ANRV373-PC60-11 ARI 25 February 2009 16:34
1
2
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210; email: kohler@chemistry.ohio-state.edu
1 Current address: Department of Chemistry, University of Wisconsin-Madison, Madison,
Wisconsin 53706
2 Permanent address: Center for Chemical Dynamics, Department of Chemistry,
Case Western Reserve University, Cleveland, Ohio 44106
Annu. Rev. Phys. Chem. 2009. 60:217–39
First published online as a Review in Advance on
November 14, 2008
The Annual Review of Physical Chemistry is online at physchem.annualreviews.org
This article’s doi:
10.1146/annurev.physchem.59.032607.093719
Copyright c 2009 by Annual Reviews.
All rights reserved
0066-426X/09/0505-0217$20.00
DNA photostability, charge transfer excited states, ultrafast spectroscopy, nonradiative decay, conical intersection, thymine dimer
Ultraviolet light is strongly absorbed by DNA, producing excited electronic states that sometimes initiate damaging photochemical reactions. Fully mapping the reactive and nonreactive decay pathways available to excited electronic states in DNA is a decades-old quest. Progress toward this goal has accelerated rapidly in recent years, in large measure because of ultrafast laser experiments. Here we review recent discoveries and controversies concerning the nature and dynamics of excited states in DNA model systems in solution. Nonradiative decay by single, solvated nucleotides occurs primarily on the subpicosecond timescale. Surprisingly, excess electronic energy relaxes one or two orders of magnitude more slowly in DNA oligo- and polynucleotides. Highly efficient nonradiative decay pathways guarantee that most excited states do not lead to deleterious reactions but instead relax back to the electronic ground state. Understanding how the spatial organization of the bases controls the relaxation of excess electronic energy in the double helix and in alternative structures is currently one of the most exciting challenges in the field.
217

ANRV373-PC60-11 ARI 25 February 2009 16:34
Photolesion: a stable photoproduct formed in DNA or RNA usually by photochemical modification of one or two bases by UV light
Electronic excitation of DNA by solar ultraviolet (UV) light can produce harmful photoproducts such as the thymine dimer. Excitation is efficient because of the substantial UV absorption cross sections of the DNA nucleobases: adenine, guanine, cytosine, and thymine ( Figure 1 ). The vast majority of excitations do not initiate photoreactions as evidenced by the quantum yields of photolesion formation, which are generally much less than 1%. The altered structures and basepairing properties of photoproducts can interfere with the work of polymerases and disrupt normal cellular processing of DNA. This interference can lead to mutations, genomic instability, and carcinogenesis (1). In organisms exposed to solar UV light, DNA constantly accrues photochemical damage that must be continually repaired. Disruption of the equilibrium between damage and a
Purines
N
H
N
O
N
NH
NH
2
Guanine (G)
N
N
H
NH
2
N
N
Adenine (A)
Pyrimidines
NH
2
N
N
H
O
Cytosine (C)
O
CH
3 NH
N
H
O
Thymine (T)
15
10
5 b
0
220 240 260 280
Wavelength (nm)
300 c
Base stack
Base pair Single strand Duplex
Figure 1
( a ) Chemical structures and ( b ) UV absorption spectra of the DNA bases. ( c ) Basic assemblies of nucleobases.
Structures were drawn using the VMD software (116).
218 Middleton et al.

ANRV373-PC60-11 ARI 25 February 2009 16:34 repair can lead to skin cancer—the cancer with the highest rate of incidence in many nations (2).
The importance of this biological problem has fueled interest in excited electronic states of nucleic acids for over 50 years.
Interest in DNA photophysics has intensified in recent years as powerful spectroscopic and computational techniques have provided unprecedented new insights into nonradiative decay mechanisms. The goal of understanding how excess electronic energy evolves in DNA at the molecular level appears increasingly within reach. This energy relaxes via a multitude of pathways that include photon emission, nonradiative transitions to the ground or intermediate electronic states, and reactive decay to photoproducts. Highly efficient nonradiative decay to the electronic ground state (S
0
) significantly lowers the rate of DNA damage, thereby reducing the workload of an organism’s repair machinery (3).
This review critically analyzes experimental, solution-phase studies of DNA excited-state dynamics. The emphasis is on developments since the 2004 review article by Crespo-Hern´andez et al. (3). We aim to sketch the map, as rough as it still is in places, of the various deactivation pathways for excited electronic states in DNA. Excellent review articles have appeared recently that can augment the discussion here and provide the interested reader with an overview of closely allied work on DNA excited states by means of computational chemistry (4) and gas-phase spectroscopy (5–7). A review of time-resolved emission experiments on DNA model systems has also appeared (8).
This review begins with photophysical studies of base monomers (Section 2), before proceeding to a discussion of excited states in base multimers (Section 3). This organization reflects the reductionist approach that we and many of our physical chemistry colleagues have pursued. Although many of the model systems have limited biological realism, they are essential stepping stones along the path to a molecular-level understanding of photodynamics in these complex, multichromophoric polymers. The success of this approach is manifest: Insights obtained from studies of single bases—the building blocks of DNA—have been indispensable for interpreting excited-state dynamics in base multimers. In Section 4, progress at mapping the photochemical pathway leading to thymine dimer formation is presented.
S
0
: state electronic ground
Base monomer: a single DNA or RNA nucleobase, nucleoside, or nucleotide isolated from other bases
Base multimer: supramolecular assembly of two or more nucleobases, including single base pairs and single- and double-stranded oligoand polynucleotides
Conical intersection
(CI): a region in the molecule’s nuclear coordinate space in which two or more potential energy surfaces become energetically degenerate
1
∗
The intense UV absorption by DNA at 260 nm arises from the strongly allowed 1 ππ ∗ transitions of the nucleobases (9, 10). Single bases in aqueous solution have small fluorescence quantum yields of ∼ 10 − 4 (11, 12), indicating that the vast majority of excited states decay nonradiatively. The first accurate measurements of 1 ππ ∗ lifetimes of DNA and RNA base monomers were made in our laboratory using the femtosecond transient absorption technique (13, 14).
Figure 2 a shows the subpicosecond decay of excited-state absorption by the 1 ππ ∗ state of 9-methyladenine.
As reviewed elsewhere (3), emission from the 1 ππ ∗ states also decays on a femtosecond timescale.
