Efficient UV-induced charge separation andrecombination in an 8-oxoguanine-containing dinucleotideYuyuan Zhanga, Jordan Dooda, Ashley A. Becksteada, Xi-Bo Lib, Khiem V. Nguyenb, Cynthia J. Burrowsb,1,Roberto Improtac,1, and Bern Kohlera,1

aDepartment of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717; bDepartment of Chemistry, University of Utah, Salt Lake City,UT 84112; and cConsiglio Nazionale delle Ricerche, Istituto di Biostrutture e Bioimmagini, 80136 Naples, Italy

Edited by Michael D. Fayer, Stanford University, Stanford, CA, and approved July 2, 2014 (received for review March 9, 2014)

During the early evolution of life, 8-oxo-7,8-dihydro-2′-deoxyguano-sine (O) may have functioned as a proto-flavin capable of repairingcyclobutane pyrimidine dimers in DNA or RNA by photoinducedelectron transfer using longer wavelength UVB radiation. To inves-tigate the ability of O to act as an excited-state electron donor,a dinucleotide mimic of the FADH2 cofactor containing O at the5′-end and 2′-deoxyadenosine at the 3′-end was studied by femto-second transient absorption spectroscopy in aqueous solution. Fol-lowing excitation with a UV pulse, a broadbandmid-IR pulse probedvibrational modes of ground-state and electronically excited mole-cules in the double-bond stretching region. Global analysis of time-and frequency-resolved transient absorption data coupled with abinitio quantum mechanical calculations reveal vibrational markerbands of nucleobase radical ions formed by electron transfer fromO to 2′-deoxyadenosine. The quantum yield of charge separation is0.4 at 265 nm, but decreases to 0.1 at 295 nm. Charge recombinationoccurs in 60 ps before the O radical cation can lose a deuteron towater. Kinetic and thermodynamic considerations strongly suggestthat all nucleobases can undergo ultrafast charge separation whenπ-stacked in DNA or RNA. Interbase charge transfer is proposed tobe a major decay pathway for UV excited states of nucleic acids ofgreat importance for photostability as well as photoredox activity.

DNA photophysics | time-resolved vibrational spectroscopy |DNA charge transfer states | ab initio calculations | photoreactivation

The RNA world hypothesis suggests that ancient life origi-nated from RNA-based oligomers due to their ability to both

store genetic information and catalyze reactions in a manner similarto protein-based enzymes (1). In proteins, the 20 canonical aminoacids are not versatile enough in their redox activity for manypurposes, and special redox cofactors, such as the dinucleotidesFADH2 and NAD(P)H, are often recruited to facilitate a desiredtransformation. Similarly, facile oxidation or reduction reactionsinvolving the canonical nucleobases could put the integrity of thegenome at risk. Recent work has shown that 8-oxo-7,8-dihy-droguanine (8-oxo-G), an oxidatively damaged form of guanine(G), is a redox-active base capable of photoinduced reversal ofthymine dimers in DNA oligomers (2, 3). In particular, continuousirradiation of substrates containing a thymine dimer and a nearby8-oxo-G using a UVB lamp decreases the amount of dimers overtime (2, 3), but direct evidence of photoinduced electron transfer(ET) has been lacking.The ability of 8-oxo-G to act as an excited-state electron donor

is plausible, but by no means assured in light of conflictingindications. On the one hand, 8-oxo-G is easier to oxidize in itselectronic ground state by ∼30 kJ·mol–1 compared with G (4),the most easily oxidized of the canonical bases, but this advan-tage may be lost for excited-state oxidation due to the lowerenergy of the first excited singlet state of 8-oxo-G. On the otherhand, the 2′-deoxynucleoside of 8-oxo-G (8-oxo-dGuo, or O) hasan excited-state lifetime of just 0.9 ± 0.1 ps at physiological pH(5)—a value that is similar to the subpicosecond lifetimes of the

