Femtosecond Two-Dimensional Infrared Spectroscopy of Adenine

Jan 10, 2011 - modes of adenine and to determine their individual line shapes. The spectra demonstrate ..... Under the broadband excitation conditions...
3 downloads 0 Views 2MB Size
Download PDF
ARTICLE pubs.acs.org/JPCB

Femtosecond Two-Dimensional Infrared Spectroscopy of Adenine-Thymine Base Pairs in DNA Oligomers Ming Yang, yukasz Szyc, and Thomas Elsaesser* Max-Born-Institut f€ur Nichtlineare Optik und Kurzzeitspektroskopie, Max-Born-Strasse 2 A, D-12489 Berlin, Germany ABSTRACT: NH and NH2 stretching excitations of adeninethymine base pairs in double-stranded DNA oligomers are studied by femtosecond two-dimensional (2D) infrared spectrosopy. The 2D spectra taken for population times of up to T = 1 ps allow for separating the NH stretching mode of thymine from the symmetric and asymmetric NH2 stretching modes of adenine and to determine their individual line shapes. The spectra demonstrate an essentially homogeneous broadening of the NH stretching band of thymine whereas the NH2 stretching modes display a pronounced, time-independent inhomogeneous broadening, pointing to disorder in the DNA structure. We observe a (downhill) vibrational energy transfer from the asymmetric NH2 stretching vibration of adenine at 3350 cm-1 to the NH stretching mode of thymine at 3200 cm-1 on a ∼500 fs time scale whereas the inverse (uphill) transfer is negligible.

1. INTRODUCTION Hydrogen-bonded base pairs are a key structural motif of deoxyribonucleic acid (DNA).1,2 Apart from their defining role for DNA double-helix structures in thermal equilibrium, their electronic and vibrational couplings are essential for the dynamics of elementary DNA excitations as well as biochemical processes. Steady-state vibrational spectroscopy has been widely applied to probe both local binding geometries and delocalized excitations in isolated base pairs and single- and double-stranded DNA oligomers.3-7 In general, the linear infrared and Raman spectra of DNA are highly complex with a high number of closely spaced and overlapping bands, in particular in the fingerprint range between approximately 1200 and 1800 cm-1. Empirical correlations between vibrational bands and the structure of DNA oligomers have been derived from such spectra and been supported by theoretical calculations, without, however, giving reliable information on microscopic molecular couplings and their role for dynamical properties of DNA. Nonlinear infrared spectroscopy with a femtosecond time resolution allows for mapping vibrational excitations on the intrinsic time scale of molecular motions and vibrational relaxation processes and for separating different modes via their nonlinear spectral features and/or their individual dynamics.8,9 Both pump-probe methods and photon-echo techniques have been applied to get insight into structure and dynamics of hydrogen bonds. In particular, two-dimensional (2D) infrared spectra have revealed vibrational couplings and dynamics of water and aqueous solutions in substantial detail.10 2D spectrocopy of base pairs and DNA oligomers has, however, remained limited. Early 2D work by Zanni and co-workers has addressed the coupling of carbonyl, CN stretching, and CC ring modes in GC pairs and oligomers containing GC pairs.11,12 The results suggest a predominant electrostatic interaction between bases with coupling strengths of the order of 10 cm-1 which have been r 2011 American Chemical Society

modeled with a transition dipole density approach. More recently, double-stranded DNA oligomers containing 23 alternating A-T base pairs in Watson-Crick geometry have been studied in a wide range of hydration levels by femtosecond pump-probe spectroscopy.13-16 Different NH and NH2 stretching excitations of adenine and thymine have been separated and distinguished from the water OH stretching band. Polarization resolved pump-probe measurements have given first information on couplings between the different NH oscillators. Another series of pump-probe studies has provided direct insight into the interaction of the phosphate groups in the DNA backbone with the surrounding water shell and established the important role of the water shell as a heat sink for vibrational excess energy.16 Femtosecond 2D infrared spectroscopy has the potential to provide more specific insight into vibrational interactions, line broadening mechanisms, and the underlying molecular coupling mechanisms. In this article, we report the first 2D infrared correlation spectra of DNA oligomers in the spectral range of the NH stretching excitations of adenine-thymine base pairs. We focus on samples of low water content to isolate the intra-DNA coupling behavior from interactions with surrounding water molecules. The different NH stretching modes are separated and their line shapes are determined. We observe anharmonic couplings between the NH oscillators on the adenine and the thymine moieties and a subpicosecond energy transfer between the modes.

2. EXPERIMENTAL TECHNIQUES We study artificial double-stranded DNA oligomers containing 23 alternating adenine-thymine (A-T) base pairs (Figure 1a) Received: September 22, 2010 Revised: December 13, 2010 Published: January 10, 2011 1262

dx.doi.org/10.1021/jp1090697 | J. Phys. Chem. B 2011, 115, 1262–1267

The Journal of Physical Chemistry B

Figure 1. (a) Sequence of alternating adenine-thymine (A-T) base pairs in the double-stranded DNA oligomers. (b) Linear infrared absorption spectrum of a DNA thin film sample at 0% RH (solid line). The spectrum is dominated by the CH stretch components below and around 3000 cm-1 and the NH and NH2 stretching absorption between 3000 and 3700 cm-1. Dash-dotted line: Spectrum of the femtosecond pulses. Inset: Molecular structure of the A-T base pair in Watson-Crick geometry.

