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The room-temperature florescence properties of DNA nucleoside and nucleotide aqueous solutions are studied by steady-state and time-resolved spectroscopy. The steady-state fluorescence spectra, although peaking in the near-UV region, are very broad, extending over the whole visible domain.

The assessment of DNA damage by means of appropriate fluorescent probes is widely spread. In the specific case of UV-induced damage, it has been suggested to use the emission of dimeric photoproducts as an internal indicator for the efficacy of spermicidal lamps. However, in the light of fundamental studies on the UV-induced processes, outlined in this review, this is not straightforward. It is by now well established that, in addition to photodimers formed via an electronic excited state, photoionization also takes place with comparable or higher quantum yields, depending on the irradiation wavelength. Among the multitude of final lesions, some have been fully characterized, but others remain unknown; some of them may emit, while others go undetected upon monitoring fluorescence, the result being strongly dependent on both the irradiation and the excitation wavelength. In contrast, the fluorescence of undamaged nucleobases associated with emission from ππ* states, localized or excitonic, appearing at wavelengths shorter than 330 nm is worthy of being explored to this end. Despite its low quantum yield, it is readily detected nowadays. Its intensity decreases due to the disappearance of the reacting nucleobases and the loss of exciton coherence provoked by the presence of lesions, independently of their type. Thus, it could potentially provide valuable information about the DNA damage induced, not only by UV radiation but also by other sanitizing or therapeutic agents., Competing Interests: The author declares no competing financial interest., (© 2024 The Author. Published by American Chemical Society.)

The DNA polarity, i.e ., the order in which nucleobases are connected together via the phosphodiester backbone, is crucial for several biological processes. But, so far, there has not been experimental evidence regarding its effect on the relaxation of DNA electronic excited states. Here we examine this aspect for two dinucleotides containing adenine and guanine: 5'-dApdG-3' and 5'-dGpdA-3' in water. We used two different femtosecond transient absorption setups: one providing high temporal resolution and broad spectral coverage (330-650 nm) between 30 fs and 50 ps, and the other recording decays at selected wavelengths until 1.2 ns. The transient absorption spectra corresponding to the minima in the potential energy surface of the first excited state were computed by quantum chemistry methods. Our results show that the excited charge transfer state in 5'-dGpdA-3' is formed with a ∼75% higher quantum yield and exhibits slower decay (170 ± 10 ps vs 112 ± 12 ps) compared to 5'-dApdG-3'.

During the past few years, several studies reported that a significant part of the intrinsic fluorescence of DNA duplexes decays with surprisingly long lifetimes (1-3 ns) at wavelengths shorter than the ππ* emission of their monomeric constituents. This high-energy nanosecond emission (HENE), hardly discernible in the steady-state fluorescence spectra of most duplexes, was investigated by time-correlated single-photon counting. The ubiquity of HENE contrasts with the paradigm that the longest-lived excited states correspond to low-energy excimers/exciplexes. Interestingly, the latter were found to decay faster than the HENE. So far, the excited states responsible for HENE remain elusive. In order to foster future studies for their characterization, this Perspective presents a critical summary of the experimental observations and the first theoretical approaches. Moreover, some new directions for further work are outlined. Finally, the obvious need for computations of the fluorescence anisotropy considering the dynamic conformational landscape of duplexes is stressed.

The paper deals with the fluorescence of guanine quadruplexes (G4) formed by association of two DNA strands d(GGGGTTTTGGGG) in the presence of K + cations, noted as OXY/K + in reference to the protozoon Oxytricha nova , whose telomere contains TTTTGGGG repeats. They were studied by steady-state and time-resolved techniques, time-correlated single photon counting, and fluorescence upconversion. The maximum of the OXY/K + fluorescence spectrum is located at 334 nm, and the quantum yield is 5.8 × 10 -4 . About 75% of the photons are emitted before 100 ps and stem from ππ* states, possibly with a small contribution of charge transfer. Time-resolved fluorescence anisotropy measurements indicate that ultrafast (<330 fs) excitation transfer, due to internal conversion among exciton states, is more efficient in OXY/K + compared to previously studied G4 structures. This is attributed to the arrangement of the peripheral thymines in two diagonal loops with restricted mobility, facilitating the interaction among them and with guanines. Thymines should also be responsible for a weak intensity excimer/exciplex emission band, peaking at 445 nm. Finally, the longest living fluorescence component (∼2.1 ns) is observed at the blue side of the spectrum. So far, high-energy long-lived emitting states had been reported only for double-stranded structures but not for G4.

