DNA molecular photonics

Synthetic DNA conjugates in which one or both ends of a short duplex is capped by a stilbene chromophore have been prepared and characterized crystallographically. Selective excitation of the chromophore can be used to initiate electron transfer processes in which a nucleobase serves as either an electron donor or an electron acceptor. These processes include hole- and electron injection and hole migration. The dynamics of these processes and its dependence on distance, driving force, and base sequence have been investigated by means of femtosecond time-resolved spectroscopy. Duplexes with identical chromophores at both ends have been used to study both the dynamics of electron transfer processes and exciton coupling between the two chromophores by means of circular dichroism spectroscopy. Duplexes with different chromophores can also be used to study distance dependence of both electron transfer and exciton coupling.     

Reductive electron transfer and transport of excess electrons in DNA

In principle, DNA-mediated charge transfer processes can be categorized as either oxidative hole transfer or reductive electron transfer. In research on DNA damage, major efforts have focused on the investigation of oxidative hole transfer or transport, resulting in insights on the mechanisms. On the other hand, the transport or transfer of excess electrons has a large potential for biomedical applications, mainly for DNA chip technology. Yet the mechanistic details of this type of charge transfer chemistry were unclear. In the last two years this mechanism has been addressed in gamma-pulse radiolysis studies with randomly DNA-bound electron acceptors or traps. The major disadvantage of this experimental setup is that the electron injection and trapping is not site-selective. More recently, new photochemical assays for the chemical and spectroscopic investigation of reductive electron transfer and electron migration in DNA have been published which give new insights into these processes. Based on these results, an electron-hopping mechanism is proposed which involves pyrimidine radical anions as intermediate electron carriers.     

Hole transfer rates in A-form DNA/2'-OMeRNA hybrid

The hole transfer rates in the DNA/DNA B-form duplex and DNA/2'-OMeRNA A-form duplex were measured which occurred in the time range of approximately 100 micros. The hole transfer rates in the A-form duplexes were slower and more strongly dependent on the temperature compared to those in the B-form duplexes, suggesting that the A-form is more rigid than the B-form duplex in this time scale.     

Long-range energy transfer in DNA

Experimental data on strand breakage in DNA following the direct deposition of ionizing energy seem to require an explanation in terms of long-range energy transfer within the DNA duplex. It is proposed here that the mechanism underlying such energy transfer might involve solitons or solitary waves. These act, at one and the same time, to provide a local environment within the DNA molecule capable of supporting delocalized electronic excitation or charge, and also enable the transportation of such regions along the molecule through the formation of a mobile “open state” in the duplex. Such a mechanism, if established, would have considerable implications for the mechanistic understanding of radiobiology.     

Charge transfer in duplex DNA containing mismatch

A theory for charge transfer between the electrode and the donor/acceptor molecule coupled through a DNA bridge in solution is developed. We explore the crossover between the coherent tunneling and the incoherent sequential transfer regimes by varying the electrode potential and discuss the effects of single-base mismatches in DNA duplex in both regimes. In the former regime a single-base mismatch in DNA duplex causes a reduction in the charge transfer rate simply by decreasing the electron tunneling matrix element, however, in the latter regime the effects are rather complicated.     

Dynamics and energetics of single-step hole transport in DNA hairpins

The dynamics of single-step hole transport processes have been investigated in a number of DNA conjugates possessing a stilbenedicarboxamide electron acceptor, a guanine primary donor, and several secondary donors. Rate constants for both forward and return hole transport between the primary and secondary donor are obtained from kinetic modeling of the nanosecond transient absorption decay profiles of the stilbene anion radical. The kinetic model requires that the hole be localized on either the primary or the secondary donor and not delocalized over both the primary and the secondary donor. Rate constants for hole transport are found to be dependent upon the identity of the secondary donor, the intervening bases, and the location of the secondary donor in the same strand as the primary donor or in the complementary strand. Rate constants for hole transport are much slower than those for the superexchange process used to inject the hole on the primary donor. This difference is attributed to the larger solvent reorganization energy for charge transport versus charge separation. The hole transport rate constants obtained in these experiments are consistent with experimental data for single-step hole transport from other transient absorption studies. Their relevance to long-distance hole migration over tens of base pairs remains to be determined. The forward and return hole transport rate constants provide equilibrium constants and free energies for hole transport equilibria. Secondary GG and GGG donors are found to form very shallow hole traps, whereas the nucleobase deazaguanine forms a relatively deep hole trap. This conclusion is in accord with selected strand cleavage data and thus appears to be representative of the behavior of holes in duplex DNA. Our results are discussed in the context of current theoretical models of hole transport in DNA.     

