RESUMO
UV irradiation induces DNA lesions particularly at dipyrimidine sites. Using time-resolved UV pump (250â nm) and mid-IR probe spectroscopy the triplet pathway of cyclobutane pyrimidine dimer (CPD) formation within TpC and CpT sequences was studied. The triplet state is initially localized at the thymine base but decays with 30â ns under formation of a biradical state extending over both bases of the dipyrimidine. Subsequently this state either decays back to the electronic ground state on the 100â ns time scale or forms a cyclobutane pyrimidine dimer lesion (CPD). Stationary IR spectroscopy and triplet sensitization via 2'-methoxyacetophenone (2-M) in the UVA range shows that the lesions are formed with an efficiency of approximately 1.5 %. Deamination converts the cytosine moiety of the CPD lesions on the time scale of 10 hours into uracil which gives CPD(UpT) and CPD(TpU) lesions in which the coding potential of the initial cytosine base is vanished.
Assuntos
Citosina/química , DNA/química , Timina/química , Sequência de Bases , Dano ao DNA/efeitos da radiação , Desaminação , Dímeros de Pirimidina/química , Teoria Quântica , Espectroscopia de Infravermelho com Transformada de Fourier , Raios UltravioletaRESUMO
The decay of electronically excited states of thymine (Thy) and thymidine 5'-monophosphate (TMP) was studied by time-resolved UV/vis and IR spectroscopy. In addition to the well-established ultrafast internal conversion to the ground state, a so far unidentified UV-induced species is observed. In D2O, this species decays with a time constant of 300 ps for thymine and of 1 ns for TMP. The species coexists with the lowest triplet state and is formed with a comparably high quantum yield of about 10% independent of the solvent. The experimentally determined spectral signatures are discussed in the light of quantum chemical calculations of the singlet and triplet excited states of thymine.
RESUMO
Base stacking in DNA is related to long-living excited states whose molecular nature is still under debate. To elucidate the molecular background we study well-defined oligonucleotides with natural bases, which allow selective UV excitation of one single base in the strand. IR probing in the picosecond regime enables us to dissect the contribution of different single bases to the excited state. All investigated oligonucleotides show long-living states on the 100-ps time scale, which are not observable in a mixture of single bases. The fraction of these states is well correlated with the stacking probabilities and reaches values up to 0.4. The long-living states show characteristic absorbance bands that can be assigned to charge-transfer states by comparing them to marker bands of radical cation and anion spectra. The charge separation is directed by the redox potential of the involved bases and thus controlled by the sequence. The spatial dimension of this charge separation was investigated in longer oligonucleotides, where bridging sequences separate the excited base from a sensor base with a characteristic marker band. After excitation we observe a bleach of all involved bases. The contribution of the sensor base is observable even if the bridge is composed of several bases. This result can be explained by a charge delocalization along a well-stacked domain in the strand. The presence of charged radicals in DNA strands after light absorption may cause reactions--oxidative or reductive damage--currently not considered in DNA photochemistry.
Assuntos
DNA/química , Fotoquímica , Oxirredução , Raios UltravioletaRESUMO
Stationary and time-resolved experiments show that 2'-methoxyacetophenone (2-M) is an interesting compound for the investigation of triplet states in thymine samples. Time-resolved emission experiments show that the fluorescence lifetime of 2-M is 660â ps. A similar time constant of 680â ps is found in transient IR experiments. The data indicate efficient intersystem crossing (≈97%) from the fluorescent singlet state to the triplet state. The lifetime of the triplet state of 2-M dissolved in D2O at room temperature and ambient oxygen concentration is 400â ns. 2-M has a strong absorption in the UV-A range and can photosensitize the triplet state of a thymidine dinucleotide with light at a wavelength of 320â nm. The experiments show that 2-M is well-suited for time-resolved experiments on the triplet-sensitizing process.
Assuntos
Fármacos Fotossensibilizantes/química , Dímeros de Pirimidina/química , Acetofenonas , Óxido de Deutério/química , Luz , Teoria Quântica , Espectrofotometria Ultravioleta , TemperaturaRESUMO
Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable ß-hairpin model peptide were initiated by two different mechanisms: temperature jump (T-jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time-resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16â µs. In the T-jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T-jump and isomerization are both determined by the same mechanism and exclude a downhill-folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization-induced folding process ends in a transition-state reaction scheme, in which a high energetic barrier of 48â kJ mol(-1) separates unfolded and folded structures.
Assuntos
Peptídeos/química , Compostos Azo/química , Dicroísmo Circular , Isomerismo , Cinética , Luz , Simulação de Dinâmica Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho , TemperaturaRESUMO
Methylated cytidine plays an important role as an epigenetic signal in gene regulation. Its oxidation products are assumed to be involved in active demethylation processes but also in damaging DNA. Here, we report the photochemical production of the 5-methyl-2'-deoxycytidine radical cation via a two-photon ionization process. The radical cation is detected by time-resolved IR spectroscopy and identified by band assignment using density functional theory calculations. Two final oxidation products are characterized with liquid chromatography coupled to mass spectrometry.
