UV-induced DNA Damage: The Role of Electronic Excited States

International audienceThe knowledge of the fundamental processes induced by the direct absorption of UV radiation by DNA allows extrapolatingconclusions drawn from in vitro studies to the in-vivo DNA photoreactivity. In this respect, the characterization ofthe DNA electronic excited states plays a key role. For a long time, the mechanisms of DNA lesion formation were discussedin terms of generic “singlet” and “triplet” excited state reactivity. However, since the beginning of the 21st century,both experimental and theoretical studies revealed the existence of “collective” excited states, i.e. excited states delocalizedover at least two bases. Two limiting cases are distinguished: Frenkel excitons (delocalized pp* states) andcharge-transfer states in which positive and negative charges are located on different bases. The importance of collectiveexcited states in photon absorption (in particular in the UVA spectral domain), the redistribution of the excitation energywithin DNA, and the formation of dimeric pyrimidine photoproducts is discussed. The dependence of the behavior of thecollective excited states on conformational motions of the nucleic acids is highlighted

Electron holes in G-Quadruplexes: The role of adenine ending groups

The study deals with four-stranded DNA structures (G-Quadruplexes), known to undergo ionization upon direct absorption of low-energy UV photons. Combining quantum chemistry calculations and time-resolved absorption spectroscopy with 266 nm excitation, it focuses on the electron holes generated in tetramolecular systems with adenine groups at the ends. Our computations show that the electron hole is placed in a single guanine site, whose location depends on the position of the adenines at the 3′ or 5′ ends. This position also affects significantly the electronic absorption spectrum of (G+ )• radical cations. Their decay is highly anisotropic, composed of a fast process (<2 µs), followed by a slower one occurring in ~20 µs. On the one hand, they undergo deprotonation to (G-H2)• radicals and, on the other, they give rise to a reaction product absorbing in the 300–500 nm spectral domai

Structures auto-assemblées de guanines étudiées par spectroscopie optique résolue en temps

Les brins d ADN riches en guanine, comme ceux présents à l'extrémité des chromosomes humains, sont capables de s associer entre eux pour former des structures G-quadruplexes, résultant de l association de quatre guanines (G-tétrade). Ces structures sont actuellement l objet d un intérêt particulier pour le développement de nouvelles thérapies anti-cancéreuses et des applications potentielles pour l électronique moléculaire. Il n existe cependant que très peu d études des propriétés photophysiques des G-quadruplexes. L'objectif de ce travail de thèse est d'étudier l influence de la structrure des G-quadruplexes sur leurs propriétés photophysiques au moyen de la spectroscopie de fluorescence résolue en temps sur une gamme temporelle allant de la centaine de femtosecondes à la centaine de nanosecondes. Nous avons examiné l effet de la taille de structures G-quadruplexes tétramoléculaires sur leurs propriétés photophysiques. Nous avons pu montrer que le caractère collectif des états pp* des guanines est renforcé lorsque le nombre de tétrades augmente et qu un transfert d'énergie ultra-rapide, en moins de 100 fs a lieu entre ces états. Nous avons ensuite mis en évidence le rôle des cations métalliques situés dans la cavité centrale des quadruplexes dans le processus de désactivation des états excités. En présence de K+, l'émission provient principalement des états délocalisée pp* des guanines, alors qu en présence de Na+, l émission dominée par la contribution d états excités à caractère de transfert de charge. Enfin, nous avons abordé l'effet de la topologie, en comparant les propriétés photophysiques des G-quadruplexes tétramoléculaires avec celles de structures formées par le repliement d un simple brin d ADN. Les différences observées peuvent s expliquer par la rigidité accrue des structures simple-brins et l'orientation relative différente des tétrades qui détermine la force du couplage électronique entre les bases.Guanine rich DNA strands have the ability to form four-stranded structures (G-quadruplexes). Their repetitive unit is the G-quartet (tetrad) where each base is connected with two others via four hydrogen bonds. These structures have a crucial role in biological aspect, as targets for anti-cancer therapies, and have great potential for applications in nanotechnology. We studied the electronic excited states of G-quadruplexes using two different techniques, fluorescence up-conversion (FU) and time-correlated single photon counting (TCSPC) , which allow probing the emissive states over six decades of time (from hundred femtoseconds to hundreds of nanoseconds). At first, we examined the effect of the size of tetramolecular G-quadruplexes on their photophysical properties. We have found that the collective behavior of Franck-Condon excited states is enhanced when the number of tetrads increases. For all systems studied, the anisotropy of the G-quadruplex, on the time scale of hundreds of femtoseconds, is lower than that of non-interacting mono-nucleotides in solution. This decrease in anisotropy is associated with an ultrafast energy transfer process between the bases. Then we demonstrated that the metal cations located in the central cavity of quadruplexes also affect their photophysical properties. In the presence of K+, emission arises mainly from delocalized pp* states (excitons), whereas in the presence of Na+, it is dominated by the contribution of charge transfer excited states. Finally, we studied the effect of conformation, comparing the properties of tetramolecular G-quadruplexes with those formed by folding a single strand (intramolecular G-quadruplexes). We have shown that the conformation of the nano-structures influences the properties of the excited Franck-Condon states as emissive states as well. These effects are attributed to different geometric arrangement of G-tetrads in tetramolecular and intramolecular quadruplexes.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Solvent Effect on the Singlet Excited-state Dynamics of 5-Fluorouracil in Acetonitrile as Compared with Water