Guanine quadruplexes are four-stranded DNA/RNA structures composed of a guanine core (vertically stacked guanine tetrads) and peripheral groups (dangling ends and/or loops). Such a dual structural arrangement of the nucleobases favors their photoionization at energies significantly lower than the guanine ionization potential. This effect is important with respect to the oxidative DNA damage and for applications in the field of optoelectronics. Photoionization quantum yields, determined at 266 nm by nanosecond transient absorption spectroscopy, strongly depend on both the type and position of the peripheral nucleobases. The highest value (1.5 × 10 -2 ) is found for the tetramolecular structure (AG 4 A) 4 in which adenines are intermittently stacked on the adjacent guanine tetrads, as determined by nuclear magnetic resonance spectroscopy. Quantum chemistry calculations show that peripheral nucleobases interfere in a key step preceding electron ejection: charge separation, initiated by the population of charge transfer states during the relaxation of electronic excited states.

The intrinsic fluorescence of nucleic acids is extremely weak compared to that of the fluorescent labels used to probe their structural and functional behavior. Thus, for technical reasons, the investigation of the intrinsic DNA fluorescence was limited for a long time. But with the improvement in spectroscopic techniques, the situation started to change around the turn of the century. During the past two decades, various factors modulating the static and dynamic properties of the DNA fluorescence have been determined; it was shown that, under certain conditions, quantum yields may be up 100 times higher than what was known so far. The ensemble of these studies opened up new paths for the development of label-free DNA fluorescence for biochemical applications. In parallel, these studies have shed new light on the primary processes leading to photoreactions that damage DNA when it absorbs UV radiation.We have been studying a variety of DNA systems, ranging from the monomeric nucleobases to double-stranded and four-stranded structures using fluorescence spectroscopy. The specificity of our work resides in the quantitative association of the steady-state fluorescence spectra with time-resolved data recorded from the femtosecond to the nanosecond timescales, made possible by the development of specific methodologies.Among others, our fluorescence studies provide information on the energy and the polarization of electronic transitions. These are valuable indicators for the evolution of electronic excitations in complex systems, where the electronic coupling between chromophores plays a key role. Highlighting collective effects that originate from electronic interactions in DNA multimers is the objective of the present Account.In contrast to the monomeric chromophores, whose fluorescence decays within a few picoseconds, that of DNA multimers persists on the nanosecond timescale. Even if long-lived states represent only a small fraction of electronic excitations, they may be crucial to the DNA photoreactivity because the probability to reach reactive conformations increases over time, owing to the incessant structural dynamics of nucleic acids.Our femtosecond studies have revealed that an ultrafast excitation energy transfer takes place among the nucleobases within duplexes and G-quadruplexes. Such an ultrafast process is possible when collective states are populated directly upon photon absorption. At much longer times, we discovered an unexpected long-lived high-energy emission stemming from what was coined "HELM excitons". These collective states, whose emission increases with the duplex size, could be responsible for the delayed fluorescence of ππ* states observed for genomic DNA.Most studies dealing with excited-state relaxation in DNA were carried out with excitation in the absorption band peaking at around 260 nm. We went beyond this and also performed the first time-resolved study with excitation in the UVA spectral range, where a very weak absorption tail is present. The resulting fluorescence decays are much slower and the fluorescence quantum yields are much higher than for UVC excitation. We showed that the base pairing of DNA strands enhances the UVA fluorescence and, in parallel, increases the photoreactivity because it modifies the nature of the involved collective excited states.

Guanine ( G ) radicals are precursors to DNA oxidative damage, correlated with carcinogenesis and aging. During the past few years, we demonstrated clearly an intriguing effect: G radicals can be generated upon direct absorption of UV radiation with energy significantly lower than the G ionization potential. Using nanosecond transient absorption spectroscopy, we studied the primary species, ejected electrons and guanine radicals, which result from photoionization of various DNA systems in aqueous solution.The DNA propensity to undergo electron detachment at low photon energies greatly depends on its secondary structure. Undetected for monomers or unstacked oligomers, this propensity may be 1 order of magnitude higher for G -quadruplexes than for duplexes. The experimental results suggest nonvertical processes, associated with the relaxation of electronic excited states. Theoretical studies are required to validate the mechanism and determine the factors that come into play. Such a mechanism, which may be operative over a broad excitation wavelength range, explains the occurrence of oxidative damage observed upon UVB and UVA irradiation.Quantification of G radical populations and their time evolution questions some widespread views. It appears that G radicals may be generated with the same probability as pyrimidine dimers, which are considered to be the major lesions induced upon absorption of low-energy UV radiation by DNA. As most radical cations undergo deprotonation, the vast majority of the final reaction products is expected to stem from long-lived deprotonated radicals. Consequently, when G radical cations are involved, the widely used oxidation marker 8-oxodG is not representative of the oxidative damage.Beyond the biological consequences, photogeneration of electron holes in G -quadruplexes may inspire applications in nanoelectronics; although four-stranded structures are currently studied as molecular wires, their behavior as photoconductors has not been explored so far.In the present Account, after highlighting some key experimental issues, we first describe the photoionization process, and then, we focus on radicals. We use as show-cases new results obtained for genomic DNA and Oxytricha G -quadruplexes. Generation and reaction dynamics of G radicals in these systems provide a representative picture of the phenomena reported previously for duplexes and G -quadruplexes, respectively.