undamaged bases (6). Rapid nonradiative decay could thusfrustrate ET despite favorable thermodynamics. Time-resolvedspectroscopy can resolve this puzzle by detecting any short-lived radicals produced by photoinduced ET. Here, we usefemtosecond time-resolved infrared (TRIR) spectroscopy todefinitively show that UV excitation of the dinucleotide d(OA)(Fig. 1) transfers an electron from O to 2′-deoxyadenosine (A)on a subpicosecond timescale to form a contact radical ion pair(exciplex) that can be unambiguously identified by comparisonwith density functional theory (DFT) calculations.The dinucleotide d(OA) was chosen as a crude mimic of

FADH2 in which 8-oxo-G replaces the dihydroflavin moiety of thecofactor that serves as the electron source for photoinitiated ETto a cyclobutane pyrimidine dimer (CPD) (7). Combining O withA offers the further advantage that the steady-state UV-visibleand IR absorption spectra have several nonoverlapping transitionsthat arise from just one of the two chromophores (Fig. 1 and Fig.S1). These spectral characteristics permit selective excitation ofO and selective detection of the localization site of an excited statevia mid-IR probing of either O or A vibrations. A vibrationalspectrum with its comparatively narrow resonances is frequentlyeasier to assign than overlapping electronic absorption spectra.Consequently, TRIR spectroscopy can often differentiate betweencharge transfer (CT) states and other excited states that may havesimilar electronic absorption spectra (8, 9).Besides its interest as a mimic of a redox cofactor, the d(OA)

dinucleotide also provides valuable insights into the role of CT

Significance

UV photons are absorbed strongly by DNA, but rarely causepermanent photodamage. Single nucleobases are protected byultrafast nonradiative decay, but excited states in single- anddouble-stranded DNA decay very differently. An intensely de-bated question is whether a UV photon can move an electronfrom one nucleobase to another along a single strand. Thisstudy demonstrates that UV absorption efficiently transfers anelectron from an oxidatively damaged guanine (8-oxo-G) toadenine in a dinucleotide mimic of the flavin cofactor FADH2,yielding radicals that decay in 60 ps. It is proposed that thephotoredox activity of 8-oxo-G, which may have repairedcyclobutane pyrimidine dimers in the RNA world, reflects theimportance of ultrafast charge separation between stackednucleobases by UV radiation.

Author contributions: C.J.B., R.I., and B.K. designed research; Y.Z., J.D., A.A.B., X.-B.L., K.V.N.,and R.I. performed research; X.-B.L. and K.V.N. contributed new reagents/analytic tools;Y.Z., X.-B.L., R.I., and B.K. analyzed data; and Y.Z., X.-B.L., C.J.B., R.I., and B.K. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: kohler@chemistry.montana.edu,burrows@chem.utah.edu, or robimp@unina.it.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1404411111/-/DCSupplemental.

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states in DNA. As shown below, the CT state of d(OA) decaysin ∼60 ps by charge recombination (CR). This decay is muchlonger than the excited-state lifetimes of either A or O asmonomers. Similarly long-lived excited states are formed in highyields whenever DNA bases are stacked with one another bothin single- and double-stranded forms (10–14). The identity ofthese long-lived states has been one of the most debated issuesin the photophysics and photochemistry of nucleic acids duringthe past decade. The results from this study suggest that a pri-mary decay channel for excited states of stacked nucleobases inDNA, whether modified or not, is ultrafast interbase ET.