Ultrafast passage between electronic states is commonplace when a wave packet moves into the vicinity of a conical intersection (CI), and it was proposed in 2000 that CIs are responsible for the subpicosecond fluorescence lifetimes of the nucleobases (14). In a pioneering computational study, Ismail et al. (15) subsequently described a nearly barrierless decay pathway from the Franck-
Condon region of cytosine to S
0 via a pair of CIs. Since then, CIs have been located for all of the natural bases and many of their derivatives at various levels of theory (4). These studies have www.annualreviews.org
• DNA Excited-State Dynamics 219

ANRV373-PC60-11 ARI 25 February 2009 16:34 a b
S n
H
2
O
D
2
O
Methanol
Acetonitrile
Visible probe
600 nm
0
2
1
*
0
UV pump
267 nm
0
1 3
UV probe
250 nm Mid-IR probe
~1600 cm –1
S
0 0 2 4
Time delay (ps)
6 8 c
1580
1590
1600
1610
1620
∆ A /10 -3
0.9
0.6
0.3
0.0
–0.3
–0.6
–0.9
–1.2
–1.5
1630
1640
0 2.5
5 7.5
Time delay (ps)
10 12.5
Figure 2
( a ) The 1 ππ ∗ state of 9-methyladenine decays within 300 fs in many solvents ( top ), but transient absorption signals at UV probe wavelengths are rate limited by solvent-dependent vibrational cooling ( bottom ). ( b ) Excitation of 9-methyladenine to the 1 ππ ∗ state at
267 nm ( step 1 ) is followed by ultrafast internal conversion to the hot ground state ( step 2 ). Vibrational cooling returns the molecule to thermal equilibrium with the solvent ( step 3 ). Visible probe pulses monitor the 1 ππ ∗ population, whereas UV probe pulses monitor the recovery of the thermalized ground state. ( c ) Mid-IR pulses can probe vibrational cooling dynamics via bleach recovery of ground-state fundamentals (1625 cm − 1 , blue ) as well as hot band decay (1590–1615 cm − 1 , red ).
220 Middleton et al.

ANRV373-PC60-11 ARI 25 February 2009 16:34 firmly established that ultrafast internal conversion (IC) occurs because CIs can be accessed from the Franck-Condon region via near-barrierless pathways. Most studies calculate minimum energy pathways through static potential energy landscapes, but dynamical studies capable of following a photoexcited wave packet as a function of time are beginning to appear (16–19).
Nucleobase CIs are often accessed via out-of-plane deformations initiated by twisting about double bonds. For pyrimidine bases, many studies have shown that torsion about the C5-C6 bond is a key deactivation step (15, 20–23). The excited-state energy is relatively insensitive to ring puckering, but the ground state is strongly destabilized by the loss of π -bond stabilization (aromaticity). As a result, the ground-state energy rises sharply along the ring-deformation coordinate, eventually meeting the comparatively flat excited-state surface in a CI. Many physical chemists will recognize this pathway from classic work on the nonradiative decay of photoexcited ethene.
Similar bond-twisting pathways have been located for purine bases (24, 25).
There is excellent experimental support for the ethene-like decay channel in pyrimidines.
First, resonance Raman experiments by Loppnow and coworkers (26–28) show that the earliest nuclear motions in the lowest 1 ππ ∗ state of several pyrimidine bases are lengthening of the C5-
C6 bond and pyramidalization at the C5 and C6 atoms. These changes, which may lie along the photochemical reaction coordinate for dimer formation (see Section 4), are also consistent with the torsional motions necessary to access the CIs identified in calculations (15, 20–25). Second, the sensitivity of pyrimidine excited-state lifetimes to C5 substitution (29) can be largely understood through the substituent’s ability to restrict torsion about this bond. Bases modified in ways that restrict torsion about the relevant bonds have greatly increased fluorescence lifetimes (30, 31).
These studies powerfully illustrate how knowledge of the nonradiative decay pathway can enable the rational design of fluorescent nucleobase derivatives.
IC: internal conversion
Vibrational cooling: the dissipation of excess vibrational energy from a molecule to the surrounding solvent until thermal equilibrium is reached
0
Many states that are dark in emission can be readily seen in transient absorption experiments. A good example is the vibrationally highly excited population formed in S
0 initial 1 ππ ∗ state. IC to S
0 following IC from an deposits more than 4 eV of energy into the vibrational modes of a given base. For thymine, this energy corresponds to a Boltzmann temperature of ∼ 2000 K. The excess vibrational energy is manifested by a strongly red-shifted S
0 absorption spectrum, which returns to equilibrium by vibrational energy transfer to surrounding solvent molecules in a process known as vibrational cooling.
Pecourt et al. (14) first showed that transient absorption signals at UV and near-UV probe wavelengths monitor vibrational cooling dynamics and decay more slowly than ones at visible wavelengths. The former signals measure how rapidly the ground-state absorption spectrum is re-established, whereas the latter ones are assigned to the decay of excited-state absorption as a result of IC. Bleach recovery signals at UV probe wavelengths are dominated by vibrational cooling. These signals exhibit a characteristic time constant of ∼ 2 ps for many single bases in aqueous solution.
Middleton et al. (32) investigated solvent effects on vibrational cooling by DNA bases. In contrast to the weak solvent dependence of the 1 ππ ∗ lifetime, these authors found that vibrational cooling is highly sensitive to the solvent ( Figure 2 ). In addition, vibrational cooling of
9-methyladenine occurred 1.7 times slower in D
2
O than H
2
O. Smaller solvent isotope effects of
1.2–1.4 were observed for 9-methyladenine, thymine, and thymidine in acetonitrile/acetonitriled
3
(33) and for 1-cyclohexyluracil in H
2
O/D
2
O (34). Because isotopic substitution primarily affects high-frequency solvent modes, these observations suggest that significant vibrational energy transfer occurs between high-frequency solute and solvent modes (32). This contrasts with the www.annualreviews.org
• DNA Excited-State Dynamics 221

ANRV373-PC60-11 ARI 25 February 2009 16:34 standard picture in which the vibrational excess energy exits a hot chromophore primarily through the lowest-frequency modes of the solute.