G-Quadruplexes are formed by guanine rich DNA/RNA sequences in the presence of metal ions, which occupy the central cavity of these four-stranded structures. We show that these metal ions have a significant effect on the photogeneration and the reactivity of guanine radicals. Transient absorption experiments on G-quadruplexes formed by association of four TGGGGT strands in the presence of K + reveal that the quantum yield of one-photon ionization at 266 nm (8.1 × 10 -3 ) is twice as high as that determined in the presence of Na + . Replacement of Na + with K + also suppresses one reaction path involving deprotonated radicals, (G-H2) • → (G-H1) • tautomerization. Such behavior shows that the underlying mechanisms are governed by dynamical processes, controlled by the mobility of metal ions, which is higher for Na + than for K + . These findings may contribute to our understanding of the ultraviolet-induced DNA damage and optimize optoelectronic devices based on four-stranded structures, beyond DNA.

G-quadruplexes ( G4 ) are four-stranded DNA/RNA structures playing a key role in many biological functions and promising for nanotechnology applications. Here, combining theoretical calculations and multiscale time-resolved fluorescence, we describe, for the first time, an ensemble of photoactivated processes involving the guanines of the G4 core. We use as showcase the G4 formed by the human telomeric sequence GGG(TTAGGG) 3 in the presence of Na + ions. According to quantum mechanical/molecular mechanics calculations, the hyperchromism at the red part of the absorption spectrum, typical of G4 structures, arises mainly from the inner Na + ions. Various relaxation pathways, leading to excited states localized on individual bases, neutral excimers, and excited charge transfer states between two guanines or a guanine and a thymine in the loop, are mapped. Their fingerprints are detected in the fluorescence anisotropies and the fluorescence decays, spanning five decades of time. Finally, a reaction funnel leading to guanine dimerization is identified.

Time-resolved circular dichroism (TR-CD) is a powerful tool for probing conformational dynamics of biomolecules over large time scales that are crucial for establishing their structure-function relationship. However, such experiments, notably in the femtosecond regime, remain challenging due to their extremely weak signals, prone to polarization artifacts. By using binol and two bridged derivatives (PL1 and PL2) as chiral prototypes, we present here the first comprehensive study of this type in the middle UV, combining femtosecond TR-CD and quantum mechanical calculations (TD-DFT). We show that excitation of the three compounds induces large variations of their transient CD signals, in sharp contrast to those of their achiral transient absorption. We demonstrate that these variations arise from both the alteration of the electronic distribution and the dihedral angle in the excited state. These results highlight the great sensitivity of TR-CD detection to signals hardly accessible to achiral transient absorption.

G-quadruplexes are four-stranded DNA structures playing a key role in many biological functions and are promising for applications in the field of nanoelectronics. Characterizing the generation and fate of radical cations (electron holes) within these systems is important in relation to the DNA oxidative damage and/or conductivity issues. This study focuses on guanine radicals in G-quadruplexes formed by association of four TGGGGT strands in the presence of Na + cations, (TG4T) 4 /Na + . Using nanosecond transient spectroscopy with 266 nm excitation, we quantitatively characterize hydrated ejected electrons and three types of guanine radicals. We show that, at an energy lower by 2.7 eV than the guanine ionization potential, one-photon ionization occurs with quantum yield of (3.5 ± 0.5) × 10 -3 . Deprotonation of the radical cations is completed within 20 μs, leading to the formation of (G-H2) • radicals, following a strongly nonexponential decay pattern. Within 10 ms, the latter undergoes tautomerization to deprotonated (G-H1) • radicals. The dynamics of the various radicals determined for (TG4T) 4 /Na + , in connection to those reported previously for telomeric G-quadruplexes TEL21/Na + , is correlated with energetic factors computed by quantum chemical methods. The faster deprotonation of radical cations in (TG4T) 4 /Na + compared to TEL21/Na + explains that irradiation of the former does not generate 8-oxodGuo, which is readily detected by high-performance liquid chromatography/mass spectrometry in the case of TEL21/Na + .

