DNA mediated charge transport: characterization of a DNA radical localized at an artificial nucleic acid base

DNA assemblies containing 4-methylindole incorporated as an artificial base provide a chemically well-defined system in which to explore the oxidative charge transport process in DNA. Using this artificial base, we have combined transient absorption and EPR spectroscopies as well as biochemical methods to test experimentally current mechanisms for DNA charge transport. The 4-methylindole radical cation intermediate has been identified using both EPR and transient absorption spectroscopies in oxidative flash-quench studies using a dipyridophenazine complex of ruthenium as the intercalating oxidant. The 4-methylindole radical cation intermediate is particularly amenable to study given its strong absorptivity at 600 nm and EPR signal measured at 77 K with g = 2.0065. Both transient absorption and EPR spectroscopies show that the 4-methylindole is well incorporated in the duplex; the data also indicate no evidence of guanine radicals, given the low oxidation potential of 4-methylindole relative to the nucleic acid bases. Biochemical studies further support the irreversible oxidation of the indole moiety and allow the determination of yields of irreversible product formation. The construction of these assemblies containing 4-methylindole as an artificial base is also applied in examining long-range charge transport mediated by the DNA base pair stack as a function of intervening distance and sequence. The rate of formation of the indole radical cation is >/=10(7) s(-)(1) for different assemblies with the ruthenium positioned 17-37 A away from the methylindole and with intervening A-T base pairs primarily composing the bridge. In these assemblies, methylindole radical formation at a distance is essentially coincident with quenching of the ruthenium excited state to form the Ru(III) oxidant; charge transport is not rate limiting over this distance regime. The measurements here of rates of radical cation formation establish that a model of G-hopping and AT-tunneling is not sufficient to account for DNA charge transport. Instead, these data are viewed mechanistically as charge transport through the DNA duplex primarily through hopping among well stacked domains of the helix defined by DNA sequence and dynamics.     

Influence of intervening mismatches on long-range guanine oxidation in DNA duplexes

A systematic investigation of the efficiency of oxidative damage at guanine residues through long-range charge transport was carried out as a function of intervening base mismatches. A series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC approximately GG approximately GT approximately GA > AA > CC approximately TT approximately CA approximately CT. For all assemblies, damage found with the Delta-Ru diastereomer was found to be greater than with the Lambda-diastereomer. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from 1H NMR measurements of the imino proton exchange rates. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, damage ratios correlate most closely with base-pair lifetimes. Competitive hole trapping at the mismatch site does not appear to be a key factor governing the efficiency of transport through the mismatch. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.     

Long-range oxidation of guanine by Ru(III) in duplex DNA

Background: Theoretical and experimental studies have demonstrated that 5′-GG-3′ sequences in DNA are ‘hot spots’ for oxidative damage, but few studies have definitively addressed whether oxidative damage to DNA may arise from a distance via long-range charge migration. Towards this end, we have prepared tethered ruthenium (Ru)-oligonucleotide duplexes and used a flash—quench strategy to demonstrate long-range charge transport through the DNA double helix.Results: DNA assemblies containing a tethered Ru(II) intercalator have been synthesized. Ru(III), generated in situ in the presence of externally bound electron-transfer quenchers, promotes base damage selectively at the 5′-G of a 5′-GG-3′ doublet located ∼ 37 Å from the binding site of the oxidant. In the absence of a guanine doublet, oxidative damage occurs equally at all guanine bases in the strand. Oxidative damage is also observed at long range for guanine in a G·A mismatch but not in a G·T mismatch.Conclusions: The present study expands the scope of long-range electron-transfer chemistry in terms of experiments, applications, and possible reactions within the cell. Here we demonstrate oxidative damage to DNA occurring with a high quantum yield over a distance of ∼37 Å using a ground-state oxidant. These results point to the equilibration of the radical across the DNA duplex to the sites of lowest energy. In addition, this charge migration is sensitive to the intervening π-stack formed by DNA base pairs and hence may be useful for the detection of mismatches.     

Direct observation of radical intermediates in protein-dependent DNA charge transport.

Charge migration through the DNA base stack has been probed both spectroscopically, to observe the formation of radical intermediates, and biochemically, to assess irreversible oxidative DNA damage. Charge transport and radical trapping were examined in DNA assemblies in the presence of a site-specifically bound methyltransferase HhaI mutant and an intercalating ruthenium photooxidant using the flash-quench technique. The methyltransferase mutant, which can flip out a base and insert a tryptophan side chain within the DNA cavity, is found to activate long-range hole transfer through the base pair stack. Protein-dependent DNA charge transport is observed over 50 A with guanine radicals formed >10(6) s(-1); hole transport through DNA over this distance is not rate-limiting. Given the time scale and distance regime, such protein-dependent DNA charge transport chemistry requires consideration physiologically.     

