On the Use of the Intrinsic DNA Fluorescence for Monitoring Its Damage: A Contribution from Fundamental Studies.

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.

Processes triggered in guanine quadruplexes by direct absorption of UV radiation: From fundamental studies toward optoelectronic biosensors.

Guanine quadruplexes (GQs) are four-stranded DNA/RNA structures exhibiting an important polymorphism. During the past two decades, their study by time-resolved spectroscopy, from femtoseconds to milliseconds, associated to computational methods, shed light on the primary processes occurring when they absorb UV radiation. Quite recently, their utilization in label-free and dye-free biosensors was explored by a few groups. In view of such developments, this review discusses the outcomes of the fundamental studies that could contribute to the design of future optoelectronic biosensors using fluorescence or charge carriers stemming directly from GQs, without mediation of other molecules, as it is the currently the case. It explains how the excited state relaxation influences both the fluorescence intensity and the efficiency of low-energy photoionization, occurring via a complex mechanism. The corresponding quantum yields, determined with excitation at 266/267 nm, fall in the range of (3.0-9.5) × 10-4 and (3.2-9.2) × 10-3 , respectively. These values, significantly higher than the corresponding values found for duplexes, depend strongly on certain structural factors (molecularity, metal cations, peripheral bases, number of tetrads …) which intervene in the relaxation process. Accordingly, these features can be tuned to optimize the desired signal.

Effect of the DNA Polarity on the Relaxation of Its Electronic Excited States.

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'.

The Ubiquity of High-Energy Nanosecond Fluorescence in DNA Duplexes.

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.

Fluorescence of Bimolecular Guanine Quadruplexes: From Femtoseconds to Nanoseconds.

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.

High-Energy Long-Lived Emitting Mixed Excitons in Homopolymeric Adenine-Thymine DNA Duplexes.

The publication deals with polymeric pA●pT and oligomeric A20●T20 DNA duplexes whose fluorescence is studied by time-correlated single photon counting. It is shown that their emission on the nanosecond timescale is largely dominated by high-energy components peaking at a wavelength shorter than 305 nm. Because of their anisotropy (0.02) and their sensitivity to base stacking, modulated by the duplex size and the ionic strength of the solution, these components are attributed to mixed ππ*/charge transfer excitons. As high-energy long-lived excited states may be responsible for photochemical reactions, their identification via theoretical studies is an important challenge.

Deprotonation Dynamics of Guanine Radical Cations.

This review is dedicated to guanine radical cations (G+ )· that are precursors to oxidatively generated damage to DNA. (G+ )· are unstable in neutral aqueous solution and tend to lose a proton. The deprotonation process has been studied by time-resolved absorption experiments in which (G+ )· radicals are produced either by an electron abstraction reaction, using an external oxidant, or by low-energy/low-intensity photoionization of DNA. Both the position of the released proton and the dynamics of the process depend on the secondary DNA structure. While deprotonation in duplex DNA leads to (G-H1)· radicals, in guanine quadruplexes the (G-H2)· analogs are observed. Deprotonation in monomeric guanosine proceeds with a time constant of ~60 ns; in genomic DNA, it is completed within 2 µs; and in guanine quadruplexes, it spans from at least 30 ns to over 50 µs. Such a deprotonation dynamics in four-stranded structures, extended over more than three decades of times, is correlated with the anisotropic structure of DNA and the mobility of its hydration shell. In this case, commonly used second-order reaction models are inappropriate for its description.

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 domain.

The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization.

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 (AG4A)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.

Fundamentals of the Intrinsic DNA Fluorescence.

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 Radicals Induced in DNA by Low-Energy Photoionization.

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.

Guanine Radicals Generated in Telomeric G-Quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions.

The study deals with the primary species, ejected electrons, and guanine radicals, leading to oxidative damage, that is generated in four-stranded DNA structures (guanine quadruplexes) following photo-ionization by low-energy UV radiation. Performed by nanosecond transient absorption spectroscopy with 266 nm excitation, it focusses on quadruplexes formed by folding of GGG(TTAGGG)3 single strands in the presence of K+ ions, TEL21/K+. The quantum yield for one-photon ionization (9.4 × 10-3) was found to be twice as high as that reported previously for TEL21/Na+. The overall population of guanine radicals decayed faster, their half times being, respectively, 1.4 and 6.7 ms. Deprotonation of radical cations extended over four orders of magnitude of time; the faster step, concerning 40% of their population, was completed within 500 ns. A reaction intermediate, issued from radicals, whose absorption spectrum peaked around 390 nm, was detected.

Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-Quadruplexes and Modify Their Reactivity.

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.

Comprehensive Study of Guanine Excited State Relaxation and Photoreactivity in G-quadruplexes.

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.

Unveiling Excited-State Chirality of Binaphthols by Femtosecond Circular Dichroism and Quantum Chemical Calculations.

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.

Populations and Dynamics of Guanine Radicals in DNA strands-Direct versus Indirect Generation.

Guanine radicals, known to be involved in the damage of the genetic code and aging, are studied by nanosecond transient absorption spectroscopy. They are generated in single, double and four-stranded structures (G-quadruplexes) by one and two-photon ionization at 266 nm, corresponding to a photon energy lower than the ionization potential of nucleobases. The quantum yield of the one-photon process determined for telomeric G-quadruplexes (TEL25/Na+) is (5.2 ± 0.3) × 10-3, significantly higher than that found for duplexes containing in their structure GGG and GG sequences, (2.1 ± 0.4) × 10-3. The radical population is quantified in respect of the ejected electrons. Deprotonation of radical cations gives rise to (G-H1)• and (G-H2)• radicals for duplexes and G-quadruplexes, respectively. The lifetimes of deprotonated radicals determined for a given secondary structure strongly depend on the base sequence. The multiscale non-exponential dynamics of these radicals are discussed in terms of inhomogeneity of the reaction space and continuous conformational motions. The deviation from classical kinetic models developed for homogeneous reaction conditions could also be one reason for discrepancies between the results obtained by photoionization and indirect oxidation, involving a bi-molecular reaction between an oxidant and the nucleic acid.

Radicals Generated in Tetramolecular Guanine Quadruplexes by Photoionization: Spectral and Dynamical Features.

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+.

Exciton Trapping Dynamics in DNA Multimers.

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-).

Multiscale time-resolved fluorescence study of a glycogen phosphorylase inhibitor combined with quantum chemistry calculations.

A fluorescence study of N1-(ß-d-glucopyranosyl)-N4-[2-acridin-9(10H)-onyl]-cytosine (GLAC), the first fluorescent potent inhibitor of glycogen phosphorylase (GP), in neutral aqueous solution, is presented herein. Quantum chemistry (TD-DFT) calculations show the existence of several conformers both in the ground and first excited states. They result from rotations of the acridone and cytosine moieties around an NH bridge which may lead to the formation of non-emitting charge-transfer states. The fingerprints of various conformers have been detected by time-resolved fluorescence spectroscopy (fluorescence upconversion and time-correlated single photon counting) and identified using as criteria their energy, polarization and relative population resulting from computations. Such an analysis should contribute to the design of new GP inhibitors with better fluorescence properties, suitable for imaging applications.