Base Stacking in Adenosine Dimers Revealed by Femtosecond Transient Absorption Spectroscopy

Excitons formed in DNA by UV absorption decay via poorly understood pathways that can culminate in mutagenic photoproducts. In order to gain insight into how base stacking influences UV excited states in DNA, five dinucleosides composed of adenosine or 2′-deoxyadenosine units joined by flexible linkers were studied by femtosecond transient absorption spectroscopy. In aqueous solution, transient absorption signals recorded at pump and probe wavelengths of 267 and 250 nm, respectively, show that UV absorption produces excimer states in all dimers that decay orders of magnitude more slowly than excitations in a single adenine nucleotide. Adding methanol as a cosolvent disrupts π–π stacking of the adenine moieties and causes the excimer states in all five dinucleosides to vanish for a methanol concentration of 80% by volume. These observations confirm that base stacking is an essential requirement for the slow decay channel seen in these and other DNA model compounds. This channel appears to be insensitive to the precise stacking conformation at the instant of photon absorption as long as the bases are cofacially stacked. Notably, circular dichroism (CD) spectra of several of the dinucleosides are weak and monomer-like and lack the exciton coupling that has been emphasized in the past as an indicator of base-stacked structure. For these dimers, the coupled transition dipole moments of the two adenines are proposed to adopt left- and right-handed arrangements upon stacking with roughly equal probability. Although the mechanism behind slow nonradiative decay in DNA is still uncertain, these results show that the signature of these states in transient absorption experiments can be a more reliable diagnostic of base stacking than the occurrence of exciton-coupled CD signals. These observations also draw attention to the important role the backbone plays in producing structures with axial (helical) chirality

Ultrafast Hydrolysis of a Lewis Photoacid

This study explores the concept that electronic excitation can dramatically enhance Lewis acidity. Specifically, it is shown that photoexcitation transforms an electron-deficient organic compound of negligible Lewis acidity in its electronic ground state into a potent excited-state Lewis acid that releases a proton from a nearby water molecule in 3.1 ps. It was shown previously (Peon et al. <i>J. Phys. Chem. A</i> <b>2001</b>, <i>105</i>, 5768) that the excited state of methyl viologen (MV²⁺) is quenched rapidly in aqueous solution with the formation of an unidentified photoproduct. In this study, the quenching mechanism and the identity of the photoproduct were investigated by the femtosecond transient absorption and fluorescence upconversion techniques. Transient absorption signals at UV probe wavelengths reveal a long-lived species with a pH-dependent lifetime due to reaction with hydronium ions at a bimolecular rate of 3.1 × 10⁹ M^–1 s^–1. This species is revealed to be a charge-transfer complex consisting of a ground-state MV²⁺ ion and a hydroxide ion formed when a water molecule transfers a proton to the bulk solvent. Formation of a contact ion pair between MV²⁺ and hydroxide shifts the absorption spectrum of the former ion by a few nm to longer wavelengths, yielding a transient absorption spectrum with a distinctive triangle wave appearance. The slight shift of this spectrum, which is in excellent agreement with steady-state difference spectra recorded for MV²⁺ at high pH, is consistent with an ion pair but not with a covalent adduct (pseudobase). The long lifetime of the ion pair at neutral pH indicates that dissociation occurs many orders of magnitude more slowly than predicted by the Smoluchowski–Debye equation. Remarkably, there is no evidence of geminate recombination, suggesting that the proton that is transferred to the solvent is conducted at least several water shells away. Although the hydrolysis mechanism has yet to be fully established, evidence suggests that the strongly oxidizing excited state of MV²⁺ triggers the proton-coupled oxidation of a water molecule. The observed kinetic isotope effect of 1.7 seen in D₂O vs H₂O is of the magnitude expected for an ultrafast concerted proton–electron transfer reaction. The ultrafast hydrolysis seen here may be a general excited-state quenching mechanism for electronically excited Lewis acids and other powerful photooxidants in aqueous solution

Base-Stacking Disorder and Excited-State Dynamics in Single-Stranded Adenine Homo-oligonucleotides