Using as a model the single adenine strand (dA) 20 , we study the ultrafast evolution of electronic excitations in DNA with a time resolution of 30 fs. Our transient absorption spectra in the UV and visible spectral domains show that internal conversion among photogenerated exciton states occurs within 100 fs. Subsequently, the ππ* states acquire progressively charge-transfer character before being completely trapped, within 3 ps, by fully developed charge-transfer states corresponding to transfer of an electron from one adenine moiety to another (A + A - ).

Telomeres, which are involved in cell division, carcinogenesis, and aging and constitute important therapeutic targets, are prone to oxidative damage. This propensity has been correlated with the presence of guanine-rich sequences, capable of forming four-stranded DNA structures (G-quadruplexes). Here, we present the first study on oxidative damage of human telomere G-quadruplexes without mediation of external molecules. Our investigation has been performed for G-quadruplexes formed by folding of GGG(TTAGGG) 3 single strands in buffered solutions containing Na + cations (TEL21/Na + ). Associating nanosecond time-resolved spectroscopy and quantum mechanical calculations (TD-DFT), it focuses on the primary species, ejected electrons and guanine radicals, generated upon absorption of UV radiation directly by TEL21/Na + . We show that, at 266 nm, corresponding to an energy significantly lower than the guanine ionization potential, the one-photon ionization quantum yield is 4.5 × 10 -3 . This value is comparable to that of cyclobutane thymine dimers (the major UV-induced lesions) in genomic DNA; the quantum yield of these dimers in TEL21/Na + is found to be (1.1 ± 0.1) × 10 -3 . The fate of guanine radicals, generated in equivalent concentration with that of ejected electrons, is followed over 5 orders of magnitude of time. Such a quantitative approach reveals that an important part of radical cation population survives up to a few milliseconds, whereas radical cations produced by chemical oxidants in various DNA systems are known to deprotonate, at most, within a few microseconds. Under the same experimental conditions, neither one-photon ionization nor long-lived radical cations are detected for the telomere repeat TTAGGG in single-stranded configuration, showing that secondary structure plays a key role in these processes. Finally, two types of deprotonated radicals are identified: on the one hand, (G-H 2 ) • radicals, stable at early times, and on the other hand, (G-H 1 ) • radicals, appearing within a few milliseconds and decaying with a time constant of ∼50 ms.

The study concerns the relaxation of electronic excited states of the DNA nucleoside deoxycytidine (dCyd) and its methylated analogue 5-methyldeoxycytidine (5mdCyd), known to be involved in the formation of UV-induced lesions of the genetic code. Due to the existence of four closely lying and potentially coupled excited states, the deactivation pathways in these systems are particularly complex and have not been assessed so far. Here, we provide a complete mechanistic picture of the excited state relaxation of dCyd/5mdCyd in three solvents-water, acetonitrile, and tetrahydrofuran-by combining femtosecond fluorescence experiments, addressing the effect of solvent proticity on the relaxation dynamics of dCyd and 5mdCyd for the first time, and two complementary quantum mechanical approaches (CASPT2/MM and PCM/TD-CAM-B3LYP). The lowest energy ππ* state is responsible for the sub-picosecond lifetime observed for dCyd in all the solvents. In addition, computed excited state absorption and transient IR spectra allow one, for the first time, to assign the tens of picoseconds time constant, reported previously, to a dark state (n O π*) involving the carbonyl lone pair. A second low-lying dark state, involving the nitrogen lone pair (n N π*), does significantly participate in the excited state dynamics. The 267 nm excitation of dCyd leads to a non-negligible population of the second bright ππ* state, which affects the dynamics, acting mainly as a "doorway" state for the n O π* state. The solvent plays a key role governing the interplay between the different excited states; unexpectedly, water favors population of the dark states. In the case of 5mdCyd, an energy barrier present on the main nonradiative decay route explains the 6-fold lengthening of the excited state lifetime compared to that of dCyd, observed for all the examined solvents. Moreover, C5-methylation destabilizes both n O π* and n N π* dark states, thus preventing them from being populated.

Adenyl radicals generated in DNA single and double strands, (dA) 20 and (dA) 20 ·(dT) 20 , by one- and two-photon ionization by 266 nm laser pulses decay at 600 nm with half-times of 1.0 ± 0.1 and 4 ± 1 ms, respectively. Though ionization initially forms the cation radical, the radicals detected for (dA) 20 are quantitatively identified as N6-deprotonated adenyl radicals by their absorption spectrum, which is computed quantum mechanically employing TD-DFT. Theoretical calculations show that deprotonation of the cation radical induces only weak spectral changes, in line with the spectra of the adenyl radical cation and the deprotonated radical trapped in low temperature glasses.