The effect of varied ion distributions on long-range DNA charge transport

Long-range oxidative damage to DNA was utilized as a probe to delineate the effects of different ion distributions on DNA charge transport. DNA assemblies were constructed, containing a tethered rhodium intercalating photooxidant, spatially separated from two 5'-GG-3' sites of oxidative damage, with either an A6-tract or a mixed DNA sequence intervening between the guanine doublets; the extent of charge transport was assessed through measurements of the ratio of yields of damage at the guanine doublet distal versus that proximal to the metal binding site. The distal/proximal damage ratios were compared after photooxidation of otherwise identical Rh-tethered assemblies, except for 32P-labeling either at the 5'- or 3'-end; this labeling difference corresponds, in the absence of charge neutralization by condensed counterions, to a shift in negative charge from one end of the duplex to the other. Both with assemblies containing the mixed sequence and the A6-tract, we observed that moving the negative charges to the proximal end of the duplex significantly decreased hole transport to the distal end. We propose that these results reflect variations in the thermodynamic potential at the proximal and distal guanine sites because of the change in charges at the termini of the oligomer. High values for the internal dielectric constant of the stacked base pairs are suggested by these data. Hence, the longitudinal polarizability of DNA may be important to consider in mechanisms for long-range DNA charge transport.     

Sequence dependence of charge transport through DNA domains

Here we examine the photooxidation of two kinetically fast electron hole traps, N4-cyclopropylcytosine (CPC) and N2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy')]3+ (dppz = dipyridophenazine) and [Rh(phi)2(bpy)]3+ (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for CPC versus CPG in DNA, CPC decomposes with photooxidation by [Rh(phi)2(bpy)]3+, while CPG undergoes ring-opening both with photoexcited [Rh(phi)2(bpy)]3+ and with [Ru(phen)(dppz)(bpy')]3+. Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both CPC and CPG, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.     

Long-range oxidative damage to DNA: effects of distance and sequence

INTRODUCTION: Oxidative damage to DNA in vivo can lead to mutations and cancer. DNA damage and repair studies have not yet revealed whether permanent oxidative lesions are generated by charges migrating over long distances. Both photoexcited *Rh(III) and ground-state Ru(III) intercalators were previously shown to oxidize guanine bases from a remote site in oligonucleotide duplexes by DNA-mediated electron transfer. Here we examine much longer charge-transport distances and explore the sensitivity of the reaction to intervening sequences. RESULTS: Oxidative damage was examined in a series of DNA duplexes containing a pendant intercalating photooxidant. These studies revealed a shallow dependence on distance and no dependence on the phasing orientation of the oxidant relative to the site of damage, 5'-GG-3'. The intervening DNA sequence has a significant effect on the yield of guanine oxidation, however. Oxidation through multiple 5'-TA-3' steps is substantially diminished compared to through other base steps. We observed intraduplex guanine oxidation by tethered *Rh(III) and Ru(III) over a distance of 200 A. The distribution of oxidized guanine varied as a function of temperature between 5 and 35 degrees C, with an increase in the proportion of long-range damage (> 100 A) occurring at higher temperatures. CONCLUSIONS: Guanines are oxidized as a result of DNA-mediated charge transport over significant distances (e.g. 200 A). Although long-range charge transfer is dependent on distance, it appears to be modulated by intervening sequence and sequence-dependent dynamics. These discoveries hold important implications with respect to DNA damage in vivo.     

DNA charge transport leading to disulfide bond formation

Here, we show that DNA-mediated charge transport (CT) can lead to the oxidation of thiols to form disulfide bonds in DNA. DNA assemblies were prepared possessing anthraquinone (AQ) as a photooxidant spatially separated on the duplex from two SH groups incorporated into the DNA backbone. Upon AQ irradiation, HPLC analysis reveals DNA ligated through a disulfide. The reaction efficiency is seen to vary in assemblies containing intervening DNA mismatches, confirming that the reaction is DNA-mediated. Interestingly, one intervening mismatch near the thiols promotes an increase in efficiency, which we attribute to increased base dynamics. Hence, here, where the reaction is on the backbone rather than within the base stack, stacking perturbations do not necessarily lead to an inhibitory effect on DNA CT.     

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.     

