Observation of Long-Lived Excited States in DNA Oligonucleotides

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Observation of Long-Lived Excited States in DNA Oligonucleotides with Significant Base Sequence Disorder Kimberly de La Harpe† and Bern Kohler*,‡ †

Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, United States, and Department of Chemistry and Biochemistry, Montana State University, P.O. Box 173400, Bozeman, Montana 59717, United States

‡

ABSTRACT Excited electronic states with lifetimes longer than those of mononucleotides have been observed in a variety of DNA and RNA base multimers. Most studies reported to date were performed on repetitive base sequences that contain a small subset of the 10 possible nearest-neighbor base pair combinations. Some have questioned whether long-lived excited states will occur in more disordered sequences including natural DNA. Here, we present evidence from femtosecond transient absorption experiments that long-lived excited states are readily observable in significant yields in single- and double-stranded forms of an 11-mer genomic DNA base sequence. Bleach recovery experiments on the separate single strands reveal that long-lived excited states are more prominent and relax more slowly in the strand richer in purine bases. In the duplex, some of the longest-lived excited states are quenched, and a substantial number of excitations decay on a subpicosecond time scale despite the presence of base stacking. SECTION Dynamics, Clusters, Excited States

U

ltrafast relaxation pathways dominate the excitedstate dynamics of single nucleobases, but much longerlived excited states are formed in DNA model compounds containing two or more bases.1,2 The yield of the long-lived states is correlated with the extent of π-π stacking between adjacent bases in a DNA strand.3,4 Most time-resolved spectroscopic studies from the last five or so years were carried out on oligo- and polynucleotides that contain just one or two different nucleobases such as A and T.5-7 These experiments have led to a picture in which initial excitonic states, which may be delocalized over a few bases, dynamically evolve to excited states localized on a pair of bases.1 Experimental4 and computational evidence8-10 favors assignment of these localized excited states to charge-transfer states, although their precise nature is still controversial. Here, we demonstrate that long-lived excited states are still significant in DNA oligonucleotides with substantial base sequence disorder, proving that they are not restricted to the simple, repetitive base sequences that have been studied to date. The first long-lived excited states detected by transient absorption were observed in single-stranded adenine homopolymers3 and later in single- and double-stranded oligonucleotides.5 Buchvarov et al. speculated that DNAs with significant base sequence disorder would favor localized, monomer-like electronic states and suppress the formation of long-lived excited states.6 Schwalb and Temps studied sequence-dependent emission from a series of single- and double-stranded nucleotides in systems with increasing sequence disorder.11 They reported that the fluorescence transients measured in fluorescence upconversion experiments

r 2011 American Chemical Society

are very sensitive to sequence. In particular, substitution of a few guanines for adenines in single-stranded d(A)20 decreased the mean fluorescence lifetime by a factor of between 2 and 5.11 This reduction was due principally to the decrease in amplitude of the longest emission decay components. Extrapolating their results, these authors concluded that fluorescent excited states with lifetimes greater than 10 ps are unlikely to be important in native DNA on account of its even greater sequence disorder.11 In fact, long emission components on the nanosecond time scale have been observed in sequence-disordered natural DNAs.12-14 This discrepancy may be due in part to differences in fluorescence detection sensitivity,1 but there is lingering uncertainty over the nature and yields of long-lived excited states in mixed-sequence DNA. In order to understand the effects of base sequence disorder on the long-lived excited states that are detected in transient absorption experiments, we undertook an investigation of an 11-mer DNA oligonucleotide with a genomic base sequence. Experiments were performed on the ras61 oligodeoxynucleotide, a structurally well-characterized sequence that contains codons 60-62 of the human n-ras proto-oncogene.15 It is composed of a purine-rich d(CGGACAAGAAG) strand (hereafter referred to as strand A) and its complementary pyrimidine-rich strand with the sequence d(CTTCTTGTCCG) Received Date: October 30, 2010 Accepted Date: December 21, 2010 Published on Web Date: January 07, 2011

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DOI: 10.1021/jz101470m |J. Phys. Chem. Lett. 2011, 2, 133–138

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Figure 1. (a) Melting temperature curves for strand A (triangles), strand B (squares), and the A 3 B duplex (circles). (b) CD spectra of single-stranded and duplex genomic DNA. The signal for the duplex shows characteristic bands for B-form DNA.