Frenkel exciton model of ultrafast excited state dynamics in AT DNA double helices

Recent ultrafast experiments have implicated intrachain base-stacking rather than base-pairing as the crucial factor in determining the fate and transport of photoexcited species in DNA chains. An important issue that has emerged concerns whether or not a Frenkel excitons is sufficient one needs charge-transfer states to fully account for the dynamics. Here we present an SU(2)  SU(2) lattice model which incorporates both intrachain and interchain electronic interactions to study the quantum mechanical evolution of an initial excitonic state placed on either the adenosine or thymidine side of a model B DNA poly(dA).poly(dT) duplex. Our calculations indicate that over several hundred femtoseconds, the adenosine exciton remains a cohesive excitonic wave packet on the adenosine side of the chain where as the thymidine exciton rapidly decomposes into mobile electron/hole pairs along the thymidine side of the chain. In both cases, the very little transfer to the other chain is seen over the time-scale of our calculations. We attribute the difference in these dynamics to the roughly 4:1 ratio of hole versus electron mobility along the thymidine chain. We also show that this difference is robust even when structural fluctuations are introduced in the form of static off-diagonal disorder.     

Investigation of excess-electron transfer in DNA double-duplex systems allows estimation of absolute excess-electron transfer and CPD cleavage rates

To investigate the parameters and rates that determine excess-electron transfer processes in DNA duplexes, we developed a DNA double-duplex system containing a reduced and deprotonated flavin donor at the junction of two duplexes with either the same or different electron acceptors in the individual duplex substructures. This model system allows us to bring the two electron acceptors in the duplex substructures into direct competition for injected electrons and this enables us to decipher how the kind of acceptor influences the transfer data. Measurements with the electron acceptors 8-bromo-dA (BrdA), 8-bromo-dG (BrdG), 5-bromo-dU (BrdU), and a cyclobutane pyrimidine dimer, which is a UV-induced DNA lesion, allowed us to obtain directly the maximum overall reaction rates of these acceptors and especially of the T=T dimer with the injected electrons in the duplex. In line with previous observations, we detected that the overall dimer cleavage rate is about one order of magnitude slower than the debromination of BrdU. Furthermore, we present a more detailed explanation of why sequence dependence cannot be observed when a T=T dimer is used as the acceptor and we estimate the absolute excess-electron hopping rates.     

Base sequence effects on transport in DNA

Given the success of the polaron model based on solvation in accounting for the width of a hole polaron on an all-adenine (A) sequence on DNA, we extend the calculations to other sequences. We find excellent agreement with the free energy differences measured by Lewis et al. (J. Am. Chem. Soc. 2000, 122, 12037-12038) between a guanine (G) cation and a pair of bases, GG, or a triple of bases, GGG, in all cases surrounded by As, by treating AGGA and AGGGA as solvated polarons. There is additional support for hole polaron formation in DNA from experiments in which oxidative damage due to injected holes is investigated in sequences involving Gs and As. Theory and comparison with transport measurements on repeated sequences involving multiple thymines (Ts) or combinations such as ATs or GCs, where C is cytosine, led to the suggestion that the basic sequences in these cases must be polarons whose wave functions have substantial amplitudes on both chains in a duplex. The size of an electron polaron in DNA is predicted to be similar to that of a hole polaron, approximately 4 or 5 bases. Although experiments have shown that polaron hopping is the dominant mode of charge transport in DNA with repeated sequences such as AGGA, further investigations, particularly of temperature dependence of site energies and transfer integrals, are needed to determine to what extent hole transport takes place by polaron hopping for arbitrary DNA sequences.     

