Ashley A. Beckstead

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.

Efficient UV-induced charge separation and recombination in an 8-oxoguanine-containing dinucleotide

Significance UV photons are absorbed strongly by DNA, but rarely cause permanent photodamage. Single nucleobases are protected by ultrafast nonradiative decay, but excited states in single- and double-stranded DNA decay very differently. An intensely debated question is whether a UV photon can move an electron from one nucleobase to another along a single strand. This study demonstrates that UV absorption efficiently transfers an electron from an oxidatively damaged guanine (8-oxo-G) to adenine in a dinucleotide mimic of the flavin cofactor FADH2, yielding radicals that decay in 60 ps. It is proposed that the photoredox activity of 8-oxo-G, which may have repaired cyclobutane pyrimidine dimers in the RNA world, reflects the importance of ultrafast charge separation between stacked nucleobases by UV radiation. During the early evolution of life, 8-oxo-7,8-dihydro-2′-deoxyguanosine (O) may have functioned as a proto-flavin capable of repairing cyclobutane pyrimidine dimers in DNA or RNA by photoinduced electron transfer using longer wavelength UVB radiation. To investigate the ability of O to act as an excited-state electron donor, a dinucleotide mimic of the FADH2 cofactor containing O at the 5′-end and 2′-deoxyadenosine at the 3′-end was studied by femtosecond transient absorption spectroscopy in aqueous solution. Following excitation with a UV pulse, a broadband mid-IR pulse probed vibrational modes of ground-state and electronically excited molecules in the double-bond stretching region. Global analysis of time- and frequency-resolved transient absorption data coupled with ab initio quantum mechanical calculations reveal vibrational marker bands of nucleobase radical ions formed by electron transfer from O to 2′-deoxyadenosine. The quantum yield of charge separation is 0.4 at 265 nm, but decreases to 0.1 at 295 nm. Charge recombination occurs in 60 ps before the O radical cation can lose a deuteron to water. Kinetic and thermodynamic considerations strongly suggest that all nucleobases can undergo ultrafast charge separation when π-stacked in DNA or RNA. Interbase charge transfer is proposed to be a major decay pathway for UV excited states of nucleic acids of great importance for photostability as well as photoredox activity.

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.

UV-Induced Proton-Coupled Electron Transfer in Cyclic DNA Miniduplexes

The excited-state dynamics of two cyclic DNA miniduplexes, each containing just two base pairs, are investigated using time-resolved infrared spectroscopy. As in longer DNA duplexes, intrastrand electron transfer induced by UV excitation triggers interstrand proton transfer in the alternating miniduplex containing two out-of-phase G·C base pairs. The resulting excited state decays on a time scale of several tens of picoseconds. This state is absent when one of the two G residues is substituted by 8-oxo-7,8-dihydroguanine, a modification that is suggested to disrupt base stacking, while maintaining base pairing. These findings demonstrate that a nucleobase tetramer arranged as two stacked base pairs accurately captures the interplay between intrastrand and interstrand decay channels. The similar signals seen in the miniduplexes and longer sequences suggest that excited states in the latter rapidly localize on two adjacent base pairs.

Excited-State Dynamics of Melamine and Its Lysine Derivative Investigated by Femtosecond Transient Absorption Spectroscopy

Melamine may have been an important prebiotic information carrier, but its excited-state dynamics, which determine its stability under UV radiation, have never been characterized. The ability of melamine to withstand the strong UV radiation present on the surface of the early Earth is likely to have affected its abundance in the primordial soup. Here, we studied the excited-state dynamics of melamine (a proto-nucleobase) and its lysine derivative (a proto-nucleoside) using the transient absorption technique with a UV pump, and UV and infrared probe pulses. For melamine, the excited-state population decays by internal conversion with a lifetime of 13 ps without coupling significantly to any photochemical channels. The excited-state lifetime of the lysine derivative is slightly longer (18 ps), but the dominant deactivation pathway is otherwise the same as for melamine. In both cases, the vast majority of excited molecules return to the electronic ground state on the aforementioned time scales, but a minor population is trapped in a long-lived triplet state.

Subnanosecond Emission Dynamics of AT DNA Oligonucleotides

UV radiation creates excited electronic states in DNA that can decay to mutagenic photoproducts. When excited states return to the electronic ground state, photochemical injury is avoided. Understanding of the available relaxation pathways has advanced rapidly during the past decade, but there has been persistent uncertainty, and even controversy, over how to compare results from transient absorption and time-resolved emission experiments. Here, emission from single- and double-stranded AT DNA compounds excited at 265 nm was studied in aqueous solution using the time-correlated single photon counting technique. There is quantitative agreement between the emission lifetimes ranging from 50 to 200 ps and ones measured in transient absorption experiments, demonstrating that both techniques probe the same excited states. The results indicate that excitations with lifetimes of more than a few picoseconds are weakly emissive excimer and charge transfer states. Only a minute fraction of excitations persist beyond 1 ns in AT DNA strands at room temperature.

Ultrafast Excited-State Deactivation of the Bacterial Pigment Violacein

The photophysical properties of the natural pigment violacein extracted from an Antarctic organism adapted to high exposure levels of UV radiation were measured in a combined steady-state and time-resolved spectroscopic study for the first time. In the low-viscosity solvents methanol and acetone, violacein exhibits low fluorescence quantum yields on the order of 1 × 10-4, and femtosecond transient absorption measurements reveal excited-state lifetimes of 3.2 ± 0.2 and 4.5 ± 0.2 ps in methanol and acetone, respectively. As solvent viscosity is increased, both the fluorescence quantum yield and excited-state lifetime of this intensely colored pigment increase dramatically, and stimulated emission decays 30-fold more slowly in glycerol than in methanol at room temperature. Excited-state deactivation is suggested to occur via a molecular-rotor mechanism in which torsion about an interring bond leads to a conical intersection with the ground state.