ResultsExperimental Results.TRIR spectra of d(OA) following excitationat 265 nm are shown in Fig. 2A. All measurements were carriedout in D2O solution at pH 7.3 because deuteration improves theIR transparency of water in the double-bond stretching regionof interest. The difference spectra consist of negative signals dueto ground-state bleaching (GSB), and positive signals that arisefrom vibrational modes of transient species. All signals disap-pear completely by 300 ps after the pump pulse, leaving onlya featureless negative offset, which originates from alteredabsorption by D2O at the elevated temperature produced by thepump pulse (15).Immediately after 265-nm excitation, three strong bleaches are

observed at the frequencies of the strongest peaks in the steady-state FTIR absorption spectrum of d(OA). Each band is labeled inFig. 2 by its dominant vibrational character as determined bynormal mode calculations. The relative amplitudes of the negativeΔA signals are in excellent agreement with the inverted FTIRspectrum, which is shown in Fig. 2A by the dot-dashed line. Strongpositive bands in the transient absorption difference spectra areseen at 1,602 and 1,684 cm−1 (marked with red arrows). Bothpositive and negative signals decay biexponentially and can beglobally fit with the same time constants (4 ± 1 ps and 60 ± 20 ps;Table 1). TRIR spectra for an equimolar mixture of 8-oxo-dGuoand AMP excited at 265 nm (Fig. 2B) display a strikingly differentpattern of initial bleach amplitudes compared with when O andA are covalently linked in the d(OA) dinucleotide (Fig. 2A).In particular, the ratios of the bleach signal of the A band at1,623 cm−1 to the O bands at 1,562 and 1,662 cm−1 are fourtimes greater in the monomer mixture than in d(OA). Also, only

short 2- to 3-ps decays are observed for the O and A monomers(Fig. S2 A and B), indicating that the 60-ps time constant isunique to the dinucleotide.TRIR spectra recorded with excitation at 295 nm, a wavelength

that is expected to excite O and not A (Fig. 1), are shown ford(OA) and the equimolar mixture in Fig. 2 C and D, respectively.A slow time constant (60 ± 30 ps) is again observed for d(OA) thatis absent in the monomer mixture (Fig. S2 C and D), but now theprominent positive features seen at 1,602 and 1,684 cm−1 followingexcitation at 265 nm are attenuated, and the relative amplitudeof this component is four times weaker (10 ± 5% vs. 40 ± 10%)for 295- vs. 265-nm excitation. Comparison of Fig. 2 A and Cclearly shows that the pattern of bleached bands seen at the earliestdelay times differs dramatically for 265- vs. 295-nm excitation. The1,623-cm−1 ground-state mode of A, which is strongly bleachedupon excitation at 265 nm, is bleached only weakly in the TRIRspectra at 295 nm (blue arrow in Fig. 2C). There is no bleachingof this band when the equimolar mixture of O and A is excitedat 295 nm (Fig. 2D), confirming that this wavelength selectivelyexcites O.

Computational Results. Vibrational spectra for ground-state O andA were computed for the 2′-deoxynucleoside pentahydrates(8-oxo-dGuo·5D2O and dAdo·5D2O, structures shown in Fig. S1J and K) using the harmonic approximation at the polarizablecontinuum model (PCM)/PBE0/6–31+G(d,p) level of theory(Fig. 3A). Assignments for 8-oxo-dGuo vibrations are based onthe dominant character and match ones in ref. 16. Inclusionof 2′-deoxyribose and explicit D2O molecules dramaticallyimproves the agreement with experiment for the ring in-planeand C=O stretching vibrations, respectively (17). Absolute in-tegrated absorption coefficients from the calculations werescaled by a constant factor that gave the best agreement withthe experimental cross-sections for the O and A fundamentals.The experimental and theoretical frequencies are compared inTable 2.

Fig. 1. UV-visible (A) and FTIR spectra (B) for d(OA) at neutral pH. Thespectra of monomeric 8-oxo-dGuo (red dashed curves) and AMP (greendotted curves) are shown for comparison. The excitation wavelengths usedin the pump-probe experiments are indicated in A by arrows.

Fig. 2. TRIR spectra at the indicated pump-probe delay times from a 5-mMsolution of the d(OA) dinucleotide (A and C) and 5 mM 8-oxo-dGuo (O) + 5 mMAMP (A) mixture (B and D) following 265-nm (A and B) and 295-nm (C and D)excitation. Red arrows point to positive signals assigned to vibrational markerbands of 8-oxo-dGuo•+ (see text). The inverted and scaled steady-state FTIRspectrum for each sample is shown by the dot-dashed gray line. Vibrationalmode assignments are included for convenience.

