-scale, provingthat ultrafast energy transfer takes place. In the present case, thesignal is too noisy to draw any conclusion. More precisely, thesignal fluctuations (0.25 ± 0.05) in Fig. 7A are comparable tothe r(t) decrease observed between 0 and 0.6 ps (from 0.28 to0.22) for AT duplexes for which emission from bright states ismore intense.17
The time zero fluorescence anisotropy of ds-11 recorded byTCSPC at 330 nm is close to that determined by FU. In Fig. 7B,we present the r(t) signals determined by TCSPC for ds-11 at330 and 420 nm; those of ds-22 are shown in Fig. ESI-5.‡Clearly lower r(t) values are observed at 420 nm, already at zero-time; negative anisotropy values have been reported recently forguanine quadruplexes which also exhibit a fluorescence band
around 420 nm.43 The difference between the two signals inFig. 7B diminishes as a function of time. Yet, at 1 ns the signalat 330 nm is slightly positive (0.01) whereas that at 420 nmapproaches zero (inset in Fig. 7B). The same value of 0.01 wasfound on the sub-nanosecond timescale for alternating GCduplexes, independently of their size.31 The spectra of thesemodel duplexes present an emission band located at energieshigher than that of the ππ* mono-nucleotide emission and decay-ing on the nanosecond time-scale. The duplex ds-11 contains asingle alternating GC sequence; therefore, it is possible that theweak amplitudes of the long-lived fluorescence componentobserved for both ds-11 and ds-22 are correlated with the sametype of electronic transition whose nature has not been eluci-dated so far.
4. Summary and concluding remarks
According to the observations based on both steady-state andtime-resolved fluorescence measurements presented here, weconclude that emission of the studied duplexes arises from threetypes of electronic excited states. (i) Bright ππ* states peakingaround 330 nm and decaying on the sub-picosecond time-scalewith an average lifetime of ca. 0.4 ps. The associated quantumyield is lower than 4 × 10−6. The radiative lifetime does notcorrespond to emission from monomeric chromophores andsuggests that complex processes take place. (ii) Excimers/exciplexes, emitting around 430 nm mainly on the sub-nanosecond time-scale. (iii) Excited states decaying mainly onthe ns time-scale and responsible for the high energy fluore-scence (λ < 330 nm). The latter excited states, whose natureremains unclear (excitonic, charge transfer, nπ*), could be corre-lated to GC pairs.31
Excimer/exciplex emission decreases upon base pairing. Thisis in line with the conclusion drawn from transient absorptionmeasurements on these systems.28 However, the lifetimesassigned to these species in the transient absorption study do notcompare with the fluorescence data. This is not surprisingbecause excited states populations in multichromophoric systemsare not expected to decay mono-exponentially. Consequently,
Fig. 6 Percentage of emitted photons per decade of time determinedfor ss-11a (blue), ss-11b (green), ds-11 (red) and ds-22 (dark red) at 305(left panels) and 420 nm (right panels) from TCSPC measurements.
Fig. 7 Fluorescence anisotropy determined for ds-11 (red) by (A) FUat 330 nm and (B) TCSPC at 330 nm (circles) and 420 nm (triangles)between 0 and 1 ps (0.6 and 1.5 ps in the inset). The FU signal corres-ponding to the stoichiometric mixture of mono-nucleotides (black) wasdetermined experimentally. The instrumental response functions areshown in grey.
transient absorption signals, recorded at 250 nm (bleaching ofthe ground state absorption) or at 570 nm, may include contri-butions from various types of excited states possibly character-ized by different molar absorption coefficients. However, theshortest time-constant determined by transient absorption isclose to the <τ> values found by FU.
Finally, the fluorescence properties of the disordered (withrespect to base sequence) oligomeric duplex studied here areradically different from those of the also disordered, but muchlonger, CT-DNA.31 This discrepancy suggests that the nearestneighbour sequence cannot account alone for the CT-DNA fluor-escence and that long range effects should play a role. The originof such effects may be related to the ground state geometry and/or the electronic coupling. An increase in the size of the duplexmay improve base pairing and base stacking. This, in turn, couldaffect trapping of the ππ* excitons by charge transfer excitedstates. The latter have been suggested to give rise to charge sep-aration followed by charge recombination to ππ* states, resultingin delayed fluorescence.27 The conditions for such complex pro-cesses are expected to depend on the occurrence of specifictracts with a given base sequence within the double helix.
Acknowledgements
The French Agency for Research (ANR-10-BLAN-0809-01) andthe Conselleria de Educacion-Generalitat Valenciana (VALi+Dprogram to I.V., no. 2010033) are acknowledged for financialsupport.