Single-step charge transport through DNA over long distances

Quantum yields for charge transport across adenine tracts of increasing length have been measured by monitoring hole transport in synthetic oligonucleotides between photoexcited 2-aminopurine, a fluorescent analogue of adenine, and N(2)-cyclopropyl guanine. Using fluorescence quenching, a measure of hole injection, and hole trapping by the cyclopropyl guanine derivative, we separate the individual contributions of single- and multistep channels to DNA charge transport and find that with 7 or 8 intervening adenines the charge transport is a coherent, single-step process. Moreover, a transition occurs from multistep to single-step charge transport with increasing donor/acceptor separation, opposite to that generally observed in molecular wires. These results establish that coherent transport through DNA occurs preferentially across 10 base pairs, favored by delocalization over a full turn of the helix.     

Charge separation in acridine- and phenothiazine-modified DNA

The formation of the long-lived, charge-separated state in DNA upon visible light irradiation is of particular interest in molecular-scale optoelectronics, sensor design, and other areas of nanotechnology. However, the efficient generation of the charge-separated state is hampered by fast charge recombination within a contact ion pair, which limits the application of DNA for photoelectrochemical sensors and devices. In this study, a series of protonated 9-alkylamino-6-chloro-2-methoxyacridine (Acr+)- and phenothiazine (Ptz)-modified DNAs were synthesized for the further understanding of the mechanism of charge separation in DNA to generate a long-lived, charge-separated state with a high quantum yield (Phi). The Acr+ serves as a photosensitizer to produce a hole on guanine (G), and the G-C base pairs were used as a hole-transporting pathway to separate a hole from Acr* (the one-electron-reduced form of Acr+) to be trapped at Ptz. Since Acr+ oxides only G upon photoexcitation, the A-T base pair can be used as a spacer between Acr+ and the G-C base pair to avoid the formation of a contact ion pair. The charge injection dynamics was investigated by steady-state fluorescence spectra and fluorescence lifetime measurements, and the Phi and the lifetime of the charge-separated state produced upon photoirradiation were assessed by nanosecond laser flash photolysis of the Acr+- and Ptz-modified DNA. A long-lived, charge-separated state was successfully formed upon visible-light irradiation, and the Phi was the highest for the DNA having a single intervening A-T base pair between Acr+ and the G-C base pair. These results clearly demonstrated that the charge separation process in DNA can be refined by putting a redox-inactive intervening base pair as a spacer between a photosensitizer and the nucleobase to be oxidized to slow down the charge recombination rate.     

Robust charge transport in DNA double crossover assemblies

BACKGROUND: Multiple-stranded DNA assemblies, encoded by sequence, have been constructed in an effort to self-assemble nanodevices of defined molecular architecture. Double-helical DNA has been probed also as a molecular medium for charge transport. Conductivity studies suggest that DNA displays semiconductor properties, whereas biochemical studies have shown that oxidative damage to B-DNA at the 5'-G of a 5'-GG-3' doublet can occur by charge transport through DNA up to 20 nm from a photo-excited metallointercalator. The possible application of DNA assemblies, in particular double crossover (DX) molecules, in electrical nanodevices prompted the design of a DNA DX assembly with oxidatively sensitive guanine moieties and a tethered rhodium photo-oxidant strategically placed to probe charge transport. RESULTS: DX assemblies support long-range charge transport selectively down the base stack bearing the intercalated photo-oxidant. Despite tight packing, no electron transfer (ET) crossover to the adjacent base stack is observed. Moreover, the base stack of a DX assembly is well-coupled and less susceptible than duplex DNA to stacking perturbations. Introducing a double mismatch along the path for charge transport entirely disrupts long-range ET in duplex DNA, but only marginally decreases it in the analogous stack within DX molecules. CONCLUSIONS: The path for charge transport in a DX DNA assembly is determined directly by base stacking. As a result, the two closely packed stacks within this assembly are electronically insulated from one another. Therefore, DX DNA assemblies may serve as robust, insulated conduits for charge transport in nanoscale devices.     

Suppression of DNA-mediated charge transport by BamHI binding

A guanine radical cation produced by one-electron DNA oxidation migrates over long distances through the DNA pi-stack. Fundamental questions regarding the likelihood of charge transport in genomic DNA, the effects of protein binding, and its biological consequences arise as the next issues of study. Electronic effects of protein binding on the efficiency of charge transport were investigated for the endonuclease BamHI-DNA complex. Direct contact of a positively charged guanidium group of BamHI to guanines in the recognition sequence 5'-GGATCC-3' completely suppressed one-electron oxidation of the guanine in the protein binding site and dramatically lowered the charge transport efficiency through the sequence. Electronically insulated guanines, by the hydrogen bonding contact of a guanidium group in BamHI, no longer function as a stepping stone in the charge transport through the DNA pi-stack.     