Figure 2. Back-to-back transient absorption signals (pump 266 nm) at the indicated probe wavelengths for single-stranded and duplex genomic DNA oligonucleotides in aqueous solution at room temperature. The 570 nm transients have been corrected for absorption by solvated electrons generated by two-photon ionization of water. Solid curves are from fits described in Table 1.

(strand B). Point mutations at the 61 codon (CAA) can lead to oncogene activation.16 The A and B strand sequences are significantly disordered. Strand Acontains seven (AA, AG, AC, GA, GG, CA, CG) of the 16 possible base doublets found in single-stranded nucleic acids. Strand B has an additional seven base doublets (GT, CG, CC, CT, TG, TC, TT) that complement those in the A strand. Only the AT and TA doublets are not represented. In contrast, single-stranded DNA composed of just two different bases contains at most four unique doublets. Double-stranded DNA built from Watson-Crick base pairs contains 10 unique nearest-neighbor base pairs or base pair doublets. The A 3 B duplex formed by thermally annealing equimolar concentrations of the A and B strands contains seven of these and has significantly more sequence disorder than the defined-sequence oligonucleotides studied to date. Strands A and B both exhibit broad melting curves, indicating that these non-self-complementary sequences are single-stranded in solution (Figure 1). On the other hand, the CD spectrum of a solution containing equal strand concentrations of A and B exhibits the characteristic B-type DNA bands, consistent with the double-stranded structure determined previously in a combined 1H NMR and constrained molecular dynamics simulation study.15 Figure 2 shows transient absorption signals recorded for strand A, strand B, and the A 3 B duplex at an excitation wavelength of 266 nm and probe wavelengths of 250 and 570 nm. The former probe wavelength monitors ground-state repopulation dynamics, while the latter probe wavelength follows excited-state relaxation.1 Even before any fitting analysis, it is clear that the signals decay in a multiexponential fashion with long-lived excited states in all three systems that persist to 100 ps and beyond. The long-lived states are particularly visible in the A strand. The long-lived signal contributions are prominent between 20 and 1000 ps after the pump pulse. Approximate mirror


symmetry is observed for most of this time window, indicating that loss of excited-state absorption at 570 nm is accompanied by an increase in absorption by ground-state molecules at 250 nm. This suggests that the dynamics seen at both wavelengths reflects the time needed for excited states to decay to the electronic ground state and not to a long-lived trap state like a triplet or 1nπ* state. At the longest delay times, a nonzero ΔA signal is still observed at both probe wavelengths. The offset, which is somewhat larger in strand B than that in either the A strand or the A 3 B duplex, is largely due to pyrimidine dimer formation, which is possible at a large number of sites in the pyrimidine-rich B strand but is impossible in the A strand. UV/vis absorption spectra recorded before and after multiple scans are shown in Figure 3. Because strand B showed significant loss of UV absorbance as expected for pyrimidine dimer formation, only the first five scans were averaged to yield the transient signals shown in Figure 2. Later scans show reduced amplitude compared to earlier ones, but the kinetics are unchanged. Figure 3 shows that the photodegradation is strongly inhibited when the B strand is paired with the A 3 B duplex, as expected for pyrimidine dimer formation. We discuss the complex dynamics seen in these experiments with two aims in mind. First, we seek to understand how excited states and decay pathways in the single strands are affected by sequence disorder. Second, we ask how excited-state dynamics is altered by base pairing in the duplex structure. It must be kept in mind that separating base pairing from base-stacking effects is challenging because the base pairing introduced upon duplex formation leads to profound structural changes. Thus, when the B strand is hybridized with the A strand, a fully base-paired B-form double helix is formed, changing the structure of the A and B strands. The A strand in

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Table 1. Best-Fit Parameters for the 266 nm Pump/250 nm Probe Signals Shown in Figure 2 a τ0 (ps), (A0)

sample

τ1 (ps), (A1)