It has been shown that in addition to formation of pyrimidine dimers, UV irradiation of DNA in the absence of photosensitizer also induces formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, but the mechanism of formation of that oxidized base has not been clearly established. In the present study, we provide an unambiguous demonstration that absorption of UVC and UVB radiation by the nucleobases induces DNA oxidation via a direct process (one-electron oxidation) and not singlet oxygen. Evidence arose from the fact that polyamine-guanine adducts that are specifically produced through the transient formation of guanine radical cation are generated following UV irradiation of DNA in the presence of a polyamine even in the absence of any photosensitizer.

The reaction intermediate in the path leading to UV-induced formation of adenine dimers A═A and AA* is identified for the first time quantum mechanically, using PCM/TD-DFT calculations on (dA)2 (dA: 2'deoxyadenosine). In parallel, its fingerprint is detected in the absorption spectra recorded on the millisecond time-scale for the single strand (dA)20 (dA: 2'deoxyadenosine).

C5-methylation of cytosines is strongly correlated with UV-induced mutations detected in skin cancers. Mutational hot-spots appearing at TCG sites are due to the formation of pyrimidine cyclobutane dimers (CPDs). The present study, performed for the model DNA duplex (TCGTA)3·(TACGA)3 and the constitutive single strands, examines the factors underlying the effect of C5-methylation on pyrimidine dimerization at TCG sites. This effect is quantified for the first time by quantum yields ϕ. They were determined following irradiation at 255, 267, and 282 nm and subsequent photoproduct analysis using HPLC coupled to mass spectrometry. C5-methylation leads to an increase of the CPD quantum yield up to 80% with concomitant decrease of that of pyrimidine(6-4) pyrimidone adducts (64PPs) by at least a factor of 3. The obtained ϕ values cannot be explained only by the change of the cytosine absorption spectrum upon C5-methylation. The conformational and electronic factors that may affect the dimerization reaction are discussed in light of results obtained by fluorescence spectroscopy, molecular dynamics simulations, and quantum mechanical calculations. Thus, it appears that the presence of an extra methyl on cytosine affects the sugar puckering, thereby enhancing conformations of the TC step that are prone to CPD formation but less favorable to 64PPs. In addition, C5-methylation diminishes the amplitude of conformational motions in duplexes; in the resulting stiffer structure, ππ* excitations may be transferred from initially populated exciton states to reactive pyrimidines giving rise to CPDs.

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.

The fluorescence properties of the 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in aqueous solution at pH 6.5 are studied by steady-state spectroscopy and femtosecond fluorescence up-conversion and compared with those of 2'-deoxyguanine (dG) and 2'-deoxyguanine monophosphate (dGMP). The steady-state fluorescence spectrum of 8-oxo-dG is composed of a broad band that peaks at 356 nm and extends over the entire visible spectral region, and its fluorescence quantum yield is twice that of dG/dGMP. After excitation at 267 nm, the initial fluorescence anisotropy at all wavelengths is lower than 0.1, giving evidence of an ultrafast internal conversion (<100 fs) between the two lowest excited ππ* states (Lb and La). The fluorescence decays of 8-oxo-dG are biexponential with an average lifetime of 0.7 ± 0.1 ps, which is about two times longer than that of dGMP. In contrast with dGMP, only a moderate dynamical shift (∼1400 vs 10,000 cm(-1)) of the fluorescence spectra of 8-oxo-dG is observed on the time scale of a few picoseconds without modification of the spectral shape. PCM/TD-DFT calculations, employing either the PBE0 or the M052X functionals, provide absorption spectra in good agreement with the experimental one and show that the deactivation path is similar to that proposed for dGMP, with a fast motion toward an energy plateau, where the purine ring keeps an almost planar geometry, followed by decay to S0, via out-of-the plane motion of amino substituent.

Human pigmentation is a complex phenomenon commonly believed to serve a photoprotective function through the generation and strategic localization of black insoluble eumelanin biopolymers in sun exposed areas of the body. Despite compelling biomedical relevance to skin cancer and melanoma, eumelanin photoprotection is still an enigma: What makes this pigment so efficient in dissipating the excess energy brought by harmful UV-light as heat? Why has Nature selected 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as the major building block of the pigment instead of the decarboxylated derivative (DHI)? By using pico- and femtosecond fluorescence spectroscopy we demonstrate herein that the excited state deactivation in DHICA oligomers is 3 orders of magnitude faster compared to DHI oligomers. This drastic effect is attributed to their specific structural patterns enabling multiple pathways of intra- and interunit proton transfer. The discovery that DHICA-based scaffolds specifically confer uniquely robust photoprotective properties to natural eumelanins settles a fundamental gap in the biology of human pigmentation and opens the doorway to attractive advances and applications.