Notes and references
1 J.-M. L. Pecourt, J. Peon and B. Kohler, Ultrafast internal conversion ofelectronically excited RNA and DNA nucleosides in water, J. Am. Chem.Soc., 2000, 122, 9348–9349.
2 J. Peon and A. H. Zewail, DNA/RNA nucleotides and nucleosides: directmeasurement of excited-state lifetimes by femtosecond fluorescence up-conversion, Chem. Phys. Lett., 2001, 348, 255–262.
3 D. Onidas, D. Markovitsi, S. Marguet, A. Sharonov and T. Gustavsson,Fluorescence properties of DNA nucleosides and nucleotides: a refinedsteady-state and femtosecond investigation, J. Phys. Chem. B, 2002, 106,11367–11374.
4 D. Markovitsi, A. Sharonov, D. Onidas and T. Gustavsson, Effect ofmolecular organisation in DNA oligomers studied by femtosecondfluorescence spectroscopy, ChemPhysChem, 2003, 4, 303–305.
5 I. Buchvarov, Q. Wang, M. Raytchev, A. Trifonov and T. Fiebig, Elec-tronic energy delocalization and dissipation in single- and double-stranded DNA, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 4794–4797.
6 N. Schwalb and F. Temps, Ultrafast electronic excitation in guanosine ispromoted by hydrogen bonding with cytidine, J. Am. Chem. Soc., 2007,129, 9272–9273.
7 N.K. Schwalb and F. Temps, Base sequence and higher-order structureinduce the complex excited-state dynamics in DNA, Science, 2008, 322,243–245.
8 W. M. Kwok, C. S. Ma and D. L. Phillips, “Bright” and “Dark” excitedstates of an alternating AT oligomer characterized by femtosecond broad-band spectroscopy, J. Phys. Chem. B, 2009, 113, 11527–11534.
9 W.-M. Kwok, C. Ma and D. L. Phillips, Femtosecond time- and wave-length-resolved fluorescence and absorption study of the excited states ofadenosine and an adenine oligomer, J. Am. Chem. Soc., 2006, 128,11894–11905.
10 L. Biemann, et al., Excited state proton transfer is not involved in theultrafast deactivation of guanine-cytosine pair in solution, J. Am. Chem.Soc., 2011, 133, 19664–19667.
11 V. Karunakaran, K. Kleinermanns, R. Improta and S. A. Kovalenko,Photoinduced dynamics of guanosine monophosphate in water from
broad-band transient absorption spectroscopy and quantum-chemicalcalculations, J. Am. Chem. Soc., 2009, 131, 5839–5850.
12 V. O. Melnikova and H. N. Ananthaswamy, Cellular and molecularevents leading to the development of skin cancer, Mutat. Res., Fundam.Mol. Mech. Mutagen., 2005, 571, 91–106.
13 B. Kohler, Nonradiative decay mechanisms in DNA model systems,J. Phys. Chem. Lett., 2010, 1, 2047–2053.
14 T. Gustavsson, R. Improta and D. Markovitsi, DNA/RNA: buildingblocks of life under UV irradiation, J. Phys. Chem. Lett., 2010, 1, 2025–2030.
15 D. Markovitsi, T. Gustavsson and I. Vayá, Fluorescence of DNAduplexes: from model helices to natural DNA, J. Phys. Chem. Lett.,2010, 1, 3271–3276.
16 B. Bouvier, T. Gustavsson, D. Markovitsi and P. Millié, Dipolar couplingbetween electronic transitions of the DNA bases and its relevance toexciton states in double helices, Chem. Phys., 2002, 275, 75–92.
17 D. Markovitsi, D. Onidas, T. Gustavsson, F. Talbot and E. Lazzarotto,Collective behavior of Franck-Condon excited states and energytransfer in DNA double helices, J. Am. Chem. Soc., 2005, 127, 17130–17131.
18 E. R. Bittner, Frenkel exciton model of ultrafast excited state dynamicsin AT DNA double helices, J. Photochem. Photobiol., A, 2007, 190,328–334.
19 A. L. Burin, J. A. Dickman, D. B. Uskov, C. F. F. Hebbard andG. C. Schatz, Optical absorption spectra and monomer interaction inpolymers: investigation of exciton coupling in DNA hairpins, J. Chem.Phys., 2008, 129, 091102.
20 A. L. Burin, M. E. Armbruster, M. Hariharan and F. D. Lewis, Sum rulesand determination of exciton coupling using absorption and circulardichroism spectra of biological polymers, Proc. Natl. Acad. Sci. U. S. A.,2009, 106, 989–994.
21 A. W. Lange and J. M. Herbert, Both intra- and interstrand charge-transferexcited states in aqueous B-DNA are present at energies comparable to,or just above, the 1ππ* excitonic bright states, J. Am. Chem. Soc., 2009,131, 3913–3922.