Single-stranded adenine homo-oligonucleotides were investigated in aqueous solution by femtosecond transient absorption spectroscopy in order to study the effect of strand length on the nature and dynamics of excited states formed by UV absorption. Global fitting analysis of bleach recovery signals recorded at a probe wavelength of 250 nm and pH 7 reveals that the same lifetimes of 2.72 and 183 ps reproduce the pronounced biexponential decays observed in all (dA)_<i>n</i> oligomers, containing between 2 and 18 residues. Although the lifetimes are invariant, the amplitudes of the short- and long-lived components depend sensitively on the number of residues. For example, the 183 ps component increases with strand length and is greater for DNA vs RNA single strands with the same number of adenines. Inhomogeneous kinetics arising from two classes of adenine bases in each oligomer best explains the observations. A subset of adenine residues produce short-lived excited states upon excitation, while absorption by the remaining adenines yields long-lived excited states that are responsible for the long-lived signal. By assuming that each short-lived excited state in the oligomer makes the same contribution to the transient absorption signal as an excited state of the adenine mononucleotide, the fraction of each type of base in the oligomer can be estimated along with the quantum yield of long-lived excited states. The fraction of oligonucleotides that yield long-lived excited states increases with oligomer length in precisely the same manner as the fraction of bases that are found in base stacks. Corroborating evidence that base stacking leads to distinct decay channels comes from experiments conducted at low pH on (dA)₂. Coulombic repulsion between the two protonated bases at pH 2 results in open, unstacked conformations causing the long-lived component seen in (dA)₂ at neutral pH to vanish completely. The fast component seen in oligomers with two or more bases is assigned to vibrational cooling following ultrafast internal conversion to the electronic ground state. This monomer-like decay channel is operative for the subset of adenine residues that are either poorly or not at all stacked with neighboring bases. This study shows that static base stacking disorder fully accounts for the length-dependent transient absorption signals. Although absorption likely creates delocalized excitons of unknown spatial extent, the results from this study suggest that long-lived excitations in single-stranded A tracts are already fully localized on no more than two bases no later than 1 ps after UV excitation

Excited State Relaxation of Neutral and Basic 8‑Oxoguanine

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) is one of the most common forms of DNA oxidative damage. Recent studies have shown that 8-oxo-dGuo can repair cyclobutane pyrimidine dimers in double-stranded DNA when photoexcited, making its excited state dynamics of particular interest. The excited state lifetimes of 8-oxo-dGuo and its anion have been previously probed using transient absorption spectroscopy; however, more information is required to understand the decay mechanisms. In this work, excited state potential energy surfaces for the neutral and deprotonated forms of the free base, 8-oxoguanine (8-oxo-G), are explored theoretically using multireference methods while the nucleoside is experimentally studied using steady-state fluorescence spectroscopy. It is determined that the neutral species exhibits ultrafast radiationless decay via easy access to conical intersections. The relatively long lifetime for the anion can be explained by the existence of sizable barriers between the Franck–Condon region and two S₁/S₀ minimum energy conical intersections. A Strickler–Berg analysis of the experimentally measured fluorescence quantum yields and lifetimes is consistent with emission from <i>ππ</i>* excited states in line with theoretical predictions

Ultrafast Excited-State Dynamics in Hexaethyleneglycol-Linked DNA Homoduplexes Made of A·T Base Pairs

Double-stranded DNA conjugates with the sequence (dA)₁₀·(dT)₁₀ and hexaethylene glycol linkers at one end (hairpin) or both ends (dumbbell) were studied in buffer solution by deep UV femtosecond transient absorption spectroscopy. These covalently constrained duplexes have greatly enhanced thermal stability compared to A·T duplex oligonucleotides that lack linkers. The conjugates eliminate the slipped-strand and end-frayed structures that form readily in unlinked (dA)_<i>n</i>·(dT)_<i>n</i> sequences, allowing the excited-state dynamics of stacked A·T base pairs to be observed without interference from structures with stacking or pairing defects. Transient absorption signals show that subpicosecond internal conversion to the electronic ground state takes place in addition to the formation of long-lived excited states having lifetimes of approximately 70 ps. Watson–Crick base-pairing slows the rate of vibrational cooling compared to monomeric bases or single-stranded DNA, possibly by reducing the total number of solute–solvent hydrogen bonds. Long-lived excited states in intact A·T base pairs decay several times more quickly than long-lived excited states observed in single-stranded (dA)_<i>n</i> sequences. These results show that base-pairing can measurably affect nonradiative decay pathways in A·T duplexes