The excited-state dynamics of 5-fluorouracil in acetonitrile has been investigated by femtosecond fluorescence upconversion spectroscopy in combination with quantum chemistry TD-DFT calculations ((PCM/TD-PBE0). Experimentally, it was found that when going from water to acetonitrile solution the fluorescence decay of 5FU becomes much faster. The calculations show that this is related to the opening of an additional decay channel in acetonitrile solution since the dark n/* excited state becomes near degenerate with the bright /* state, forming a conical intersection close to the Franck-Condon region. In both solvents, a S1-S0 conical intersection, governed by the out-of-plane motion of the fluorine atom, is active, allowing an ultrafast internal conversion to the ground state

Effect of C5-Methylation of Cytosine on the UV-Induced Reactivity of Duplex DNA: Conformational and Electronic Factors

International audienceC5-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, performedfor 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

Base Pairing Enhances Fluorescence and Favors Cyclobutane Dimer Formation Induced upon Absorption of UVA Radiation by DNA

[EN] The photochemical properties of the DNA duplex (dA)(20) center dot (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 similar to 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.We thank Mrs. Si. Karpati and M. Perron for their help, Dr. R. lmprota for helpful discussions, and the French Agency for Research (ANR PCV07_ 194999) for financial support. I.V. acknowledges the Conselleria de Educacion-Generalitat Valenciana (VALi+D program, No. 20100331).Banyasz, A.; Vayá Pérez, I.; Changenet-Barret, P.; Gustavsson, T.; Douki, T.; Markovitsi, D. (2011). Base Pairing Enhances Fluorescence and Favors Cyclobutane Dimer Formation Induced upon Absorption of UVA Radiation by DNA. Journal of the American Chemical Society. 133:5163-5165. doi:10.1021/ja110879m5163516513

Influence of the spacer on the photoreactivity of flurbiprofen-tyrosine dyads

[EN] The photoreactivity of diastereomeric dyads containing (S)- or (R)-flurbiprofen (FBP) and (S)-Tyr, either directly linked (1) or separated by a cyclic spacer (3) has been investigated. The main feature is a remarkable intramolecular quenching of FBP fluorescence, especially in 1. The process is clearly configuration dependent, being more efficient for the (R,S)- diastereomer in 1 and for the (S,S)-analogue in 3. Noteworthy, exciplex emission is detected in the 380-500 nm region in the case of 3. Fluorescence decay kinetics from the femtosecond to the nanosecond time-domains provides evidence for the dynamic nature of the quenching. In agreement with the steady-state and time-resolved observations, molecular modelling points to a more favourable geometric arrangement of the two interacting chromophores in 1 than in 3.Financial support from the Spanish Government (CTQ2013-47872-C2-1-P), EU (PCIG12GA-2012-334257, LASERLAB-EUROPE grant agreement no. 284464, EU FP7, and MSCA- 657465) and Generalitat Valenciana (PROMETEOII/2013/005) is gratefully acknowledged.Vayá Pérez, I.; Gustavsson, T.; Markovitsi, D.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2016). Influence of the spacer on the photoreactivity of flurbiprofen-tyrosine dyads. Journal of Photochemistry and Photobiology A: Chemistry. 322:95-101. https://doi.org/10.1016/j.jphotochem.2016.03.006S9510132

Excited State Pathways Leading to Formation of Adenine Dimers

International audienceThe reaction intermediate in the path leading to UV-induced formation ofadenine dimers A=A and AA* is identified for the first time quantum mechanically, usingPCM/TD-DFT calculations on (dA)2 (dA: 2′deoxyadenosine). In parallel, its fingerprint isdetected in the absorption spectra recorded on the millisecond time-scale for the singlestrand (dA)20 (dA: 2′deoxyadenosine)

Molecular spectroscopy: Complexity of excited-state dynamics in DNA

Absorption of ultraviolet light by DNA is known to lead to carcinogenic mutations, but the processes between photon absorption and the photochemical reactions are poorly understood. In their study of the excited-stated dynamics of model DNA helices using femtosecond transient absorption spectroscopy1, Crespo-Hernández et al. observe that the picosecond component of the transient signals recorded for the adenine–thymine oligonucleotide (dA)18(dT)18 is close to that for (dA)18, but quite different from that for (dAdT)9(dAdT)9; from this observation, they conclude that excimer formation limits excitation energy to one strand at a time. Here we use time-resolved fluorescence spectroscopy to probe the excited-state dynamics, which reveals the complexity of these systems and indicates that the interpretation of Crespo-Hernández et al. is an oversimplification. We also comment on the pertinence of separating base stacking and base pairing in excited-state dynamics of double helices and question the authors' assignment of the long-lived signal component found for (dA)18(dT)18 to adenine excimers