Excited states are classified as dark or bright according to whether they are reached by transitions from S
0 states, all nucleobases have excited states with
3 ππ ∗ that have small or large oscillator strengths, respectively. In addition to the bright
1
1 ππ ∗ n π ∗ character as well as triplet states ( 3 n π ∗ and
) (3, 10). These states are dark and have been extremely difficult to characterize by conventional spectroscopic techniques. Although excitation of S
0 molecules overwhelmingly populates the bright 1 ππ ∗ states, dark states can be subsequently reached via IC. Direct spectroscopic evidence of the dark states has been accumulating recently for pyrimidine bases, as reviewed in the following subsections.
2.3.1.
1 n π
∗ states.
There is ongoing debate about whether passage to S
0 occurs directly through a single CI or indirectly through two or more CIs in a cascade involving an intermediate 1 n π ∗ state (35–38). This debate, which has largely concerned deactivation pathways for gas-phase nucleobases, has become highly relevant to solution-phase dynamics since the surprising discovery that a large fraction of the population excited to the lowest 1 ππ ∗ state decays to the lowest-energy
1 n π ∗ state in water (39) and other solvents (34).
Hare et al. discovered by means of transient absorption measurements that the initial excitedstate population bifurcates for 1-cyclohexyluracil in a variety of solvents (34) and for various pyrimdine bases in aqueous solution (39). For 1-cyclohexyluracil, 60% of excited molecules decay on an ultrafast timescale to S
0
, and the remainder relax orders of magnitude more slowly via a long-lived trap state (34). The trap state is dark, as indicated by the absence of long-time emission from pyrimidine bases (23, 40, 41), and is assigned to the lowest-energy between 10% and 50% of photoexcited pyrimidine bases decay via a 1 n π
1
∗ n π ∗ state (34). In water, state (39). The lifetime of the 1 n π ∗ state of 1-cyclohexyluracil is highly sensitive to solvent, ranging from 26 ps in water to
3.2 ns in acetonitrile (34). In contrast, 1 ππ ∗ lifetimes depend only modestly on the solvent (32, 42).
The discovery that electronic energy relaxes on a picosecond timescale much of the time in photoexcited pyrimidine bases shows that ultrafast IC is not the sole factor responsible for
DNA’s photostability. In water, Hare et al. (39) observed lifetimes of between 10 and 150 ps for different pyrimidine bases. Additionally, they reported that the 1 n π ∗ lifetime is significantly longer for pyrimidine nucleosides than for free bases (39). This is the only known example in which sugar substitution alters the lifetime of an excited nucleobase. Hare et al. (39) also proposed that excess vibrational energy in the 1 n π ∗ state accelerates IC to the ground state. According to this hypothesis, ribosyl substitution extends the 1 n π ∗ lifetime by reducing the excess vibrational energy that promotes nonradiative decay.
Femtosecond time-resolved infrared (IR) experiments are providing many new insights into
DNA excited states, including the elusive dark states (43–45). UV pump/IR probe experiments provide a particularly powerful way to study excited-state dynamics (46). Electronic absorption bands frequently overlap, making transients from UV/UV or UV/visible experiments difficult to interpret. In contrast, vibrational bands are narrower and have much greater structural sensitivity.
Quinn et al. (44) measured lifetimes of 33 and 37 ps for a band at 1574 cm − 1 in dCyd (2 deoxycytidine) and dCMP (2 -deoxycytidine 5 -monophosphate), respectively. These decays agree within experimental uncertainty with the 1 n π ∗ lifetime of CMP (cytidine 5 -monophosphate) measured in Hare et al.’s (39) UV/UV experiments. We have detected a broad vibrational band at 1760 cm − 1 due to a carbonyl stretch in the 1 n π ∗ state of 1-cyclohexyluracil ( Figure 3 ). The
222 Middleton et al.

ANRV373-PC60-11 ARI 25 February 2009 16:34 a
0
0
Methanold
1
Acetonitrile b
0.2
0.1
0
0
–0.2
–0.4
1602 cm –1
1712 cm –1
1775 1750 1725 1700 1675
Wave number (cm –1 )
0 10 20 30 40 100
Time delay (ps)
1000
Figure 3
Mid-IR transient absorption is advantageous for probing dark-state dynamics. ( a ) Transient IR difference spectra of 1-cyclohexyluracil show a broad positive band at 1760 cm methanold
1
( top ) and acetonitrile ( bottom
The bleach recovery signal at 1712 cm
− 1
). ( b lowest triplet state of thymine in acetonitriled
3
( top
− 1 assigned to the
) The long-lived transient at 1602 cm
− 1
1 n π ∗ state in is assigned to the
), which is fully formed just 10 ps after photoexcitation.
for 1-cyclohexyluracil in acetonitrile exhibits complex kinetics
( bottom ). The fastest decay component results from vibrational cooling following ultrafast internal conversion from the 1 ππ ∗ state, and is followed by an ∼ 3-ns decay, which is assigned to the lowest state. The constant offset at long times is assigned to the lowest triplet state.
1 n π ∗ lifetime of this band in acetonitrile and methanol is in good agreement with previous UV/UV measurements (34). The band is blue-shifted with respect to both the ground-state carbonyl stretches, but the underlying reasons are not understood.
2.3.2. Triplets.
Hare et al. (34) observed that the long-time 1-cyclohexyluracil signals are quenched in the presence of oxygen and assigned them to the lowest triplet state. Observing intersystem crossing (ISC) dynamics is difficult in aqueous solution because triplet yields are less than a few percent (47). However, yields are much greater in less polar, aprotic solvents (34, 48).
Based on the observation of vibrational cooling by hot triplet states, Hare et al. (34) concluded that the triplet states are formed within the first few picoseconds after photoexcitation, even in solvents in which long-lived 1 n π ∗ states are found. The same conclusion was reached in a later study of
ISC by pyrimidine bases in water (39). The most compelling evidence for rapid ISC comes from
UV/IR experiments on thymine in acetonitrile that directly monitored the prompt appearance of vibrational bands assigned to the 3 ππ ∗ state (45).
The appearance of triplet states after no more than a few picoseconds seems to indicate that
ISC takes place from the short-lived 1 ππ ∗ state, as suggested by some theoretical studies (49, 50).