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PCM/time-dependent (TD)-M052X calculations on the en-tire d(OA) dinucleotide show that a state with well-defined CTcharacter lies close in energy to the locally excited states of Oand A (Table S1). Geometry optimization of this state leads toa minimum with strong CT character and a small interbaseseparation (see computational details in SI Text). The vibrationalspectra of O•+ (8-oxo-dGuo•+·5D2O) and A•− (dAdo•−·5D2O),two possible species created by UV excitation, are shown in Fig. 3B.Vibrational modes of the radical cation O•+ are generally blue-shifted compared with its unoxidized form, whereas the singlevibrational band of A•− in our window of interest is red-shifted(Table 2). Fig. 3C displays the difference spectrum obtained bysubtracting the spectra in Fig. 3 A and B. Fig. S3 displays similardifference spectra calculated for the excited state of the entiredinucleotide.

Kinetic Modeling. An excellent global fit to the time- and fre-quency-dependent TRIR spectra for d(OA) (Fig. 2) was ob-tained using two exponential time constants and a long timeoffset to account for heating of the solvent. Target analysis (18)was used to identify a kinetic model that best accounts for theobserved signals. A model with two parallel decay channels gavethe most reasonable predictions (Figs. S4 and S5). In this model,initial excited states of d(OA) created by the femtosecond pumppulse branch faster than our experimental time resolution to twostates that decay monoexponentially with the lifetimes de-termined by global fitting.The difference spectrum of the long-lived state [i.e., its spe-

cies-associated difference spectrum (SADS)] (18) determined byglobal analysis of the TRIR spectra recorded with a pumpwavelength of 265 nm is shown by the dashed curve in Fig. 4A.The SADS derived from the d(OA) TRIR spectra excited at295 nm is identical in appearance (dotted curve in Fig. 4A).The excellent match of the two SADS indicates that the sametransient species is present at the longest delay times for 265-and 295-nm excitation. Furthermore, this common spectrum is inexcellent agreement with the calculated difference spectrum ofthe O and A radical ions shown in Fig. 3C, which has beenreproduced in Fig. 4A. The SADS of the short-lived state alongwith other details are presented in Figs. S4–S6.

DiscussionThe remarkable agreement between the experimental differ-ence spectrum of the 60-ps state and the calculated differencespectrum obtained from the redox pairs O•+/O and A/A•− (Fig.4A) indicates that UV excitation of d(OA) transfers an electronfrom O to A, mimicking the first step of photolyase-catalyzedrepair of CPDs. Ultrafast spectroscopic measurements on thephotolyase system suggest that ET from the excited state of theisoalloxazine moiety of FADH− to the 5′ thymine residue of thedimer occurs via the adenine base situated between the iso-alloxazine and the thymine (7). In the present studies, interbasecharge separation is supported by a calculation performed onthe entire dinucleotide, which predicts that the CT state is theglobal minimum among all excited states, and that O•+ and A•−

interact weakly (see computational details in SI Text). The latter

observation validates the computational approach in which theradical ion pair is treated as two separate monomers.The excellent agreement between the ground-state FTIR spec-

trum of d(OA) (Fig. 1B) and the calculated spectrum (Fig. 3A)validates the level of theory and the inclusion of explicit D2Omolecules and the ribose group. To confirm that this computa-tional approach is suitable for modeling vibrational spectra ofradical ions, the vibrational difference spectrum of O•+ wasrecorded by two-photon ionization of an aqueous solution of theO monomer (dot-dashed curve in Fig. 4C). The calculated differ-ence spectrum (solid curve in Fig. 4C) is in excellent agreementwith the experiment. The positive features in the experimentalspectrum at 1,602 and 1,684 cm−1 are assigned to νamino and νC6=Omodes of O•+, respectively. These modes have a similar spacing,but are red-shifted by ∼20 cm−1 compared with the vibrationalmarker bands of the guanine radical cation in D2O at ∼1,620and ∼1,700 cm−1 (9, 19).The kinetic steps in the parallel model used to analyze the