τ2 (ps), (A2)

τ3 (ps), (A3)

A¥

strand A CGGACAAGAAG 0.43 ( 0.15, (0.62 ( 0.14) 3.4 ( 0.3, (-1.47 ( 0.08) 64 ( 30, (-0.45 ( 0.20) 300 ( 100, (-0.51 ( 0.21) -0.13 ( 0.02 strand B CTTCTTGTCCG

0.43 ( 0.15, (0.43 ( 0.11) 3.4 ( 0.3, (-0.64 ( 0.08) 27 ( 6, (-0.28 ( 0.04)

-0.06 ( 0.01

0.43 ( 0.15, (0.59 ( 0.12) 3.4 ( 0.3, (-1.32 ( 0.10) 27 ( 6, (-0.49 ( 0.07) 440 ( 260 (-0.16 ( 0.03) -0.09 ( 0.02 P3 a The fitting function i=0 Ai exp(-t/τi) þ A¥ was analytically convoluted with a Gaussian function (FWHM = 300 fs) to model the instrument response function. Identical values in the same column were linked during global fitting. All uncertainties are twice the standard error.

A 3 B duplex

decay by p percent from its time-zero value.19 The τ75 values for the A, B, and A 3 B transients at 250 nm are 114, 26, and 19 ps (all values (3 ps), respectively, supporting the conclusion that a greater fraction of excited states relax more slowly in the A strand than those in the others. Although the B strand exhibits a relatively long-lived decay component (τ2 = 27 ( 6 ps), not unlike those assigned to 1 nπ* states in pyrimidine mononucleotides,20 the fact that the same component is seen at 570 nm, a wavelength where the 1nπ* states show no absorption,20 indicates that it must have a different origin. We propose that the different lifetimes seen in the A versus B strands reflect the different lifetimes associated with charge-transfer-like excited states formed in the base doublets present in each. Of course, individual decay constants need not correspond to individual base doublets but could represent an average of similar lifetimes. AA and AG dinucleosides have particularly longlived excited states on the order of 100 ps,4 while shorter lifetimes are seen in systems with TT5 and CC doublets.19 Longer lifetimes are observed in base doublets thought to have higher-energy charge-transfer states.4 The presence of long-lifetime components in the A strand and in the A 3 B duplex suggests that energy funneling or transfer from highenergy exciplex states to lower-lying ones may not occur at significant rates. Of course, this could depend strongly on the presence of base stacking (and therefore the coupling) between two different base doublets. The τ1 decay of 3.4 ( 0.3 ps has the largest amplitude of all decay components in all three systems, reflecting the pronounced bleach recovery observed at 250 nm during the first 10 ps. During the same interval, the 570 nm transients decay somewhat more rapidly. For several hundred femtoseconds after t=0, the bleach signals at 250 nm actually increase slightly in absolute value (Figure 2, inset). For this reason, fits to all three of the 250 nm transients require a subpicosecond decay constant (τ0 = 0.43 ( 0.15 ps) with a positive amplitude. Similar dynamics were observed previously at the same probe wavelength for 9-methyladenine in H2O solution and assigned to hot ground-state absorption by a high-energy absorption band after ultrafast relaxation to the electronic ground state.21 The observed short-time dynamics at 250 nm thus strongly suggest that there is a significant amount of population relaxation to S0 in less than 1 ps. Assuming that the signals at 250 nm are dominated by ground-state absorption, then the rapid decrease of the τ1 component suggests that more than half of all excited states decay on an ultrafast time scale in all three systems. Previously, we presented evidence that unstacked bases present in base multimers contribute to this ultrafast relaxation and

Figure 3. Absorption spectra taken before the transient absorption measurements (solid), after 5 scans (- - -), and after 10 scans ( 3 3 3 ). Each scan represents approximately 8 min of irradiation by the 1 mW pump beam at 266 nm.