Charge-transfer excitons in DNA

There have been a number of theoretical treatments of excitons in DNA, most neglecting both the intrachain and interchain wavefunction overlaps of the electron and hole, treating them as Frenkel excitons. Recently, the importance of the intrachain and interchain coupling has been highlighted. Experiments have shown that in (dA)n oligomers and in duplex (dA)n.(dT)n, to be abbreviated (A/T), where A is adenine and T is thymine, the exciton wavefunction is delocalized over several bases. In duplexes it is possible to have charge-transfer (CT) excitons. Theoretical calculations have suggested that CT excitons in DNA may have lower energy than single chain excitons. In all the calculations of excitons in DNA, the polarization of the surrounding water has been neglected. Calculations have shown, however, that polarization of the water by an excess electron or a hole in DNA lowers its energy by approximately 1/2 eV, causing it to become a polaron. It is therefore to be expected that polarization charge induced in the surrounding water has a significant effect on the properties of the exciton. In what follows, we present calculations of some properties CT excitons would have in an A/T duplex taking into account the wavefunction overlaps, the effect of the surrounding water, which results in the electron and hole becoming polarons, and the ions in the water. As expected, the CT exciton has lowest energy when the electron and hole polarons are directly opposite each other. By appropriate choice of the dielectric constant, we can obtain a CT exciton delocalized over the number of sites found in photoinduced absorption experiments. The absorption threshold that we then calculate for CT exciton creation in A/T is in reasonable agreement with the lowest singlet absorption deduced from available data.     

Direct chemical evidence for charge transfer between photoexcited 2-aminopurine and guanine in duplex DNA

Photoexcited 2-aminopurine (Ap*) is extensively exploited as a fluorescent base analogue in the study of DNA structure and dynamics. Quenching of Ap* in DNA is often attributed to stacking interactions between Ap* and DNA bases, despite compelling evidence indicating that charge transfer (CT) between Ap* and DNA bases contributes to quenching. Here we present direct chemical evidence that Ap* undergoes CT with guanine residues in duplex DNA, generating oxidative damage at a distance. Irradiation of Ap in DNA containing the modified guanine, cyclopropylguanosine (CPG), initiates hole transfer from Ap* followed by rapid ring opening of the CPG radical cation. Ring opening accelerates hole trapping to a much shorter time regime than for guanine radicals in DNA; consequently, trapping effectively competes with back electron transfer (BET) leading to permanent CT chemistry. Significantly, BET remains competitive, even with this much faster trapping reaction, consistent with measured kinetics of DNA-mediated CT. The distance dependence of BET is sharper than that of forward CT, leading to an inverted dependence of product yield on distance; at short distances product yield is inhibited by BET, while at longer distances trapping dominates, leading to permanent products. The distance dependence of product yield is distinct from forward CT, or charge injection. As with photoinduced charge transfer in other chemical and biological systems, rapid kinetics for charge injection into DNA need not be associated with a high yield of DNA damage products.     

Femtosecond electron-transfer reactions in mono- and polynucleotides and in DNA

Quenching of redox active, intercalating dyes by guanine bases in DNA can occur on a femtosecond time scale both in DNA and in nucleotide complexes. Notwithstanding the ultrafast rate coefficients, we find that a classical, nonadiabatic Marcus model for electron transfer explains the experimental observations, which allows us to estimate the electronic coupling (330 cm(-1)) and reorganization (8070 cm(-1)) energies involved for thionine-[poly(dG-dC)](2) complexes. Making the simplifying assumption that other charged, pi-stacked DNA intercalators also have approximately these same values, the electron-transfer rate coefficients as a function of the driving force, DeltaG, are derived for similar molecules. The rate of electron transfer is found to be independent of the speed of molecular reorientation. Electron transfer to the thionine singlet excited state from DNA obtained from calf thymus, salmon testes, and the bacterium, micrococcus luteus (lysodeikticus) containing different fractions of G-C pairs, has also been studied. Using a Monte Carlo model for electron transfer in DNA and allowing for reaction of the dye with the nearest 10 bases in the chain, the distance dependence scaling parameter, beta, is found to be 0.8 +/- 0.1 A(-1). The model also predicts the redox potential for guanine dimers, and we find this to be close to the value for isolated guanine bases. Additionally, we find that the pyrimidine bases are barriers to efficient electron transfer within the superexchange limit, and we also infer from this model that the electrons do not cross between strands on the picosecond time scale; that is, the electronic coupling occurs predominantly through the pi-stack and is not increased substantially by the presence of hydrogen bonding within the duplex. We conclude that long-range electron transfer in DNA is not exceptionally fast as would be expected if DNA behaved as a "molecular wire" but nor is it as slow as is seen in proteins, which do not benefit from pi-stacking.     