Charge-transfer and spin dynamics in DNA hairpin conjugates with perylenediimide as a base-pair surrogate

A perylenediimide chromophore (P) was incorporated into DNA hairpins as a base-pair surrogate to prevent the self-aggregation of P that is typical when it is used as the hairpin linker. The photoinduced charge-transfer and spin dynamics of these hairpins were studied using femtosecond transient absorption spectroscopy and time-resolved EPR spectroscopy (TREPR). P is a photooxidant that is sufficiently powerful to quantitatively inject holes into adjacent adenine (A) and guanine (G) nucleobases. The charge-transfer dynamics observed following hole injection from P into the A-tract of the DNA hairpins is consistent with formation of a polaron involving an estimated 3-4 A bases. Trapping of the (A 3-4) (+*) polaron by a G base at the opposite end of the A-tract from P is competitive with charge recombination of the polaron and P (-*) only at short P-G distances. In a hairpin having 3 A-T base pairs between P and G ( 4G), the radical ion pair that results from trapping of the hole by G is spin-correlated and displays TREPR spectra at 295 and 85 K that are consistent with its formation from (1*)P by the radical-pair intersystem crossing mechanism. Charge recombination is spin-selective and produces (3*)P, which at 85 K exhibits a spin-polarized TREPR spectrum that is diagnostic of its origin from the spin-correlated radical ion pair. Interestingly, in a hairpin having no G bases ( 0G), TREPR spectra at 85 K revealed a spin-correlated radical pair with a dipolar interaction identical to that of 4G, implying that the A-base in the fourth A-T base pair away from the P chromophore serves as a hole trap. Our data suggest that hole injection and transport in these hairpins is completely dominated by polaron generation and movement to a trap site rather than by superexchange. On the other hand, the barrier for charge injection from G (+*) back onto the A-T base pairs is strongly activated, so charge recombination from G (or even A trap sites at 85 K) most likely proceeds by a superexchange mechanism.     

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.     

A mechano-electronic DNA switch

We report a new kind of DNA nanomachine that, fueled by Hg(2+) binding and sequestration, couples mechanical motion to the multiply reversible switching of through-DNA charge transport. This mechano-electronic DNA switch consists of a three-way helical junction, one arm of which is a T-T mismatch containing Hg(2+)-binding domain. We demonstrate, using chemical footprinting and by monitoring charge-flow-dependent guanine oxidation, that the formation of T-Hg(2+)-T base pairs in the Hg(2+)-binding domain sharply increases electron-hole transport between the other two Watson-Crick-paired stems, across the three-way junction. FRET measurements are then used to demonstrate that Hg(2+) binding/dissociation, and the concomitant increase/decrease of hole transport efficiency, are strongly linked to specific mechanical movements of the two conductive helical stems. The increase in hole transport efficiency upon Hg(2+) binding is tightly coupled to the movement of the conductive stems from a bent arrangement toward a more linear one, in which coaxial stacking is facilitated. This switch offers a paradigm wherein the performance of purely mechanical work by a nanodevice can be conveniently monitored by electronic measurement.     

Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is >10(7) s(-1).     

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.     

193 NM LIGHT INDUCES SINGLE STRAND BREAKAGE OF DNA PREDOMINANTLY AT GUANINE

Irradiation of DNA with 193 nm light results in monophotonic photoionization, with the formation of a base radical cation and a hydrated electron (φ_P1 = 0.048–0.065). Although >50% of the photoionization events initially occur at guanine in DNA, migration of the “hole” from the other bases to guanine occurs to yield predominantly its radical cation or its deprotonated form. From sequence analysis, the data reveal that 193 nm light induces single strand breaks (ssb) in double-stranded DNA preferential 3’ to a guanine residue. However, it has previously been reported that 193 nm light yields very low yields of ssb (<2% of the yield of eaq). The distribution of these ssb at guanine is nonrandom, showing a dependence on the neighboring base moiety. The efficiency of ssb formation at nonguanine sites is estimated to be at least one order of magnitude lower. The preferred cleavage at guanine is consistent with migration and localization of the electron loss center at guanine. It is argued that singlet oxygen and the photoionized phosphate group of the sugar moiety are not major precursors to ssb. At present, the mechanisms of strand breakage are not known although a guanine radical or one of its products remain potential precursors.