the duplex likely has longer helical regions then it does as a single strand. Even greater changes are expected for the pyrimidine-rich strand B, which, as a single strand, is likely to form a random-coil structure with few helical base stacks as in the case of (dT)n.1 We sought additional insight from least-squares fitting of the transients. Fitting multiple exponentials can be a numerically ill-posed problem when decay constants are similar in magnitude, and it can be difficult to determine the appropriate number of decay components.17 Often, it is possible to reduce the χ2 of a least-squares fit by including more parameters, but this may have little physical significance. An elegant and well-tested strategy to avoid overfitting is to select the model that minimizes the corrected Akaike information criterion (AICc).18 Using this strategy, which we will describe in more detail in a later publication, we globally fit the 250 nm transients to sums of up to four exponentials with time constants linked in various ways across the set of three transients. The parameters from the model with the minimum AICc value are given in Table 1. The conclusion that four exponentials are needed to fit the A and A 3 B transients while only three are needed to fit the B transient is robust. Furthermore, the shortest time constants, τ0 and τ1, are well-determined, but the values of the longer time constants, τ2 and especially τ3, are subject to greater uncertainty, as judged from the fact that quite different values were obtained from alternative models with AICc values that were nearly as small as the lowest observed value. A method for quantifying decay rates that is modelindependent and therefore avoids the pitfalls of multiexponential fitting is to determine the time τp required for the signal to


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suggested that most excitations in base stacks decay more slowly.4 However, the single-stranded systems studied in ref 3 have significant numbers of both stacked and unstacked bases, making it difficult to determine whether ultrafast nonradiative decay occurs only when bases are unstacked. Only a minor fraction of bases in the A 3 B duplex studied here are expected to be unstacked. Terminal base pairs are much more likely to be open (“end fraying”) than interior ones, and the terminal GC base pairs in the A 3 B duplex will be open much less often than terminal AT base pairs.22,23 If 10% of terminal GC base pairs are open at 25 C,23 then fewer than 2% of all bases should be unstacked, assuming full stacking of interior bases. In fact, an excess of one strand is estimated to be a more important source of unstacked bases. If one strand is present in 10% excess, then up to 5% of all bases could be unstacked, assuming that there is no residual stacking in an unassociated single strand. This upper limit of 5% unstacked bases in the A 3 B duplex cannot account for the fact that nearly half of all excitations decay on an ultrafast time scale, suggesting that ultrafast internal conversion to the electronic ground state is still significant for this well-stacked system. The authors of a computational study have suggested that subpicosecond, monomer-like channels can operate in basestacked assemblies.24 More effort is needed to understand the possibility that base stacking may be a necessary but not a sufficient condition for long-lived excited states in DNA. The transient absorption signals shown in Figure 2 were measured in back-to-back experiments on equal absorbance solutions. Experimental conditions were maintained as constant as possible between scans such that the same number of initial excited states was formed in each of the samples. It is therefore interesting to compare changes in amplitudes associated with the various lifetimes. Although it is difficult to partition excitation probability in a multichromophoric assembly like duplex DNA, we neglect base-pairing interactions and assume that the probability of producing an excitation on the A versus the B strand is given by the extinction coefficients of the separate strands. According to formulas published by Tataurov et al.,25 strands A and B have nearly equal extinction coefficients at the pump wavelength of 266 nm; therefore, we compare the A 3 B signal with the average A and B strand signals in Figure 4. The weighted sum of the separate signals crudely reproduces the duplex signal. The overall agreement suggests that any quenching introduced by base pairing is a limited effect and that excited-state decay is determined more by intra- than by interstrand effects, as discussed earlier for more regular sequences made of A and T.5 By subtracting the average of the A and B strand signals from the A 3 B duplex signal (black curve in upper panel of Figure 4), differences do emerge, however. The duplex signal has larger absolute value near time zero than the average signal, but the opposite is true between 10 and 200 ps. The net result is that the amplitude of the 3.1 ps decay (A1 in Table 1) is about 30% greater than expected by averaging the signals from the separate strands. This is significant because there is even greater base stacking in the duplex than in the individual strands as discussed above. Increased stacking by B strand bases could explain the increased amplitude of the τ2 decay time in the A 3 B duplex compared to what is observed in the


