TCA IAAAAAAGMGTCTA -AIT CTTTATTCCCAGAT
5.1 INTRODUCTION
Numerous spectroscopic and biochemical experiments have shown that the base stack of DNA can mediate charge transport (CT) reactions (1-4). Chemically well- defined assemblies, consisting of DNA duplexes with covalently bound oxidants, have been particularly useful in exploring the effects of base stacking perturbations (5-8), intervening DNA sequence (9, 10), and donor-acceptor distance (11-13) on CT. Long- range oxidative DNA damage has been demonstrated over a distance of 200 Å (14, 15).
Indeed DNA either packaged in nucleosome core particles (16) or inside the cell nucleus (17) has been found to be susceptible to long-range oxidative damage.
DNA-mediated CT from a distance to generate oxidative damage was first demonstrated in an assembly containing a tethered, intercalating phenanthrenequinone diimine (phi) complex of Rh(III) (18). In this assembly, photoinduced oxidative damage of the 5’-G of 5’-GG-3’ sites was observed; this damage pattern is considered the
hallmark of CT and long-range oxidative damage has now been confirmed using a variety of pendant oxidants (19-23). Hence, the focus of research has shifted from whether or not DNA CT occurs at all to mechanistically how does charge migrate through DNA.
Utilizing yield measurements of oxidative DNA damage as a function of intervening sequence, Giese, Jortner, and coworkers offered experimental support for a model involving a mixture of hopping and tunneling (24, 25). Also based on oxidative yield determinations, Schuster and coworkers have proposed phonon-assisted polaron hopping between guanine bases (26). In this model the formation of polarons, localized structural distortions of DNA that stabilize the cation radical, allows for charge delocalization over regions of sequence; propagation of these polarons through the helix is aided by phonons.
In addition to biochemical experiments to determine yields of oxidative damage, experiments using some oxidants permit spectroscopic analysis to determine rates of CT or to monitor CT radical intermediates. These studies can be especially useful in
addressing the mechanistic considerations of DNA CT. Using rigid stilbene-modified hairpins, Lewis and coworkers observed a steep distance dependence of CT rates by monitoring formation of the stilbene radical anion (13). Recent work by Kawai, Majima, and coworkers examined the yields of the charge-separated state in DNA hairpins
modified to contain a naphthalene diimide (NDI) acceptor and a phenothiazine donor (12). By monitoring the formation and decay of the NDI radical anion, the yield of the charge-separated state was slightly dependent on the number of intervening A-T base pairs; however, the rate of charge recombination was found to be strongly dependent upon the number of intervening A-T steps. The authors propose an adenine hopping model to account for these and other previous experimental observations. Using
photoexcited 2-aminopurine (*Ap), a fluorescent nucleobase analog, our laboratory found that rates of intrastrand base-base CT between *Ap and G were ~1010 s-1 (27). Very recent experiments, also monitoring quenching of *Ap, have shown that base dynamics play a significant role in modulating propagation of charge through DNA (28, 29). In these systems, higher yields of CT are observed with increasing temperature; these higher temperatures provide increased base fluctuations which in turn allow access to more CT- active conformations.
DNA assemblies containing dipyridophenazine (dppz) complexes of Ru(II) have been particularly useful for probing CT, because they allow both for spectroscopic studies to monitor formation of DNA radicals on a short time scale and for biochemical
analysis to determine the yield of oxidative damage occurring on a longer time scale.
With these ruthenium complexes, a flash/quench technique is typically utilized (Figure 5.1) (30, 31). The cycle is initiated by visible light, which excites the intercalated Ru(II) complex. This excited Ru(II) complex, *Ru(II), is then quenched by a nonintercalating electron acceptor, Q, such as [Ru(NH3)6]3+ or [Co(NH3)5Cl]2+, so as to form Ru(III) in situ. It is this Ru(III) species that can oxidize guanines from a distance. The oxidized guanine radical can then undergo further reaction with H2O and/or O2 to form a family of oxidative products, Gox (32). The flash/quench technique, coupled with transient
absorption spectroscopy, has been applied effectively in characterizing the resultant neutral guanine radical in duplex DNA; deprotonation of the cation radical must occur faster than the 10-7 s time scale of the experiment (31). We have also utilized
flash/quench experiments in characterizing radical products in peptide/DNA assemblies (33) and a protein/DNA complex (7).
Assemblies containing 4-methylindole (M) as the electron donor as well as a tethered Ru intercalator were constructed to explore long-range DNA CT
spectroscopically and biochemically (34). The methylindole moiety is particularly amenable as an artificial base in these studies because of its relatively low oxidation potential (1 V vs. NHE) and the strong absorptivity at 600 nm of its cation radical.
Formation of the M cation radical over 17-37 Å away from the tethered intercalating oxidant occurs with a rate ≥ 107 s-1 and is found to be coincident with quenching of the ruthenium excited state to form the Ru(III) oxidant; CT is not rate limiting. Furthermore, in these assemblies, where the intervening DNA bridge was composed solely of A-T base
pairs, efficient CT is observed over 37 Å with no intervening guanines. Thus, a model of charge hopping strictly among guanines seems unlikely.
We have found that DNA CT is exquisitely sensitive to DNA base pair stacking, both statically and dynamically. Based on spectroscopic and biochemical experiments using Ru and Rh intercalating oxidants, along with the temperature dependent base-base CT chemistry (28, 29), we have proposed a model (29) for CT involving
conformationally gated charge hopping among DNA domains. Domains over which charge may be delocalized are defined by sequence and dynamics; a domain size of ~ 4 base pairs has been characterized in assemblies containing repetitive tracts of adenines.
CT among these DNA domains is conformationally gated: increased base dynamics permits access to CT-active configurations and hence facilitates CT.
Recently, additional assemblies containing a pendant Ru oxidant and M as the charge donor were constructed to examine spectroscopically the effects of sequence at the injection site on charge propagation to yield oxidative damage (35). Sequences contained either a G or inosine (I) at the injection site, where I is ~ 200 mV harder to oxidize than G. Interestingly, despite the higher oxidation potential, a larger signal at 600 nm, indicative of a higher cation radical yield, is observed for sequences containing I at the injection site. Biochemical analyses suggest these differences depend upon the extent of radical delocalization at the injection site by allowing (with G) or inhibiting (with I) a non-productive back reaction with reduced quencher.
Recent photophysical experiments have also demonstrated that back electron transfer (BET) can play a significant role in diminishing the yield of charge propagation out to a distant site (36, 37). If BET with the reduced DNA-bound photooxidant is faster
than trapping of the guanine radical by H2O and/or O2, fast BET can prevent formation of a permanent guanine lesion. Perhaps the clearest example is with thionine, a potent photooxidant (~ 2 V vs. NHE) that produces no detectable DNA damage owing to its fast rate of BET (38). As another example, in identical DNA duplexes containing different tethered oxidants, different yields of oxidative damage at a distal 5’-GG-3’ double guanine site as compared to a proximal 5’-GG-3’ site are observed (39). These differences in relative oxidative damage are attributed to variations in BET.
The consequences of rapid BET demonstrate that in DNA reverse CT must be as carefully considered as in studies of forward CT. Given radical migration in both directions through DNA, yields of oxidative damage will necessarily reflect some extent of charge equilibration prior to radical localization and trapping. To explore this issue, we have prepared duplexes containing a tethered dppz complex of Ru(II), the nucleoside analogue methylindole, and a second radical trap, a GGG site; the two trapping sites have been varied in position with and without an intervening stacking perturbation to limit migration between these low potential sites. Using these assemblies, here we report results demonstrating charge equilibration across the DNA duplex.