However, Hare et al.’s (34) result showing that 60% of the 1 ππ ∗ population returns directly to the ground state in all solvents, independent of the triplet quantum yield, make this mechanism unlikely. In contrast, the 1 n π ∗ yield depends on the solvent and is inversely proportional to the triplet yield (34), suggesting that ISC to the does not occur during the entire 1 n π ∗
3 ππ ∗ state occurs from the 1 n π ∗ state. Because ISC lifetime, Hare et al. (34) proposed that ISC to the triplet state occurs only in 1 n π ∗ molecules with excess vibrational energy. In this model, vibrational cooling in the 1 n π ∗ state rapidly reduces the internal energy, and ISC effectively halts within a few picoseconds. This model explains the low triplet yields observed in hydrogen-bonding solvents
ISC: intersystem crossing www.annualreviews.org
• DNA Excited-State Dynamics 223

ANRV373-PC60-11 ARI 25 February 2009 16:34 with their high vibrational cooling rates, compared to polar, aprotic solvents, in which vibrational cooling occurs more slowly (34).
CT: charge transfer
Base stacking: stabilizing interaction resulting from π overlap of the aromatic rings of adjacent nucleobases
Base pairing: association of two nucleobases by hydrogen bonding
The spatial organization of the bases in DNA creates new photophysical decay pathways not found in base monomers. Precisely how interbase couplings, conformational heterogeneity, and the unique environment of the double helix give rise to these new pathways is poorly understood and the subject of intense investigation. Much recent work has sought to understand how base pairing and base stacking, the dual architectural motifs of the DNA double helix ( Figure 1 c ), influence relaxation pathways. In the following subsections, we critically review experimental and theoretical progress at elucidating the effects of these interactions on the relaxation of excess electronic energy in nucleic acids.
Physical chemists have been fascinated for decades by the possibility of photoinduced proton transfer in nucleic acid base pairs. It was proposed that light-induced proton motion between
DNA strands could induce mutations (51, 52), but this has never been observed experimentally
(3). Recently, a photoprotective role has been proposed for proton transfer (53–55). Calculations by
Sobolewski & Domcke (54) suggest that ultrafast decay to the electronic ground state is mediated by aborted transfer of a single proton from guanine to cytosine. In this mechanism, an interbase charge transfer (CT) state causes an electron to move from guanine to cytosine, triggering spontaneous proton transfer in the same direction along the middle of the three guanine-cytosine hydrogen bonds (54, 55). Progress along the proton transfer coordinate leads to a CI with S
0
, causing the excited molecule to return to S
0
(54, 55).
This intriguing and influential proposal has gained some experimental support. Abo-Riziq et al. (56) studied isolated guanine-cytosine base pairs in the gas phase by resonance-enhanced multiphoton ionization spectroscopy. Broad UV spectra with line widths of several hundred wave numbers were measured for isolated Watson-Crick guanine-cytosine base pairs, but only sharp resonances ( ν < 1 cm − 1 ) were seen for non-Watson-Crick structures (56). The authors proposed that Sobolewski & Domcke’s (54) ultrafast nonradiative decay channel is unique to the Watson-
Crick guanine-cytosine base pair. Calculations subsequently supported this conclusion (55).
Single base pairs are unstable in aqueous solution, but they can be prepared in nonpolar solvents from suitably derivatized nucleobases. Schwalb & Temps (57) studied fluorescence decays from a modified guanine-cytosine base pair in chloroform using the femtosecond upconversion technique.
They measured a lifetime of 0.355 ps for the Watson-Crick base pair, whereas the fastest decay components observed for the solvated guanine and cytosine derivatives were 0.67 and 0.84 ps, respectively. They invoked Sobolewski & Domcke’s (54) quenching mechanism as a possible explanation for their observations. Time-resolved observation of the underlying proton motions is needed to confirm this explanation.
In aqueous solution, single-stranded sequences can form partially ordered helices ( Figure 1 c ), which are conformationally similar to the strands in a double helix (58). Single-stranded DNAs are ideal for studying the effects of base stacking on excited-state dynamics in the absence of base pairing (59, 60). Excited states of a single-stranded polynucleotide can decay orders of magnitude
224 Middleton et al.

ANRV373-PC60-11 ARI 25 February 2009 16:34 a b poly(A)
A form poly(dA)
B form
570 nm
0
0
250 nm
0
1729 cm –1
AMP poly(A) poly(dA)
0 2 4 6 8
2 3 4 5
10
Time delay (ps)
100
2 3 4 5
Figure 4
Conformation and excited-state dynamics in single-stranded adenine homopolymers. ( a ) Poly(A) forms an A-type helix, whereas poly(dA) adopts a B-type helix (both views are along the helix axis). The bottom panels show the base stacking between two adjacent bases in the single strands. ( b ) The excited-state-absorption signals probed at 570 nm ( top ) and bleach signals at 250 nm ( middle ) show a long-lived state in poly(A) and poly(dA) in addition to the short-lived state observed in AMP. The visible and UV signals decay with similar lifetimes and show an approximate mirror symmetry. The UV pump/IR probe signal ( bottom ) also shows slow bleach recovery of a ground-state vibrational mode. Structures were generated using the UCSF Chimera software (117).
more slowly (59–62) than excitations in the monomeric building blocks (Section 2) or in single base pairs (Section 3.1). In single-stranded adenine homopolymers both ultrafast ( τ ≈ 1 ps) and more slowly decaying components are observed ( Figure 4 ) (59). Crespo-Hern´andez & Kohler
(59) suggested that the fast and slow signal components correspond to excitations in unstacked and stacked base regions, respectively. Poorly stacked bases are hypothesized to decay via the monomerlike pathways described in Section 2. Disrupting base stacking thermally or via a denaturing cosolvent attenuates the long-lived signals, showing clearly that they arise in domains of two or more stacked bases (59, 63).
UV pump/UV probe experiments have provided dramatic new insights into base multimer excited-state dynamics (60, 64–66). Probing at UV wavelengths monitors the time needed to repopulate the ground state following excitation by the pump pulse. This technique first revealed the existence of a slow decay pathway from the 1 ππ ∗ state of single pyrimidine bases (Section
2.3). The transients at 250 nm in Figure 4 b show approximate mirror symmetry with the transients at 570 nm, indicating definitively that ground-state recovery occurs on two timescales. The
250-nm transients exhibit the ∼ 2-ps decay time that is the signature of ultrafast vibrational cooling following ultrafast decay to S
0
(60). This is convincing evidence that the ∼ 1-ps decay of the excited-state-absorption signal at 570 nm results from ultrafast ground-state repopulation.