DNA methylation, occurring at the 5 position of cytosine, is a natural process associated with mutational hotspots in skin tumors. By combining experimental techniques (optical spectroscopy, HPLC coupled to mass spectrometry) with theoretical methods (molecular dynamics, DFT/TD-DFT calculations in solution), we study trinucleotides with key sequences (TCG/T5mCG) in the UV-induced DNA damage. We show how the extra methyl, affecting the conformational equilibria and, hence, the electronic excited states, increases the quantum yield for the formation of cyclobutane dimers while reducing that of (6-4) adducts.

Excited-state proton transfer has been hypothesized as a mechanism for UV energy dissipation in eumelanin skin pigments. By using time-resolved fluorescence spectroscopy, we show that the previously proposed, but unresolved, excited-state intramolecular proton transfer (ESIPT) of the eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA) occurs with a time constant of 300 fs in aqueous solution but completely stops in methanol. The previously disputed excited-state proton transfer involving the 5- or 6-OH groups of the DHICA anion is now found to occur from the 6-OH group to aqueous solvent with a rate constant of 4.0 × 10(8) s(-1).

The study addresses interconnected issues related to two major types of cycloadditions between adjacent thymines in DNA leading to cyclobutane dimers (T<>Ts) and (6-4) adducts. Experimental results are obtained for the single strand (dT)(20) by steady-state and time-resolved optical spectroscopy, as well as by HPLC coupled to mass spectrometry. Calculations are carried out for the dinucleoside monophosphate in water using the TD-M052X method and including the polarizable continuum model; the reliability of TD-M052X is checked against CASPT2 calculations regarding the behavior of two stacked thymines in the gas phase. It is shown that irradiation at the main absorption band leads to cyclobutane dimers (T<>Ts) and (6-4) adducts via different electronic excited states. T<>Ts are formed via (1)ππ* excitons; [2 + 2] dimerization proceeds along a barrierless path, in line with the constant quantum yield (0.05) with the irradiation wavelength, the contribution of the (3)ππ* state to this reaction being less than 10%. The formation of oxetane, the reaction intermediate leading to (6-4) adducts, occurs via charge transfer excited states involving two stacked thymines, whose fingerprint is detected in the fluorescence spectra; it involves an energy barrier explaining the important decrease in the quantum yield of (6-4) adducts with the irradiation wavelength.

Excited-state interactions between (S)- or (R)-flurbiprofen ((S)- or (R)-FBP) and thymidine (dThd) covalently linked in dyads 1 or 2 have been investigated. In both dyads, the only emitting species is (1)FBP*, but with a lower fluorescence quantum yield (φ(F)) and a shorter fluorescence lifetime (τ(F)) than when free in solution. These results indicate that dynamic quenching occurs either by electron transfer or via exciplex formation, with FBP as the charge-donating species. In acetonitrile, both mechanisms are favored, while in dioxane exciplex formation is predominating. Isomer 1 displays lower values of φ(F) and τ(F) than its analogue 2, indicating that the relative spatial arrangement of the chromophores plays a significant role. The triplet quantum yields (φ(T)) of 1 and 2 are significantly higher than the expectations based solely on (1)FBP*-dThd intersystem crossing quantum yields (φ(ISC)), with φ(T) (1) > φ(T) (2). This can be explained in terms of intramolecular charge recombination at the radical ion pairs and/or the exciplexes, which would be again dependent on geometrical factors. The triplet lifetimes (τ(T)) of (3)FBP*-dThd and free (3)FBP* are similar, indicating the lack of excited-state interactions at this stage. The FBP-dThd dyads could, in principle, constitute appropriate model systems for the elucidation of the excited-state interactions in noncovalent DNA-ligand complexes.

Transfer of the electronic excitation energy in calf thymus DNA is studied by time-resolved fluorescence spectroscopy. The fluorescence anisotropy, after an initial decay starting on the femtosecond time scale, dwindles down to ca. 0.1. The in-plane depolarized fluorescence decays are described by a stretched exponential law. Our observations are consistent with one-dimensional transfer mediated by charge-transfer excited states.

The photochemical properties of the DNA duplex (dA)(20)·(dT)(20) are compared with those of the parent single strands. It is shown that base pairing increases the probability of absorbing UVA photons, probably due to the formation of charge-transfer states. UVA excitation induces fluorescence peaking at ∼420 nm and decaying on the nanosecond time scale. The fluorescence quantum yield, the fluorescence lifetime, and the quantum yield for cyclobutane dimer formation increase upon base pairing. Such behavior contrasts with that of the UVC-induced processes., (© 2011 American Chemical Society)