Figure 4. Top panel: Difference of the A 3 B duplex signal and the weighted average of signals from strands A and B. Bottom panel: Comparison of signal from the A 3 B duplex with the average of strand A and strand B signals.

isolated B strand. Figure 4 and the parameters in Table 1 show that the longest decay component (τ3), while still seen in the duplex, is less prominent than that in the A strand, even after accounting for competitive absorption by the B strand. Further experiments on a richer set of sequences are needed to clarify the origin of this quenching in this and in similarly complex oligonucleotides. In summary, excited electronic states with lifetimes significantly longer than those of mononucleotides are present in significant yields in a single-stranded, mixed-sequence DNA oligonucleotide and in its complementary sequence despite substantial base sequence disorder. In the duplex formed from these strands, ultrafast internal conversion accounts for a somewhat larger fraction of all decay events, but long-lived excited states are still observed. To the best of our knowledge, this is the first demonstration of an ultrafast nonradiative decay pathway in a DNA model system in which all bases are well-stacked with neighbors. Emission by bright states seen in fluorescence upconversion measurements may not be particularly long-lived in mixed-sequence oligonucleotides,11 but experiments that use sensitive timecorrelated photon counting technique detection have revealed long-lived emission in synthetic26 and natural DNA13 that extends out to nanosecond time scales. It will be important to search for possible connections between these long-lived emissive states and the long-lived excited states that have been shown by this transient absorption study to be formed efficiently in mixed-sequence DNA.

EXPERIMENTAL SECTION Femtosecond pump and probe pulses for transient absorption measurements were derived from the output of an amplified Ti:Sapphire laser system (Mantis/Legend, Coherent, Inc., Santa Clara, CA). The pump pulses at 266 nm were obtained by third-harmonic generation. Probe pulses at 250 nm were obtained from an optical parametric amplifier (OPerA, Coherent, Inc.), and pulses at 570 nm were produced by white light continuum generation in a 1-cm path length water-filled cell. At the sample, the polarization of the pump pulses was set at the magic angle relative to the probe

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pulses. The sample was held between two calcium fluoride windows with a 1.2-mm spacer in a home-built spin cell that was spun at several hundred rotations per minute to avoid reexcitation of the sample by successive pump pulses. After the sample, the probe beam was spectrally isolated using a double-grating monochromator and detected with a PMT. Transient absorption signals were recorded at 21 C. Signals recorded at 570 nm were corrected for two-photon ionization of the solvent using a procedure described previously.3 Transient absorption signals were globally fit to a sum of exponentials convoluted with a Gaussian instrument response using IGOR Pro 5.04 software. The ammonium-salt forms of the oligodeoxynucleotides 50 -d(CGGACAAGAAG)-30 and 50 -d(CTTCTTGTCCG)-30 were obtained as lyophilized powders from Midland Certified Reagent Company (Midland, TX). Samples were dissolved in 0.1 M NaCl, 0.025 M Na2HPO4/KH2PO4 buffer (pH 6.9) and thermally annealed at 70 C. Duplexes were formed by mixing equimolar solutions of the corresponding single strands, followed by annealing. Extinction coefficients from ref 25 were used to determine strand concentrations. Solution concentrations were adjusted to obtain an absorbance of 1.0 at the pump wavelength of 266 nm. CD spectra were collected using an Aviv (Pistacaway, NJ) model 202 circular dichroism spectropolarimeter. CD spectra were recorded every 10 C from 20 to 80 C following a 5-min equilibration period. The path length was 1 cm, and solutions had a concentration of 0.1 mM per nucleotide. To generate melting curves, CD dynode signals were converted to absorption spectra using a standard routine in the Aviv software. The converted absorption values at 260 nm were plotted as a function of temperature and normalized.

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AUTHOR INFORMATION Corresponding Author: (16)

*To whom correspondence should be addressed. E-mail: kohler@ chemistry.montana.edu.

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ACKNOWLEDGMENT This research was made possible by a grant

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REFERENCES

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from the National Science Foundation (CHE-1005447).

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Observation of Long-Lived Excited States in DNA Oligonucleotides

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