Bleach-signal amplitudes can be used to estimate the yield or fraction of excitations that decay by a given channel (60, 65, 67, 68). On the basis of these yields, Crespo-Hern´andez et al. (60) determined that most excitations in base-stacked regions of DNA decay to long-lived states, whereas excitations in unstacked bases decay by ultrafast IC. Earlier studies (reviewed in 3) established www.annualreviews.org
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ANRV373-PC60-11 ARI 25 February 2009 16:34
As discussed elsewhere (3, 115), it is sometimes mistakenly assumed that an excimer is only formed when an electronically excited molecule encounters a second, unexcited one. An excimer or exciplex is an excited electronic state with strong charge transfer character (70) independent of how it is formed. Excimers and exciplexes are observed in aromatic crystals and photopolymers (70) in which diffusion is not required to bring the interacting molecules together. Excimers in this case can be formed from different initial states, including Frenkel excitons.
DNA exciplexes can also be called interbase charge transfer states (65), keeping in mind that there may be significant configuration interaction with the excitonic state formed by the interaction between transition dipoles of the two bases.
Excimer/exciplex: an excited electronic state with substantial charge transfer character involving two identical
(excimer) or different
(exciplex) molecules that pico- and nanosecond timescale emission is seen in DNA polymers. However, these timeresolved emission experiments could not determine whether the slow relaxation amounted to a major or a minor decay channel. Crespo-Hern´andez et al.’s (60) measurements were the first to show that most excited states in single-stranded DNAs decay to S
0 on the picosecond timescale.
This established that most relaxation occurs faster than the nanosecond timescale seen in some emission experiments (69) and slower than the femtosecond timescale seen in experiments on base monomers (Section 2.1).
Crespo-Hern´andez et al. (60) assigned the long-lived states observed in single-stranded oligonucleotides to intrastrand excimers in which excitation is shared by two neighboring bases
(see the sidebar). This conclusion is supported by the excellent kinetic agreement (described in 59) between the long-time transient absorption signals for single-stranded adenine tracts and timeresolved emission signals measured by Plessow et al. (69) for single-stranded (dA)
15
. Time-resolved emission spectra from the 15-mer (69) clearly show the broadened and red-shifted emission that is a hallmark of excimer/exciplex states (70). In fact, excimer/exciplex states are hardly a new concept in DNA photophysics, having been observed in DNA di- and polynucleotides in cryogenic glasses in the 1960s (71).
Transient absorption experiments in other laboratories have confirmed the existence of longlived excited states in DNA oligomers (61, 62). Kwok et al. (61) studied long-lived excited states in
(dA)
20 by femtosecond transient absorption and the femtosecond Kerr-gated fluorescence technique. On the basis of red-shifted emission signals that decayed synchronously with transient absorption signals, these authors also assigned the long-lived states to excimers (61). However, they proposed that two distinct excimers are formed with lifetimes of 4.3 ps and 182 ps. The latter lifetime is in reasonable agreement with the (dA)
18 lifetime reported by Crespo-Hern´andez et al.
(60). Kwok et al. (61) assign the long-lived component to an excimer-like state because they argue that it involves more than two bases as opposed to the short-lived excimer, which they believe is localized on just two bases. Experimental evidence that the long-lived excimer-like state actually spans more than two bases was never presented. Kwok et al.’s (61) suggestion that an initially localized excitation progressively delocalizes with time is also somewhat counterintuitive for multichromophoric systems. Experiments by Takaya et al. (65) (see Section 3.5) instead suggest that the long-lived states in adenine tracts are localized on just two stacked bases.
Base pairing in solvated DNA is usually accompanied by base stacking, which has led us to investigate excited states in DNA strands joined by hydrogen bonds (60, 64, 66, 72). Crespo-Hern´andez
226 Middleton et al.

ANRV373-PC60-11 ARI 25 February 2009 16:34 et al. (60) reported that the long-lived states in duplex (dA)
18
· (dT)
18 decay with essentially identical kinetics as those seen in single-stranded (dA)
18
. Adenine-thymine base pairing thus neither inhibits the formation nor hastens the decay of excimers in the adenine strand. Longlived states seen in the alternating duplex (dAdT)
9
· (dAdT)
9 were also assigned to intrastrand exciplexes (60).
The similar kinetics in (dA)
18
· (dT)
18 and in (dA)
18 provide evidence that the initial Franck-
Condon excited states decay to excitations localized on just one of the two strands in duplex DNA
(60). Base stacking limits excitation energy to one strand at a time in the B-form double helix, possibly explaining the preponderance of intrastrand photoproducts and enabling repair using the undamaged strand as a template (60). The concept of strand-localized excited states has been tested in an innovative theoretical study by Bittner (73), who was the first to study the interplay between excitonic and CT states in photoexcited DNA (see Section 3.5).
The conclusion that adenine-thymine base pairs do not introduce rapid quenching channels for excess electronic energy has been extended recently to duplexes comprising guanine-cytosine base pairs (64, 66). As illustrated in Figure 5 , ground-state recovery occurs more slowly in hairpins and duplex structures with guanine-cytosine base pairs than in an equimolar mixture of the 5 -mononucleotides of guanine and cytosine. This suggests that Sobolewski & Domcke’s (54) fast quenching channel is not relevant to base-stacked DNA. Long-lived excited states have now been observed in many duplex and nonduplex structures, including the acid double-stranded form of poly(A) (59), the hemiprotonated duplex [or, possibly, tetraplex (74)] structure of poly(dC) a
CpG c
0
CMP + GMP d(C
4
G
4
) d(C
4
G
4
)
GpC
0 d(C
5
T
4
G
5
) d(C
5
A
4
G
5
)
0
B-form Z-form
Z-form
B-form b
0
H
+
Hoogsteen
Watson-Crick
Watson-Crick base pairing
Hoogsteen base pairing
0 10 20
Time delay (ps)
30 40
Figure 5
Conformation and excited-state dynamics of duplex guanine-cytosine DNA. ( a ) The alternating sequence d(GC)
9
· d(GC)
9 adopts both
B- and Z-helix conformations. Base-pair overlaps are shown for the 5 -CpG-3 and 5 -GpC-3 steps (viewed 5 to 3 down the helix axis; the colored base pairs are above the ones in gray ). ( b ) The complementary guanine ( blue ) and cytosine ( red ) bases can form both
Watson-Crick and Hoogsteen base pairs. ( c ) Transient absorption signals (266-nm pump/250-nm probe) show long-lived states in various guanine-cytosine DNAs. Different helix conformations ( green ) and base pairing ( blue ) motifs of d(GC)
9
· d(GC)
9 result in nearly identical kinetics. Figure adapted from Reference 66. Structures were generated using the UCSF Chimera software (117).