Thymine Dimer Photoreversal in Purine-Containing Trinucleotides

Cyclobutane–pyrimidine dimer yields in UV-irradiated DNA are controlled by the equilibrium between forward and reverse photoreactions. Past studies have shown that dimer yields are suppressed at sites adjacent to a purine base, but the underlying causes are unclear. In order to investigate whether this suppression is the result of repair by electron transfer from a neighboring nucleobase, the yields and dynamics of the reverse reaction were studied using trinucleotides containing a <i>cis</i>–<i>syn</i> dimer (T<>T) flanked on the 5′ or the 3′ side by adenine or guanine. The probability of forming an excited state on T<>T or on the purine base was varied by tuning the irradiation wavelength between 240 and 280 nm. Cleavage quantum yields decrease by an order of magnitude over this wavelength range and are less than 1% at 280 nm, a wavelength that excites the purine base with more than 95% probability. Conditional quantum yields of cleavage for the trinucleotides given excitation of T<>T are similar in magnitude to the quantum yield of cleavage of unmodified T<>T. These results indicate that within experimental uncertainty all photoreversal in these single-stranded substrates is the result of direct electronic excitation of T<>T. Photolyase-like repair of T<>T due to electron transfer from an adjacent purine is negligible in these substrates. Instead, the observed variation in photoreversal quantum yields for adenine- versus guanine-flanked <i>cis</i>–<i>syn</i> dimer could be due to uncertainties in absorption cross sections or to a modest quenching effect by the purine on the excited state of T<>T. Pump–probe measurements reveal that the excited-state lifetimes of A or G in the dimer-containing trinucleotides are unperturbed by the neighboring dimer, indicating that electron transfer from purine base to T<>T is not competitive with rapid excited-state deactivation. Pump–probe measurements on unmodified T<>T in aqueous solution indicate that cleavage is most likely complete on a picosecond or subpicosecond time scale

UV-Induced Proton Transfer between DNA Strands

UV radiation creates excited states in DNA that lead to mutagenic photoproducts. Photoexcitation of single-stranded DNA can transfer an electron between stacked bases, but the fate of excited states in the double helix has been intensely debated. Here, photoinduced interstrand proton transfer (PT) triggered by intrastrand electron transfer (ET) is detected for the first time by time-resolved vibrational spectroscopy and quantum mechanical calculations. Long-lived excited states are shown to be oppositely charged base pair radical ions. In two of the duplexes, the base pair radical anions are present as tautomers formed by interstrand PT. Charge recombination occurs on the picosecond time scale preventing the accumulation of damaging radicals or mutagenic tautomers

Interligand Electron Transfer in Heteroleptic Ruthenium(II) Complexes Occurs on Multiple Time Scales

The time-dependent localization of the metal-to-ligand charge transfer (MLCT) excited states of ruthenium­(II) complexes containing 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen) ligands was studied by femtosecond transient absorption spectroscopy. Time-resolved anisotropy measurements indicate that the excited state hops randomly among the three ligands of each complex by subpicosecond interligand electron transfer (ILET). Although the bpy- and phen-localized ³MLCT states have similar energies and steady-state emission spectra, pronounced differences in their excited-state absorption spectra make it possible to observe changes in excited state populations using magic angle transient absorption measurements. Analysis of the magic angle signals shows that the excited electron is equally likely to be found on any of the three ligands approximately 1 ps after excitation, but this statistical distribution subsequently evolves to a Boltzmann distribution with a time constant of approximately 10 ps. The apparent contradiction between ultrafast ILET revealed by time-dependent anisotropy measurements and the slower ILET seen in magic angle measurements on the tens of picoseconds time scale is explained by a model in which the underlying rates depend dynamically on excess vibrational energy. The insight that ILET can occur over multiple time scales reconciles contradictory literature observations and may lead to improved photosensitizer performance

Ultrafast Formation of a Delocalized Triplet-Excited State in an Epigenetically Modified DNA Duplex under Direct UV Excitation

Epigenetic modifications impart important functionality to nucleic acids during gene expression but may increase the risk of photoinduced gene mutations. Thus, it is crucial to understand how these modifications affect the photostability of duplex DNA. In this work, the ultrafast formation (<20 ps) of a delocalized triplet charge transfer (CT) state spreading over two stacked neighboring nucleobases after direct UV excitation is demonstrated in a DNA duplex, d(G5fC)9•d(G5fC)9, made of alternating guanine (G) and 5-formylcytosine (5fC) nucleobases. The triplet yield is estimated to be 8 ± 3%, and the lifetime of the triplet CT state is 256 ± 22 ns, indicating that epigenetic modifications dramatically alter the excited state dynamics of duplex DNA and may enhance triplet state-induced photochemistry

Excited-State Dynamics of DNA Duplexes with Different H‑Bonding Motifs

The excited-state dynamics of three distinct forms of the d­(GC)₉·d­(GC)₉ DNA duplex were studied by combined time-resolved infrared experiments and quantum mechanical calculations. In the B- and Z-forms, bases on opposite strands form Watson–Crick (WC) base pairs but stack differently because of salt-induced changes in backbone conformation. At low pH, the two strands associate by Hoogsteen (HG) base pairing. Ultraviolet-induced intrastrand electron transfer (ET) triggers interstrand proton transfer (PT) in the B- and Z-forms, but the PT pathway is blocked in the HG duplex. Despite the different decay mechanisms, a common excited-state lifetime of ∼30 ps is observed in all three duplex forms. The ET–PT pathway in the WC duplexes and the solely intrastrand ET pathway in the HG duplex yield the same pair of π-stacked radicals on one strand. Back ET between these radicals is proposed to be the rate-limiting step behind excited-state deactivation in all three duplexes