Strong localization of positive charge in DNA induced by its interaction with environment

We investigate a quantum state of positive charge in DNA. A quantum state of electron hole is determined by the competition of the pi-stacking interaction b sharing a charge between different base pairs and the interaction lambda with the local environment which attempts to trap charge. To determine which interaction dominates, we investigate charge quantum states in various (GC)(n) sequences choosing DNA parameters that satisfy experimental data for the balance of charge transfer rates G(+) <--> G(n)(+), n = 2, 3. We show that experimental data can be consistent with theory only assuming b G(n)(+), n > or = 4 and comparing the experimental results with our predictions.     

Fluorescence measurements of duplex DNA oligomers under conditions conducive for forming M-DNA (a metal-DNA complex)

M-DNA (a metal complex of DNA with millimolar concentrations of Zn2+, Co2+, or Ni2+ and basic pH) has been proposed to undergo electron transfer over long distances along the helix and has generated interest as a potential building block for nanoelectronics. We show that DNA aggregates form under solvent conditions favorable for M-DNA (millimolar zinc and pH = 8.6) by fluorescence correlation spectroscopy. We have performed steady-state F?rster resonance energy transfer (FRET) experiments with DNA oligomers conjugated with 6-carboxyfluorescein and tetramethylrhodamine to the opposite ends of double-stranded DNA (dsDNA) molecules. Enhanced acceptor emission is observed for distances larger than expected for identical DNA molecules with no zinc. To avoid intermolecular FRET, the fluorescently labeled dsDNA is diluted with a 100-fold excess of unlabeled dsDNA. The intramolecular FRET efficiency increases 25-fold for a 30-mer doubly labeled duplex DNA molecule upon addition of millimolar concentrations of zinc ions. Without zinc, this oligomer has less than 1% FRET efficiency. This dramatic increase in the FRET efficiency points to either significant changes in the F?rster radius or fraying of the ends of the DNA helices. The latter hypothesis is supported by our experiments with a 9-mer that show dissociation of the duplex by zinc ions.     

Electron transfer processes in DNA: mechanisms, biological relevance and applications in DNA analytics

In principle, DNA-mediated charge transfer processes can be categorized as oxidative hole transfer and reductive electron transfer. With respect to the routes of DNA damage most of the past research has been focused on the investigation of oxidative hole transfer or transport. On the other hand, the transport or transfer of excess electrons has a large potential for biomedical applications, mainly for DNA chip technology.     

Crossover from superexchange to hopping as the mechanism for photoinduced charge transfer in DNA hairpin conjugates

The mechanism and dynamics of photoinduced charge separation and charge recombination have been investigated in synthetic DNA hairpins possessing donor and acceptor stilbenes separated by one to seven A:T base pairs. The application of femtosecond broadband pump-probe spectroscopy, nanosecond transient absorption spectroscopy, and picosecond fluorescence decay measurements permits detailed analysis of the formation and decay of the stilbene acceptor singlet state and of the charge-separated intermediates. When the donor and acceptor are separated by a single A:T base pair, charge separation occurs via a single-step superexchange mechanism. However, when the donor and acceptor are separated by two or more A:T base pairs, charge separation occurs via a multistep process consisting of hole injection, hole transport, and hole trapping. In such cases, hole arrival at the electron donor is slower than hole injection into the bridging A-tract. Rate constants for charge separation (hole arrival) and charge recombination are dependent upon the donor-acceptor distance; however, the rate constant for hole injection is independent of the donor-acceptor distance. The observation of crossover from a superexchange to a hopping mechanism provides a "missing link" in the analysis of DNA electron transfer and requires reevaluation of the existing literature for photoinduced electron transfer in DNA.     