22 F. Santoro, V. Barone and R. Improta, Excited states decay of the A-TDNA: A PCM/TD-DFT study in aqueous solution of the (9-Methyl-adenine)2·(1-methyl-thymine)2 stacked tetramer, J. Am. Chem. Soc.,2009, 131, 15232–15245.
23 E. B. Starikov, G. Cuniberti and S. Tanakam, Conformationdependence of DNA exciton parentage, J. Phys. Chem. B, 2009, 113,10428–10435.
24 I. Vayá, T. Gustavsson, F. A. Miannay, T. Douki and D. Markovitsi,Fluorescence of natural DNA: from the femtosecond to the nanosecondtime-scales, J. Am. Chem. Soc., 2010, 132, 11834–11835.
25 A. Anders, DNA fluorescence at room temperature excited by means of adye laser, Chem. Phys. Lett., 1981, 81, 270–272.
26 J. P. Ballini, P. Vigny and M. Daniels, Synchrotron excitation of DNAfluorescence: decay time evidence for excimer emission at room temp-erature, Biophys. Chem., 1983, 18, 61–65.
27 I. Vayá, T. Gustavsson, T. Douki, Y. Berlin and D. Markovitsi, Electronicexcitation energy transfer between nucleobases of natural DNA, J. Am.Chem. Soc., 2012, 134, 11366–11368.
28 K. de La Harpe and B. Kohler, Observation of long-lived excited states inDNA oligonucleotides with significant base sequence disorder, J. Phys.Chem. Lett., 2011, 2, 133–138.
29 D. Onidas, T. Gustavsson, E. Lazzarotto and D. Markovitsi, Fluorescenceof the DNA double helices (dAdT)n. (dAdT)n studied by femtosecondspectroscopy, Phys. Chem. Chem. Phys., 2007, 9, 5143–5148.
30 D. Onidas, T. Gustavsson, E. Lazzarotto and D. Markovitsi, Fluorescenceof the DNA double helix (dA)20.(dT)20 studied by femtosecond spec-troscopy – effect of the duplex size on the properties of the excited states,J. Phys. Chem. B, 2007, 111, 9644–9650.
31 I. Vayá, F.A. Miannay, T. Gustavsson and D. Markovitsi, High energylong-lived excited states in DNA double strands, ChemPhysChem, 2010,11, 987–989.
32 D. Markovitsi, et al., UVB/UVC induced processes in model DNAhelices studied by time-resolved spectroscopy: pitfalls and tricks,J. Photochem. Photobiol., A, 2006, 183, 1–8.
33 D. F. Lewis, X. Liu, Y. Wu and X. Zuo, Stepwise evolution of the struc-ture and the electronic properties of DNA, J. Am. Chem. Soc., 2003, 125,12729–12731.
34 D. Markovitsi, T. Gustavsson and A. Banyasz, Absorption of UV radi-ation by DNA: Spatial and temporal features, Mutat. Res., Rev. Mutat.Res., 2010, 704, 21–28.
35 A.V. Tataurov, Y. You and R. Owczarzy, Predicting ultraviolet spectrumof single stranded and double stranded deoxyribonucleic acids, Biophys.Chem., 2008, 133, 66–70.
36 J. Eisinger, M. Gueron, R.G. Shulman and T. Yamane, Excimer fluor-escence of dinucleotides polynucleotides and DNA, Proc. Natl. Acad.Sci. U. S. A., 1966, 55, 1015–1020.
37 G. Olaso-Gonzalez, M. Merchan and L. Serrano-Andres, The role ofadenine excimers in the photophysics of oligonucleotides, J. Am. Chem.Soc., 2009, 131, 4368–4377.
38 F. Santoro, V. Barone, A. Lami and R. Improta, The excited electronicstates of adenine-guanine stacked dimers in aqueous solution: a PCM/TD-DFT study, Phys. Chem. Chem. Phys., 2010, 12, 4934–4948.
39 R. Improta and V. Barone, Interplay between “neutral” and “charge-transfer” excimers rules the excited state decay in adenine-rich poly-nucleotides, Angew. Chem., Int. Ed., 2012, 12016–12019.
40 E. Emanuele, D. Markovitsi, P. Millié and K. Zakrzewska, UV spectraand excitation delocalisation in DNA: influence of the spectral width,ChemPhysChem, 2005, 6, 1387–1392.
41 F. A. Miannay, in Chemistry, Université Paris Sud N°9709, Orsay,2009.
42 T. Fiebig, Exciting DNA, J. Phys. Chem. B, 2009, 113, 9348–9349.43 Y. Hua, et al., Cation effect on the electronic excited states of guanine