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(72), the guanine quadruplex (75), and in duplexes and hairpins composed of guanine-cytosine base pairs (64, 66).
Frenkel exciton: an excited state of a multichromophoric system produced by dipolar coupling of the neutral excited states of individual molecules
Gustavsson, Markovitsi, and coworkers (8) have pioneered the time-resolved study of fluorescence from DNA oligomers and polymers using the femtosecond upconversion technique. Interestingly, these experiments yield fluorescence decays with time constants of no more than a few picoseconds
(76–80). This rapidly decaying emission, which has many of the characteristics of emission from the 1 ππ ∗ excited states of base monomers, has been assigned to Frenkel exciton states formed by the coupling of 1 ππ ∗ states of proximal nucleobases (81–84). It has recently been suggested that some of the emission may be from 1 ππ ∗ states localized on single bases (79, 80).
Time-resolved fluorescence anisotropy experiments provide evidence for rapid IC or intrabrand scattering among the excitonic states (85), which differ in their polarization properties (82). For example, the fluorescence anisotropy of poly(dA) · poly(dT) decays by approximately 30% 1 ps after excitation (85). Markovitsi et al. (8) proposed that the bottom of the exciton band is reached in
100 fs because rise times are not observed at any emission wavelength.
A fascinating recent question concerns the size of the excitons: What is the extent of delocalization, at the instant of excitation and at later times? Excitonic eigenstates calculated with an idealized helix geometry are delocalized over the entire length of a double helix containing 20 base pairs (81). The inclusion of conformational (off-diagonal) disorder using structures sampled from molecular dynamics (MD) simulations reduces the spatial extent to between four and eight base pairs (82). Finally, the addition of homogeneous broadening (diagonal disorder) to model calculations further lessens the delocalization (83). Nearly half the excitons in (dCdG)
5
· (dCdG)
5 are calculated to be localized on a single base with the remainder “delocalized over at least two bases” (83). Fiebig and coworkers (62) estimated a 1/e delocalization length of 3.3
± 0.5 bases in
DNA adenine tracts from their transient absorption experiments. Kadhane et al. (86) concluded from their interesting analysis of the circular dichroism spectra of adenine tracts that excitons corresponding to long-wavelength transitions ( λ > 200 nm) are delocalized over no more than two bases.
Some workers have suggested that the long-lived excitations in base-stacked DNAs are Frenkel excitons (62, 87, 88), but this hypothesis is poorly supported by experiment (61, 64–67). Calculations have shown that the lowest-energy exciton states of DNA have much lower oscillator strengths than those at higher energy (81–83, 88). It has been suggested that the longer radiative lifetimes of the low-energy exciton states are responsible for the long excited-state lifetimes seen in DNA (87, 88). A serious weakness of this analysis is that the lifetime of an excited state is determined by the radiative lifetime and the total rate of nonradiative decay. The latter rate is not predictable from exciton theoretical models that deal only with oscillator strengths and energies
(67).
A simple consideration shows that radiative decay cannot be the only relaxation channel for
DNA excitons, regardless of their location in the exciton band: According to calculations, the bright 1 ππ ∗ states of single bases have radiative lifetimes of several nanoseconds (90, 91). Any exciton built from the 1 ππ ∗ states, and that has a lower oscillator strength, must therefore have a radiative lifetime even longer than a few nanoseconds. An excited-state lifetime of 10–100 ps in
DNA is therefore a certain indicator that the rate of nonradiative decay greatly exceeds the rate of radiative decay. Exciplex states reached by the decay of the initial excitons, whatever their precise degree of delocalization, provide the best explanation for long-lived excited states in DNA base multimers, as we describe next.
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Femtosecond fluorescence upconversion provides only a partial view of the dynamics of emissive states in DNA. Emission at times greater than approximately 10 ps after photoexcitation is often too weak to be detected in femtosecond upconversion experiments (77) but can be observed in timecorrelated single-photon counting measurements because of this technique’s longer gate window.
These more sensitive measurements have shown that emission extends out to the nanosecond timescale (reviewed in 3, 8). The need to switch from femtosecond upconversion to time-correlated single-photon counting is a consequence of the CT character of the long-lived excited states, which causes a precipitous drop in the radiative decay rate.
Transient absorption measurements with UV probing show that repopulation of the ground state occurs considerably more slowly in photoexcited DNAs than is suggested by the rapid loss of fluorescence seen in the femtosecond upconversion experiments (64). These experiments are simply sensitive to different excited states (67). The bright Frenkel exciton states rapidly decay to excimer/exciplex states, which have much lower radiative transition rates (64, 65). These comparatively dark states are responsible for the long-lived emission that extends over pico- and nanosecond timescales. The dark character of these states explains why fluorescence quantum yields of DNA oligo- and polynucleotides do not differ substantially from those observed for mononucleotides, despite the presence in the former systems of large yields of relatively long-lived excited states (60).
Experiments by Takaya et al. (65) on a series of dinucleoside monophosphate compounds decisively support the hypothesis that excitons in DNA rapidly decay to exciplexes, which then decay on a picosecond timescale by charge recombination. Takaya et al. (65) observed long-lived excited states in minimal stacks of just two nucleobases ( Figure 6 a ). The decay rates of the longlived states decrease with increasing energy of the charge-separated state formed by transferring an electron from one of a pair of stacked bases to the other ( Figure 6 b ). Takaya et al. (65) also showed that identical long-time decays are seen for the adenine dimer and for the homopolymer containing hundreds of bases. This is strong evidence that, regardless of sequence length, initial excitons trap to a common state that is localized on just two bases.
Recent experiments have shown that DNA exciplex lifetimes vary sensitively with base sequence (64, 65) but are insensitive to base-pairing motif and helix conformation (66). The similar charge-recombination dynamics observed for Hoogsteen base-paired DNA and Watson-Crick base-paired DNA ( Figure 5 c ) suggests yet again that the ultrafast quenching pathway discussed in Section 3.1 for single Watson-Crick base pairs is not accessed in DNAs composed of stacked base pairs.