Assuntos
DNA/química , Desoxicitidina/análogos & derivados , Radicais Livres/química , Cátions/química , Dano ao DNA , Desoxicitidina/química , Oxirredução , Espectrofotometria InfravermelhoRESUMO
The importance of a backbone: The mechanism of formation of Dewar lesions has been investigated by using femtosecond IR spectroscopy and abâ initio calculations of the exited state. The 4πâ electrocyclization is rather slow, occurs with an unusual high quantum yield, and--surprisingly--is controlled by the phosphate backbone.
Assuntos
Dano ao DNA/efeitos da radiação , DNA/genética , Ciclização , DNA/química , Isomerismo , Conformação de Ácido Nucleico , Fosfatos/química , Teoria Quântica , Espectrofotometria Infravermelho , Raios UltravioletaRESUMO
The ability of aqueous salt solutions to form hydrates by cooling them at ambient pressure is probed by infrared (IR) spectroscopy by examining the structure of the spectra in the OH-stretching region (3000-3800 cm(-1)). A collection of 75 organic and inorganic salts in saturated solutions are examined. We have found a correlation between the enthalpy of solution of the salt and its ability to form a hydrate, namely, that the salt's enthalpy of solution is lower than the standard enthalpy of fusion of ice (6 kJ/mol). This observation can serve as an empirical rule that determines whether a salt will form a hydrate upon cooling from its aqueous solution.
Assuntos
Sais/química , Termodinâmica , Água/química , SoluçõesRESUMO
We report on the first time-resolved study of the OH stretching vibration in NaCl dihydrate with the use of two-color IR spectroscopy. The sample is characterized by conventional FTIR spectroscopy. The water molecules bound in the hydrate show two well separated absorption bands at 3426 cm(-1) and 3541 cm(-1). The transient data display an ultrafast heating of the polycrystalline ice-hydrate samples after excitation of the OH stretching vibration and its transient relaxation. The relaxation time of the low-frequency OH stretching band in the NaCl hydrate is measured to be 6.8 +/- 1 ps. The dynamics are significantly slower than those measured in neat water. This fact, together with the reproducible crystalline environment reveals the potential of aqueous hydrates for a systematic investigation of the OH stretching vibration in varying hydrogen bonding environments.
RESUMO
UV-induced formation of the cyclobutane pyrimidine dimer (CPD) lesion is investigated by stationary and time-resolved photosensitization experiments. The photosensitizer 2'-methoxyacetophenone with high intersystem crossing efficiency and large absorption cross-section in the UV-A range was used. A diffusion controlled reaction model is presented. Time-resolved experiments confirmed the validity of the reaction model and provided information on the dynamics of the triplet sensitization process. With a series of concentration dependent stationary illumination experiments, we determined the quantum efficiency for CPD formation from the triplet state of the thymine dinucleotide TpT to be 4 ± 0.2%.
RESUMO
UV-induced Dewar lesion formation is investigated in single- and double-stranded oligonucleotides with ultrafast vibrational spectroscopy. The quantum yield for the conversion of the (6-4) lesion to the Dewar isomer in DNA strands is reduced by a factor of 4 in comparison to model dinucleotides. Time resolved spectroscopy reveals a fast process in the excited state with spectral characteristics of bases which are adjacent to the excited (6-4) lesion. These kinetic components have large amplitudes and indicate that an additional quenching channel acts in the stranded DNA systems and reduces the Dewar formation yield. Presumably relaxation evolves via a charge transfer to the neighboring guanine and the paired cytosine participates in a double-stranded oligomer. Changes in the decay of the relaxed excited electronic state of the (6-4) chromophore point to modifications in the excited state energy landscape which may lead to an additional reduction of the Dewar formation yield.
Assuntos
Pareamento de Bases , Dano ao DNA , DNA/química , Cromatografia Líquida de Alta Pressão , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
UV excitation of the DNA single strand (dT)18 leads to electronically excited states that are potential gateways to DNA photolesions. Using time-resolved infrared spectroscopy we characterized a species with a lifetime of â¼100 ps and identified it as a charge separated excited state between two thymine bases.
Assuntos
DNA de Cadeia Simples/química , Timina/análise , Conformação de Ácido Nucleico , Espectrofotometria Infravermelho , Raios UltravioletaRESUMO
The decay of triplet states and the formation of cyclobutane pyrimidine dimers (CPDs) after UV excitation of the all-thymine oligomer (dT)18 and the locked dinucleotide TLpTL were studied by nanosecond IR spectroscopy. IR marker bands characteristic for the CPD lesion and the triplet state were observed from â¼1 ns (time resolution of the setup) onward. The amplitudes of the CPD marker bands remain constant throughout the time range covered (up to 10 µs). The triplet decays with a time constant of â¼10 ns presumably via a biradical intermediate (lifetime â¼60 ns). This biradical has often been invoked as an intermediate for CPD formation via the triplet channel. The present results lend strong support to the existence of this intermediate, yet there is no indication that its decay contributes significantly to CPD formation.