TRIR data are assigned in Fig. 5. According to our model, theCT state is populated faster than the experimental time resolu-tion (<0.5 ps) in competition with the appearance of vibration-ally hot nucleobases produced by ultrafast internal conversion.The latter species undergo vibrational cooling on a timescale ofseveral picoseconds. For excitation at 265 nm, the pattern ofbleached bands at early times is consistent with the creation of an

Fig. 3. Calculated vibrational spectra for monomeric species by PCM/PBE0/6–31+G(d,p) and the harmonic approximation. (A) Ground-state 8-oxo-dGuo·5D2O (O) and dAdo·5D2O (A) spectra; (B) ground-state 8-oxo-dGuo•+·5D2O(O•+) and dAdo•−·5D2O (A•−) spectra; (C) difference spectrum obtained from theabovemonomeric species. Molecular structures andmode assignments are shownfor convenience (R, 2′-deoxyribose; explicit D2O not shown). All transition fre-quencies are broadened by a Gaussian function with 20 cm−1 FWHM.

Table 1. Global fit parameters for the d(OA) GSB signals

λpump A1 τ1, ps A2 τ2, ps

265 nm* 0.6 ± 0.1 4 ± 1 0.4 ± 0.1 60 ± 20295 nm† 0.9 ± 0.4 3 ± 1 0.10 ± 0.05 60 ± 30

Normalized traces were fit to A1e−t=τ1 +A2e−t=τ2 after subtracting thelong-time signal due to solvent heating.*Fit to bleach signals for O (νC6=O) at 1,662 cm−1, O (νamino) at 1,562 cm−1,and A (νrings) at 1,623 cm−1 (Fig. S2 A and B).†Fit to bleach signals for O (νC6=O) at 1,662 cm−1 and O (νamino) at 1,562 cm−1

(Fig. S2 C and D).

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exciton that is delocalized over both O and A (see discussion ofshort-time dynamics in SI Text for more details). The rapid ap-pearance of the CT state is supported by the absence of anysignal growth at frequencies where O•+ and A•− absorb. Oncepopulated, the CT state decays monoexponentially with a life-time of ∼60 ps. Because there is no spectral signature of a furtherstate, the CT state is proposed to decay to the ground state ofd(OA) by CR.ET from O to A is considerably more efficient for 265- vs.

295-nm excitation as seen from the relative amplitudes of thelong-lived signal in the kinetic traces in Fig. S2 A and C. Thenormalized GSB signals at several different frequencies areidentical (Fig. S2 B and D), indicating that the bleach signals arefree of excited-state and/or hot ground-state absorption. In thiscase, the amplitude of the slow decay component (A2 in Table 1)is an estimate of the fraction of all excited states that decay to theCT state. Our calculations indicate that the shorter wavelengthpopulates predominantly the bright, locally excited La state ofA, which couples strongly with the CT state (see computationaldetails in SI Text), which may explain the fourfold larger CT yieldupon 265-nm excitation. Strong coupling between the La state ofA and the CT state also suggests that the CT state is populatedextremely rapidly.Nucleobase radical ions have enhanced acid/base properties

compared with the parent compounds (20), but proton transferoccurs too slowly to compete with CR in d(OA). Notably, theexperimental difference spectrum (Fig. 4A) disagrees with thespectrum calculated assuming that O is present as a neutralradical (Fig. 4B, gray curve). Reported deprotonation rates forG•+ at neutral pH range from 3.5 × 105 s−1 (21) to 1.8 × 107 s−1 (22).O•+ (pKa = 6.6) is a weaker acid than G•+ (pKa = 3.9) (23),suggesting that it would lose a proton at a still slower rate. A•− isprotonated by a water molecule at a rate of 1.4 × 108 s−1 (20).These reactions are too slow to compete with CR in 60 ps. RapidCR and the prevention of follow-on reactions that would per-manently alter O support the finding that O in an oligodeoxy-nucleotide is able to catalyze the reversal of a thymine dimer forat least five turnover cycles with no detectable degradation of the8-oxo-G base (2).