A detailed experimental and computational study of the absorption and fluorescence spectra of 5-aminouracil (5 AU) and 6-aminouracil (6 AU) in aqueous solution is reported. The lowest energy band of the steady-state absorption spectra of 5 AU is considerably red-shifted, noticeably less intense, and broader than its counterpart in uracil (U). On the contrary, the 6 AU lowest energy absorption peak is close in energy to that of U, but it is much narrower and the transition is much more intense. The emission properties of 5 AU, 6 AU, and U are also very different. Both amino-substituted compounds exhibit indeed a much larger Stokes shift as compared to U, and the emission band of 5 AU is much narrower than that of 6 AU. Those features are fully rationalized with the help of PCM/TD-PBE0 calculations in aqueous solution and MS-CASPT2/CASSCF calculations in the gas phase. A stable minimum on the potential energy surface of the lowest energy bright state is found for 5 AU, both in the gas phase and in aqueous solution. For 6 AU a barrierless path leads to the conical intersection with the ground electronic state, but a nonplanar plateau region is predicted in aqueous solution, which is responsible for the very large Stokes shift. Some general considerations on the excited-state dynamics of uracil derivatives are also reported.

The fluorescence of calf thymus DNA is studied by steady-state and time-resolved spectroscopy combining fluorescence upconversion and time-correlated single photon counting. The fluorescence spectrum is very similar to that of a stoichiometric mixture of monomeric chromophores, arising from bright pi pi* states, and contrasts with the existing picture of exciplex emission in natural DNA. Yet, the DNA fluorescence decays span over five decades of time, with 98% of the photons being emitted at times longer than 10 ps. These findings, in association with recent studies on model duplexes, are explained by the involvement of dark states, possibly related to charge separation, serving as a reservoir for the repopulation of the bright pi pi* states.

The paper presents quantum yield results for the [2+2] and 6-4 photodimerization of TT steps in several DNA structures, including hairpins where the context dependence of the photodimerization yield is determined, and it develops a theoretical model that correctly describes the trends in dimerization yield with DNA structure. The DNA conjugates considered include dT(20), dA(20)dT(20), and three alkane-linked hairpins that contain a single TT step. The theoretical modeling of the [2+2] process is based on CASSCF electronic structure calculations for ethylene + ethylene, which show that photoexcitation of low-lying excited states leads to potential surfaces that correlate without significant barriers to a conical intersection with the ground state surface at geometries close to the dimer structure. The primary constraint on dimerization is the distance d between the two double bonds, and it is found that d < 3.52 A leads to quantum yield trends that match the observed trends within a factor of 3. Constraints on the dihedral angle between the two double bonds are not as important, and although it is possible to generate better dimerization yield predictions for some structures by including these constraints, the best overall picture is obtained with no constraint. For 6-4 dimerization, a distance g < 2.87 A and no constraint on dihedral angle provide an accurate description of the yield.

The room-temperature fluorescence of 2'-deoxyguanosine 5'-monophosphate (dGMP) in aqueous solution is studied by steady-state and time-resolved fluorescence spectroscopy. The steady-state fluorescence spectrum of dGMP shows one band centered at 334 nm but has an extraordinary long red tail, extending beyond 700 nm. Both the fluorescence quantum yield and the relative weight of the 334 nm peak increase with the excitation wavelength. The initial fluorescence anisotropy after excitation at 267 nm is lower than 0.2 for all emission wavelengths, indicating an ultrafast S(2) --> S(1) internal conversion. The fluorescence decays depend strongly on the emission wavelength, getting longer with the wavelength. A rise time of 100-150 fs was observed for wavelengths longer than 450 nm, in accordance with a gradual red shift of the time-resolved spectra. The results are discussed in terms of a relaxation occurring mainly on the lowest excited (1)pi pi*-state surface toward a conical intersection with the ground state, in line with recent theoretical predictions. Our results show that the excited-state population undergoes a substantial "spreading out" before reaching the CI, explaining the complex dynamics observed.

The DNA double helix poly(dGdC).poly(dGdC) is studied by fluorescence upconversion spectroscopy with femtosecond resolution. It is shown that the excited-state relaxation of the duplex is faster than that of the monomeric components dGMP and dCMP. This contrasts with the behavior of duplexes composed exclusively of adenine-thymine base pairs, for which an overall lengthening of the fluorescence lifetimes with respect to that of an equimolar mixture of dAMP and TMP was reported previously. Despite the difference in the excited-state deactivation rate between the two types of duplexes, the signature of ultrafast energy transfer is present in both of them. It is attested by the decrease of fluorescence anisotropy decay of the duplexes on the subpicosecond time scale, where molecular motions are inhibited, and is corroborated by the fact that their steady-state fluorescence spectra do not change with the excitation wavelength. Energy transfer involves excited states delocalized over at least two bases, whose existence is revealed by the UV absorption spectrum of the duplex, clearly different from that of an equimolar spectrum of dGMP and dCMP.