De La Harpe et al. (66) studied d(GC)
9
· d(GC)
9 in B- and Z-form duplex structures by transient absorption. Despite significantly different base stacking ( Figure 5 a ), the bleach signals for Band Z-forms recover with similar lifetimes of 6.4
± 0.6 ps and 7.6
± 0.8 ps, respectively (66)
( Figure 5 c ). Earlier, Crespo-Hern´andez & Kohler (59) observed identical lifetimes for poly(A) and poly(dA) despite their different helix conformations ( Figure 4 a ). These results indicate that exciplex lifetimes are nearly independent of ground-state base-stacking geometries. This is at first surprising given the exquisite sensitivity to conformation of the rates of the charge-shift reactions studied in DNA charge transport (92–96). De La Harpe et al. (66) have proposed that the electronic coupling between the hole and the electron, which are separated by a relatively small distance in an exciplex, may be too great to be significantly altered by helix conformation. Although exciplex lifetimes appear to depend only weakly on secondary structure, photoproduct formation is highly dependent on local conformational properties, as described in Section 4.
Figure 7 summarizes the current understanding of the various photophysical pathways in DNA.
This figure shows the profound effect that base stacking is believed to have on both the nature of www.annualreviews.org
• DNA Excited-State Dynamics 229

ANRV373-PC60-11 ARI 25 February 2009 16:34 a
0
CpG
CMP + GMP
N
NH
2
N
HO
N
N
H
H
O
O
H
H
OH
ApU
HN
O
O
P
O
O N
O
H
O
H
OH
H
H
OH b
C – pG +
0
10 11
8
7
6
5
4
3
A + pU –
ApA
AMP
ApC
AMP + CMP A + pC –
2
0
ApG
AMP + GMP
ApU
AMP + UMP
10 10
8
7
6
5
A – pG + A + pA – A + pG –
0 10 100 1000 0
Time delay (ps)
10 100 1000 7.8
8.2
8.6
IP – EA (eV)
9.0
Figure 6
( a ) Ground-state recovery signals for a series of RNA dinucleosides and equimolar mixtures of their respective monomers. The structure of ApU is shown. ( b ) The correlation ( dashed line ) between the decay rate of the long-lived states of the dinucleosides and the thermodynamic driving force for charge recombination ( IP
−
EA ) indicates electron transfer in the Marcus inverted region. Figure adapted from Reference 65.
the initial excitations (excitons versus localized excited states) and the subsequent decay pathways.
In base-stacked contexts, excitation initially populates Frenkel exciton states that are likely spread over no more than two stacked bases or stacked base pairs. These states decay rapidly to longlived excimer/exciplex states formed between two π -stacked bases. Because of their substantial
CT character, these excimer/exciplex states are dark and make a small contribution to the total fluorescence. Recent electronic structure calculations by Santoro et al. (97) on π -stacked adenines support this model.
Previous sections describe photophysical nonradiative decay pathways for DNA excited states that terminate in S
0
. In this section, we return to our initial motivation of connecting excited-state dynamics and photochemical outcomes. Space limitations only allow discussion of the thymine dimer—the main mutagenic photoproduct in DNA (98). This mainly intrastrand photoproduct is formed by (2 + 2) cycloaddition of the C5-C6 double bonds of adjacent thymines to give a cyclobutane ring ( Figure 8 a ).
Because of its inherent structural specificity, time-resolved IR spectroscopy is a promising approach for elucidating photochemical decay channels. Using this method, Schreier et al. (99)
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+
B
–
+
B
–
<1 ps
B
B
+
–
Excimer/exciplex
3–200 ps
Purines and pyrimidines
1 ππ*
0.2–1 ps Pyrimidines only
1 nπ*
<10 ps
UV
267 nm
3 ππ*
10–150 ps
μs
Electronic ground state
Figure 7
The fate of excess electronic energy deposited in DNA by UV light is governed by base-stacking conformation. ( Left ) Stacked bases are excited to an exciton state that decays to an exciplex state in less than
1 ps. The exciplex state returns to the ground state on a timescale of 10–100 ps via charge recombination.
( Right ) UV excitation of unstacked or poorly stacked bases creates localized ground state within 1 ps.
1 n π ∗
1 ππ ∗ states, which decay to the states have been observed in unstacked pyrimidine bases, but it is unknown whether these states can be populated in the presence of base stacking.
observed that IR marker bands specific to the cis-syn thymine dimer are fully formed less than 1 ps after excitation of single-stranded (dT)
18 at 266 nm. Subpicosecond dimer formation strongly suggests that the reaction occurs directly from the 1 ππ ∗ state (60). This seems to end the decadesold debate about the multiplicity of the precursor excited state (100). Kwok et al. (101) recently claimed that a triplet state with a lifetime of 140 ps is responsible for dimer formation in (dT)
20
, but this conclusion is suspect in view of the subpicosecond kinetics seen by Schreier et al. (99).
Both theory (102–104) and experiment (26–28) support a singlet-state pathway to the thymine dimer. Loppnow and coworkers (26–28) have analyzed absorption spectra and resonance Raman excitation profiles for pyrimidine base monomers in solution. The observed Raman intensities reveal elongation of the C5-C6 bond and increased pyramidalization at C5 and C6. These nuclear motions are consistent with the expected reaction coordinate for dimer formation and suggest that the photochemistry is determined by initial dynamics near the Franck-Condon region of the lowest-lying 1 ππ ∗ state. Such motions may predispose adjacent pyrimidine bases for dimer formation. Recently, Boggio-Pasqua et al. (103) have located and characterized the CI that putatively enables ultrafast dimer formation from an initial singlet excited state. A computational study by
Blancafort & Migani (104) suggests that the reaction may proceed via a singlet excited state that is unique to π -stacked bases.