On the Generality of Photoinduced CT in Nucleobase Stacks. Themain finding of this study is that absorption of a UVB or UVCphoton by the d(OA) dinucleotide transfers an electron fromO to A, producing a radical ion pair; this supports the role ofO as a flavin mimic, but this study also adds to evidence thatphotoinduced ET occurs generally and with relatively highquantum yield between any pair of stacked nucleobases

(11, 24–26). The latter conclusion rests on strong thermodynamicand kinetic similarities between CT states in other dinucleotidesand in d(OA).Although O is easily oxidized, the Gibbs energy change for

photoinduced ET (ΔGET) depends not only on the oxidation half-reaction, but also on the reduction potential of the acceptor, theenergy of the excited state, and the electrostatic work of bringingfree ions to the short separation at which ET takes place (27). Thework term appropriate for charge separation favors forward ET.However, even if this term is neglected, as is commonly done (28),combining the large singlet-state energies of the canonical DNAbases (29) with experimental reduction potentials (30) predictsthat photoinduced ET is thermodynamically allowed for five of thesix possible heterodimers made from A, G, C, and T. In fact, theuse of the relaxed singlet excited state energy likely under-estimates the energy available for an ET reaction that proceeds onan ultrafast timescale before the thermalization of excess vibronicenergy (31). If the available excited-state energy is equal to theenergy of the 265-nm photon (4.68 eV), then photoinduced ET ispredicted to be exergonic for all dinucleotides made from A, G, C,and T, including the four homodimers, with ΔGET ranging be-tween −0.27 and −1.05 eV (Table S2). Importantly, ΔGET for ETfrom O to A induced by a 295-nm photon (−0.58 eV; Table S2)falls in the middle of this range and is less favorable than ET from

Fig. 4. Comparison of experimental and calculated vibrational differencespectra of d(OA). (A) Long-lived SADS for 265-nm (dashed) and 295-nm (dot-ted, shown after scaling) excitation obtained from global fitting comparedwith the theoretical difference spectrum from Fig. 3C (solid) calculated at thePCM/PBE0/6–31+G(d,p) level with explicit D2O molecules; (B) the differencespectra calculated with 8-oxo-dGuo(–DN7)

• (gray) at the same level of theory;(C) comparison of the experimental difference spectrum of 8-oxo-dGuo•+ (dot-dashed) obtained by two-photon ionization of 8-oxo-dGuo and the theoreticalspectrum (solid).

Table 2. Experimental and calculated [PCM/PBE0/6–31+G(d,p)]vibrational frequencies in inverse centimeters for the groundstates of 8-oxo-dGuo·5D2O (O), 8-oxo-dGuo•+·5D2O (O•+),dAdo·5D2O (A), and dAdo•−·5D2O (A•−)

Species νamino* νC4C5† νrings‡ νC6=O§ νC8=O{

O (expt.) 1,562 1,601 ― 1,662 1,704O (calc.) 1,566 1,609 ― 1,655 1,717O•+ (expt.) 1,602jj ― ― 1,684jj ―

O•+ (calc.) 1,602 1,473 1,649 1,677 1,763A (expt.) ― ― 1,623 ― ―

A (calc.) ― ― 1,625 ― ―

A•− (expt.) ― ― ― ― ―

A•− (calc.) ― ― 1,565 ― ―

calc., calculated; expt., experimental.*Vibrations localized on the C2–ND2 group.†Pyrimidine ring in-plane vibration with dominant C4 = C5 stretching.‡Ring in-plane vibration.§C6 = O stretch.{C8 = O stretch.jjBand maximum from the TRIR spectrum.