The fluorescence of the DNA double-stranded oligomer (dA)20 x (dT)20 is studied at room temperature by fluorescence up-conversion at times shorter than 10 ps. The profile of the up-conversion spectra is similar to that of the steady-state fluorescence spectrum, showing that the majority of the photons are emitted within the probed time scale. At all the probed wavelengths, the fluorescence decays are slower than those of the monomeric chromophores dAMP and TMP. The fluorescence anisotropy decays show strong wavelength dependence. These data allow us to conclude that energy transfer takes place in this double helix and that this process involves exciton states. The spectral and dynamical properties of the oligomer are compared to those of the polymer poly(dA) x poly(dT), composed of about 2000 base pairs, reported previously. The oligomer absorption spectrum is characterized by a smaller hypsochromic shift and weaker hypochromism compared to the polymer. Moreover, the fluorescence decays of (dA)20 x (dT)20 are twice as fast as those of poly(dA) x poly(dT), and its fluorescence anisotropy decays more slowly. These differences are the fingerprints of a larger delocalization of the excited states induced by an increase in the size of the duplex.

The excited-state properties of uracil, thymine, and nine other derivatives of uracil have been studied by steady-state and time-resolved spectroscopy. The excited-state lifetimes were measured using femtosecond fluorescence upconversion in the UV. The absorption and emission spectra of five representative compounds have been computed at the TD-DFT level, using the PBE0 exchange-correlation functional for ground- and excited-state geometry optimization and the Polarizable Continuum Model (PCM) to simulate the aqueous solution. The calculated spectra are in good agreement with the experimental ones. Experiments show that the excited-state lifetimes of all the compounds examined are dominated by an ultrafast (<100 fs) component. Only 5-substituted compounds show more complex behavior than uracil, exhibiting longer excited-state lifetimes and biexponential fluorescence decays. The S(0)/S(1) conical intersection, located at CASSCF (8/8) level, is indeed characterized by pyramidalization and out of plane motion of the substituents on the C5 atom. A thorough analysis of the excited-state Potential Energy Surfaces, performed at the PCM/TD-DFT(PBE0) level in aqueous solution, shows that the energy barrier separating the local S(1) minimum from the conical intersection increases going from uracil through thymine to 5-fluorouracil, in agreement with the ordering of the experimental excited-state lifetime.

Absorption of UV radiation by DNA bases is known to induce carcinogenic mutations. The lesion distribution depends on the sequence around the hotspots, suggesting cooperativity between bases. Here we show that such cooperativity may intervene at the very first step of a cascade of events by formation of Franck-Condon states delocalized over several bases and subsequent energy transfer faster than 100 fs. Our study focuses on the double helix poly(dA).poly(dT), whose fluorescence, induced by femtosecond pulses at 267 nm, is probed by the upconversion technique and time-correlated single photon counting, over a large time domain (100 fs to 100 ns). The time-resolved fluorescence decays and fluorescence anisotropy decays are discussed in relation with the steady-state absorption and fluorescence spectra in the frame of exciton theory.

The present communication deals with the excited states of the alternating DNA oligomer (dCdG)5.(dCdG)5 which correspond to the UV absorption band around 260 nm. Their properties are studied in the frame of the exciton theory, combining molecular dynamics simulations and quantum chemistry data. It is shown that the dipolar coupling undergoes important variations with the site and the helix geometry. In contrast, the energy of the monomer transitions within the double helix is not sensitive to the local environment. It is thus considered to be distributed over Gaussian curves whose maximum and width are derived from the experimental absorption spectra of nucleosides in aqueous solution. The influence of the spectral width on the excited state delocalization and the absorption spectra is much stronger than that of the oligomer plasticity. About half of the excited states are delocalized over at least two bases. Many of them result from the mixing of different monomer states and extend on both strands. The trends found in the simulated spectra, when going from non-interacting monomers to the duplex, are in agreement with experimental observations. Conformational changes enhance the diversity of the states which can be populated upon excitation at a given energy. The states with larger spatial extent are located close to the maximum of the absorption spectrum.

The formation of thymine dimers in the single-stranded oligonucleotide, (dT)20, is studied at room temperature by laser flash photolysis using 266 nm excitation. It is shown that the (6-4) adduct is formed within 4 ms via a reactive intermediate. The formation of cyclobutane dimers is faster than 200 ns. The overall quantum yield for the (6-4) formation is (3.7 +/- 0.3) x 10-3, and that of the cyclobutane dimers is (2.8 +/- 0.2) x 10-2. No triplet absorption is detected, showing that either the intersystem crossing yield decreases by 1 order of magnitude upon oligomerization (<1.4 x 10-3) or the triplet state reacts with unit efficiency in less than 200 ns to yield cyclobutane dimers.