Because dimerization occurs faster than whole-base motions, the reaction probability is hypothesized to depend mainly on the relative orientation of the bases at the instant of excitation
(99). There is evidence from crystal studies that dimerization of suitably stacked thymines is highly efficient, with a quantum yield of essentially unity. Schreier et al. (99) therefore proposed that the low quantum yield of dimerization observed in DNA results because the average twist angle of
36 ◦ between successive base pairs is too large for reaction to occur. This paradigm explains past experiments showing that pyrimidine dimer formation depends sensitively on DNA conformation www.annualreviews.org
• DNA Excited-State Dynamics 231

ANRV373-PC60-11 ARI 25 February 2009 16:34 a b
<1 ps
O
UV light
<300 nm
O
N
HN
C6
O
N
C5 d
CH
3
H
N
C5’
CH
3
C6’
O
7
6
5
4
3
10
Water
Dimerizable conformations d
30 40
| η | (degrees)
50 60 70 e
7
6
5
4
40% (v/v) ethanol
Dimerizable conformations
Side view Top view
3
10 30 40
| η | (degrees)
50 60 70
Figure 8
( a ) Thymine dimer formation occurs in less than 1 ps for favorably orientated thymine steps. ( b ) The two parameters used for conformational analysis: the distance, d , between C5-C6 bond centers and the improper torsion angle, η . Figure reprinted with permission from Reference 109. ( c ) Conformational distributions for dTpdT in water ( blue ) and in 40% (v/v) ethanol ( red ). Dimerizable conformations lie within the green region. ( d ) The mean dimerizable structure ( blue ) overlapped at the 5 end with the thymine dimer structure from Reference 111 shown in green. ( e ) The mean dimerizable structure ( blue ) overlapped at the 5 base with the ideal B-form structure ( black ). Structures in panel a were generated using the VMD software (116). Structures in panels d and e were generated using the UCSF Chimera software (117).
(105–107). It will be important to investigate whether other DNA photoreactions are also under ground-state control (108).
The hypothesis that conformations in the electronic ground state control thymine dimer yields has been explored in two MD studies (109, 110). Law et al. (109) proposed a two-parameter model for identifying reactive thymine-thymine steps based on the distance ( d ) and the torsion angle ( η )
232 Middleton et al.

ANRV373-PC60-11 ARI 25 February 2009 16:34 between the two C5-C6 double bonds ( Figure 8 b ). They used MD simulations to sample the conformational space of thymidylyl-(3 ,5 )-thymidine in various solvent conditions ( Figure 8 c ).
Dimerizable conformations were defined as ones with d < 3.63 ˚A and η < 48.2
◦ ( Figure 8 b – d ).
These conformations occurred at a frequency approximately equal to the experimentally observed quantum yields for dimer formation in each solvent. In a closely related study, Johnson & Wiest
(110) applied analogous geometrical criteria to estimate populations of reactive conformations from MD simulations of (dT)
18
.
Law et al. (109) showed that the mean dimerizable structure is similar in all cosolvent systems studied. This structure shares many conformational characteristics with the nuclear magnetic resonance–derived (111) structure of the cis-syn dimer itself ( Figure 8 d ). The heuristically determined model thus appears to have successfully identified structural relationships important for the actual reaction pathway (109). The vertical separation between the two bases is similar in B-form
DNA and in the mean dimerizable structure, but the twist angle of the latter structure is reduced greatly ( Figure 8 e ). Alignment of the C5-C6 double bonds, and not their separation, appears to be the main obstacle to efficient dimer formation in double-stranded DNA. This may explain why dimers form with similar quantum yields in single- and double-stranded DNA (112, 113):
Single-stranded DNA has reduced base stacking but is more flexible and can more readily achieve the undertwisted conformation that promotes dimerization (114).
Research into DNA excited states is thriving. Time-resolved spectroscopic methods now exist for directly observing excess energy flow in DNA. Significant advances in the understanding of excited-state decay pathways in base monomers and multimers have occurred as a result of theoretical and experimental efforts. Despite rapid progress during the past decade, many challenging questions remain. For example, the roles of base pairing and base stacking in mediating electronic energy relaxation, although coming into focus, are still uncertain. Controversies about the nature of the initial excitons in DNA remain to be settled. Very little is known about the dynamics of excitons and exciplexes in mixed-sequence DNA, in which it is conceivable that a low-energy site could dissociate or trap these excitations. As more attention is focused on understanding the dynamics of oligo- and polynucleotide systems, the gap between theory and experimental results in base multimers will close. This work will also bring us closer to a fully molecular accounting of the formation mechanisms for DNA photoproducts as a function of base sequence and local DNA conformation.
SUMMARY POINTS
1. CIs are responsible for the ultrafast fluorescence lifetimes of DNA monomers. Synergistic work by experimentalists and theorists has identified the nuclear motions responsible for ultrafast nonradiative decay to the ground state.
2. Ground-state recovery signals from UV-pump/UV-probe transient absorption measurements reveal the dynamics of dark excited states and provide estimated quantum yields for the various decay pathways.
3. For single pyrimidine bases in solution, the excited-state population bifurcates in the bright 1 ππ ∗ state, with 60% returning to the ground state and 40% first passing through a 1 n π ∗ state. ISC to the triplet state is proposed to take place on a picosecond timescale from the vibrationally excited 1 n π ∗ state.
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4. Decay pathways for excess electronic energy differ dramatically in stacks of two or more bases compared with monomeric bases. Initial excitons in stacked bases rapidly trap to form long-lived excited states in high yields. These excimer/exciplex states have significant CT character.
5. The decay of excimer/exciplex states by charge recombination may play a dominant role in the photostability of DNA by guaranteeing that most excited states do not lead to deleterious reactions but instead relax back to the electronic ground state.
6. DNA exciplex lifetimes vary sensitively with base sequence but have so far proven to be insensitive to base pairing and helix conformation.
7. Thymine dimerization occurs in less than 1 ps, indicating that a short-lived singlet state is the reactive precursor state. The low quantum yield for dimerization stems from the small number of dipyrimidine base steps with suitable conformation for dimerization at the moment of excitation.
The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.
We thank our colleagues and collaborators, who have shared our enthusiasm for this topic, and apologize to those whose work we were unable to cover owing to space limitations. We are indebted to the students and postdocs who have worked on this project since the late 1990s. Our research on DNA excited states has been generously supported by grants from the National Institutes of
Health and the National Science Foundation. B.K. thanks the University of Aarhus for a visiting professorship that facilitated the completion of this manuscript.
3. Presents a comprehensive review of DNA photophysics through 2003.
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Annual Review of
Physical Chemistry
Volume 60, 2009
viii
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Contents ix