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G to A, C, or T upon 265-nm excitation. These considerationssuggest that photoinduced ET between pairs of the canonicalDNA bases is thermodynamically every bit as likely as betweenO and the others.In addition to these thermodynamic similarities, there are

strong kinetic parallels between the excited-state dynamics ofd(OA) and model compounds made of natural bases. The 60-pslifetime of the radical ion pair state of d(OA) is in the range oflifetimes previously reported for the long-lived excited statesdetected in DNA/RNA dinucleotides (11, 24–26) and oligonu-cleotides (10, 26, 32). Earlier, Takaya et al. (11) proposed thatthe long-lived excited states are exciplex states with substantialCT character that decay via CR on timescales of 10–100 ps,depending on the thermodynamic driving force. These long-livedexcited states are only observed when π−π stacking brings two ormore bases into van der Waals contact (10, 11, 32). This minimalseparation enables ET to proceed on a femtosecond timescale(33), allowing charge separation to compete with the subpico-second dynamics responsible for the ultrashort excited-statelifetimes of single bases.In further support of the similarities between d(OA) and

compounds containing natural bases, we note that the CD spec-trum of d(OA) (Fig. S1A) is nearly identical to that of d(GA) (34),indicating that both have similar electronic structure and dis-tributions of conformers. Dinucleotides are relatively flexible andadopt a range of conformations in aqueous solution, which maydiffer from the prevalent ones in double-stranded DNA. However,the dynamics of the long-lived exciplex states have been shown tobe identical for different initial stacking conformations (14). Fi-nally, although d(OA) is single-stranded, long-lived excited stateshave been detected in duplex DNAs made from all four bases(12, 35). The similar dynamics observed in double- and in single-stranded forms (10, 13) suggests that base pairing perturbs, butdoes not fundamentally alter, the charge separation that we pro-pose occurs generally between π-stacked nucleobases.The striking excitation wavelength dependence of the CT state

yield seen in our experiments suggests that the energetics andcoupling between the excitonic and CT states at the FC regionplay an important role in the excited-state dynamics. Similar tod(OA), calculations on canonical base stacks predict that CTstates lie in close proximity to the bright excitonic states in theFC region (36, 37). Furthermore, dynamical calculations predictthat even a small amount of vibronic coupling between these

states can populate the CT state, independent of their relativeenergy (38). The higher quantum yield observed at shorter ex-citation wavelength is also consistent with subpicosecond chargeseparation that occurs before vibronic relaxation.The creation of a radical ion pair by UV light in d(OA) or in a

general DNA sequence is reminiscent of the creation of a boundpolaron pair in polymer blends (39, 40), establishing an un-expected link between DNA photophysics and photoprocesses inorganic solar cells. The factors that determine whether such anion pair can escape to form free charges may also governwhether nucleobase radicals live long enough to initiate dam-aging photoreactions. The high quantum yields of charge sepa-ration noted here for d(OA), and in other systems with modifiedbases (26), greatly exceed the highest DNA photoproduct yields,suggesting that CR, which occurs in d(OA) with a time constantof 60 ps, reverses transient damage before follow-on reactionscan take place, including proton transfer with the solvent.However, reversal of a CPD following one-electron reductionoccurs in less than 100 ps (7), suggesting that photoreactivationmay be possible despite the generally short lifetimes of exciplexesor radical ion pairs in DNA.We suggest that the efficient population of the CT state and its

subsequent decay by CR result from the intimate positioning ofacceptors next to donors by π−π stacking of the bases in a DNAstrand; this may explain how a molecular architecture that pro-duces high yields of CT states can nevertheless exhibit high pho-tostability. Notably, this same paradigm raises questions for futureinvestigations about whether the facile ET from O to a neighboringbase on the same strand can repair a CPD that is located severalbases away from O or even on the opposite strand. These studieswill reveal whether the abundant radicals formed in nucleic acidsby UV radiation can contribute to photoreactivation without in-creasing the risk of photodamage to the genome.