Electron Tunneling in DNA

The reorganization energy that accompanies interfacial or through-strand electron tunneling in DNA is remarkably similar to that of a protein, and the attenuation factor for electron transfer between intercalated reagents also remains protein-like. These factors ensure rapid, but short-range electron tunneling through the duplex (shown schematically).     

Selective one-electron oxidation of duplex DNA oligomers: reaction at thymines

The one-electron oxidation of duplex DNA generates a nucleobase radical cation (electron "hole") that migrates long distances by a hopping mechanism. The radical cation reacts irreversibly with H2O or O2 to form oxidation products (damaged bases). In normal DNA (containing the four common DNA bases), reaction occurs most frequently at guanine. However, in DNA duplexes that do not contain guanine (i.e., those comprised exclusively of A/T base pairs), we discovered that reaction occurs primarily at thymine and gives products resulting from oxidation of the T-C5 methyl group and from addition to its C5-C6 double bond. This surprising result shows that it is the relative reactivity, not the stability, of a nucleobase radical cation that determines the nature of the products formed from oxidation of DNA. A mechanism for reaction is proposed whereby a thymine radical cation may either lose a proton from its methyl group or H2O/O2 may add across its double bond. In the latter case, addition may initiate a tandem reaction that converts both thymines of a TT step to oxidation products.     

Highly selective DNA biosensor based on the long-range electron transfer of indigo carmine through DNA duplex

We have developed a highly selective DNA biosensor. It was based on the long-range electron transfer (LRET) from the electroactive dye indigo carmine (IC) through the DNA duplex on a glassy carbon electrode. Voltammetric experiments showed that IC interacts with dsDNA through a typical intercalative mode with a relatively strong affinity of 2.3(±0.6)?×?10⁶?M^-1. If incubated with DNA in IC solution, no response was observed with the ssDNA-modified probe electrode. However, a pair of well-defined redox peak was observed with a DNA-hybridized electrode, proving the presence of LRET on the biosensor. The biosensor also can differentiate complementary sequences, non-complementary sequences, and even the mutated sequences with single-base mismatches at different sites.

Charge separation in a ruthenium-quencher conjugate bound to DNA

A novel tris heteroleptic dipyridophenazine complex of ruthenium(II), [{Ru(phen)(dppz)(bpy'-his)}{Ru(NH3)5}]5+, containing a covalently tethered ruthenium pentammine quencher coordinated through a bridging histidine has been synthesized and characterized spectroscopically and biochemically in a DNA environment and in organic solvent. Steady-state and time-resolved luminescence measurements indicate that the tethered Ru complex is quenched relative to the parent complexes [Ru(phen)(dppz)(bpy')]2+ and [Ru(phen)(dppz)(bpy'-his)]2+ in DNA and acetonitrile, consistent with intramolecular photoinduced electron transfer. Intercalated into guanine-containing DNA, [{Ru(phen)(dppz)(bpy'-his)}{Ru(NH3)5}]5+, upon excitation and intramolecular quenching, is capable of injecting charge into the duplex based upon the EPR detection of guanine radicals. DNA-mediated charge transport is also indicated using a kinetically fast cyclopropylamine-substituted base as an electron hole trap. Guanine damage is not observed, however, in measurements using the guanine radical as the kinetically slower hole trap, indicating that back electron-transfer reactions are competitive with guanine oxidation. Moreover, transient absorption measurements reveal a novel photophysical reaction pathway for [{Ru(phen)(dppz)(bpy'-his)}{Ru(NH3)5}]5+ in the presence of DNA that is competitive with the intramolecular flash-quench process. These results illustrate the remarkably rich redox chemistry that can occur within a bimolecular ruthenium complex intercalated in duplex DNA.