ConclusionsUV excitation of d(OA) creates a radical ion pair with a lifetimeof 60 ps that is unambiguously assigned to interbase CT throughthe vibrational signature of this state in the double-bondstretching region. The observation of ET from O to a π-stackedneighbor, which is observed here, to our knowledge for the firsttime, supports the hypothesis that O can repair CPDs in a pho-tolyase-like manner (2, 3). Strikingly, the quantum efficiency ofreaching the CT state is four times higher for excitation withUVC than with UVB photons. This observation is important fordeveloping models to explain how CT states, which generallyhave low oscillator strengths, are reached from the initial exci-tons. These results and ones from recent studies (24–26) suggestthat interbase ET is a primary event whenever nucleic acidsabsorb UV light.

MethodsThe UV-pump/broadband-mid-IR-probe experimental setup has been de-scribed in detail elsewhere (41). Broadband mid-IR probe pulses centered at6,150 nm were generated with an optical parametric amplifier (OPA)(TOPAS-C+NDFG; Light Conversion). The probe beam was split into “signal”and “reference” portions to reduce pulse-to-pulse noise. Both probe beamspassed through the sample and were detected by a dual-row 64-elementHgCdTe array (Infrared Systems Development).

The 265- and 295-nm pump pulses were generated by a second OPA. Thepump fluence at the sample was attenuated to between 1.7 and 2.0 mJ·cm−2.The relative polarization of the pump and probe pulses was set to the magicangle (54.7°). Kinetics traces were fit to exponential functions in Igor Prosoftware, and the reported uncertainties are 2σ. Global fitting of the TRIRspectra was done using programs described in ref. 42.

A 5.0-mM d(OA) solution was prepared in 50 mM phosphate buffer and100 mM NaCl in D2O. D2O solutions of 3′-AMP (Sigma-Aldrich) and 8-oxo-dGuo (Berry and Associates, Inc.) were prepared with the same buffer andsalt concentrations. The synthesis (43) and characterization of d(OA) aredescribed in SI Text and Figs. S7–S9. The sample solution under study(total volume of 2.0 mL) was recirculated through a flow cell (Harrick Sci-entific Products, Inc.) with a 100-μm path length.

Fig. 5. Kinetic schemes for excited-state decay of d(OA) in aqueous solu-tion. The relative population of each decay channel from the fitting analysisis also indicated (Table 1). The initial steps in gray occur faster than the in-strument response time.

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Geometry optimizations and vibrational frequencies of monomeric mol-ecules and radicals were calculated using DFT with the PBE0 functional (44)and the 6–31+G(d,p) basis set. All labile hydrogen atoms were substituted bydeuterium atoms. The 2′-deoxyribose group and five explicit D2O moleculeswere also included (see Dataset S1 for the Cartesian coordinates). A polar-izable continuum model was used to model the bulk solvent effect (45). Forcalculations on the entire dinucleoside monophosphate, the ground-stategeometry and vibrational frequencies were obtained using both PBE0 andM052X (46) functionals. The excited-state geometry and vibrational fre-quencies were obtained by their time-dependent extensions (47). Due tothe cost of the computation, a smaller 6–31G(d) basis set was used, and

explicit D2O molecules were not included. All calculated vibrational fre-quencies were multiplied by 0.972.

ACKNOWLEDGMENTS. Work at Montana State University was supportedby National Science Foundation Grant CHE-1112560 and National Aero-nautics and Space Administration Grant NNX12AG77G, and at theUniversity of Utah by National Science Foundation Grant CHE-1152533.The femtosecond TRIR spectrometer was constructed with generousfunding from the M. J. Murdock Charitable Trust; R.I. was supported bythe Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIURGrants PRIN-2010ERFKXL and FIRB- RBFR08DUX6-003), and French Agencyfor Research Grant ANR-12-BS08-0001-01.

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