in Watson-Crick geometry (inset of Figure 1b, supplier Thermo Scientific, HPLC, desalted). To generate thin films of high optical quality, the sodium counterions of the oligomers were replaced by cetylmethylammonium chloride (CTMA), a surfactant forming complexes with DNA. The ∼30 μm thick films were made by the procedure described in detail in refs17 and 18 and were cast onto 1 mm thick CaF2 substrates. The DNA concentration in the films was approximately 1.5  10-2 M. The film samples were integrated into a homebuilt humidity cell which was connected to a reservoir. By putting various agents into the reservoir, we were able to control the relative humidity (RH) in the cell and the film sample.13,19 In the following, we concentrate on measurements performed at 0% RH (agent P2O5), corresponding to a water concentration of 2 water molecules per base pair. Such water molecules are predominantly located close to the phosphate groups in the backbone of the DNA oligomers. The 2D spectra are derived from heterodyne detected 3-pulse photon echoes.20-26 A parametric infrared source driven by a regeneratively amplified Ti:sapphire laser system (repetition rate 1 kHz) provides pulses of up to 7 μJ energy tunable from 2800 to 4000 cm-1 (spectral pulse width >250 cm-1). The pulse duration was 50 ( 5 fs. Two phase-locked pulse pairs are generated with diffractive optics.24 They serve for generating photon echo signals as a function of the coherence time τ, the delay between pulse 1 and 2, and the population time T, the delay between pulse 2 and 3 interacting with the DNA sample. The resulting photon echo signal is heterodyned with pulse 4, the local oscillator, and detected by spectral interferometry. Details of the photon echo setup and the procedures applied for data analysis have been presented in refs 25 and 26. In the following, we present 2D spectra measured with pulses that were centered at 3250 cm-1 (Figure 1b). Two different polarization schemes were applied, parallel linear polarizations of all 4 pulses (||||) as well as parallel polarizations for pulses 1 and 2 and perpendicular polarizations for pulses 3 and 4 (||^^). The fraction of excited NH stretching oscillators was less than 1%.

3. RESULTS The infrared absorption spectrum of the DNA films between 2700 and 3800 cm-1 is shown in Figure 1b. The strong bands

ARTICLE

between 2800 and 3000 cm-1 are due to the different CH stretching modes of the DNA oligomers and the CTMA counterions. The weaker, spectrally broad band between 3000 and 3700 cm-1 represents the superposition of the NH and NH2 stretching bands of the A-T base pairs and the OH stretching band of the residual water molecules. The linear absorption spectrum does not allow for a clear separation of the different contributions whereas 2D spectra give specific insight into the lineshapes and couplings of the different bands. Absorptive 2D correlation spectra are shown in Figure 2. In Figure 2a, the sum of the rephasing and nonrephasing 2D signal23 for a population time T = 25 fs is plotted as a function of the excitation frequency ν1 (ordinate) and the detection frequency ν3 (abscissa) for the (||||) polarization configuration. In Figure 2b, spectra measured with the (||^^) configuration are presented for the population times indicated. The spectrum in Figure 2a exhibits strong positive peaks P1 and P2 on the ν1 = ν3 diagonal which are due to the v = 0 to 1 transitions of the different oscillators. Such components are complemented by (positive) cross peaks P3 around (ν1,ν3) = (3350,3200) cm-1 and P4 around (3200,3350) cm-1. The spectrally broad negative signal found at low detection frequencies ν3 is red-shifted compared to the diagonal peaks and mainly due to the v = 1 to 2 transitions of the oscillators. The different components of the 2D spectrum display a substantial spectral overlap. Nevertheless, different line shapes of the peaks are distinguished clearly. The diagonal signal below ν1 = ν3 = 3300 cm-1 (P1) consists of a component elongated along the diagonal which is superimposed by a strong peak close to ν1 = ν3 = 3200 cm-1 that is tilted toward the ν1 axis. Above ν1 = ν3 = 3300 cm-1 (P2), one observes an elliptic shape elongated along the diagonal. 2D spectra recorded at longer population times T of up to 1 ps (not shown) display a decay of the peak amplitudes but minor changes of the individual line shapes. To characterize the different components in more detail, we determined the center lines (CL) for the different peaks (thick solid lines in Figure 2a). The CL are derived from slices through the 2D spectra along the ν3 axis taken at a fixed ν1. The CL connect the frequency positions (ν1,ν3) at which the respective slice displays a local maximum. It has been shown for weakly anharmonic systems that the slope of the CL as a function of population T is proportional to the frequency fluctuation correlation function.27 In Figure 2a, the CL of the diagonal peaks reveal the diagonal and tilted orientations of the two components below ν1 = ν3 = 3300 cm-1 (P1) and the diagonal elongation of P2. The CL of the cross peak P3 is located at a fixed ν3 ≈ 3200 cm-1 and parallel to the ν1 axis. P4 does not display a local maximum but rather a broad shoulder for which a CL cannot be extracted. All CL remain unchanged up to our longest population times T of 1 ps. The 2D spectra measured in the (||^^) configuration allow for a better separation of the partly overlapping contributions because of the smaller relative amplitude of the diagonal components. In Figure 2b, the two cross peaks are clearly distinguished from the diagonal signals. The cross peak P3 at (3350,3200) cm-1 is elongated along the ν1 axis while the cross peak P4 at (3200,3350) cm-1 has a shape close to circular. The frequency positions and the CL (not shown) of the different peaks agree well with the spectrum shown in Figure 2a. In Figure 3, we summarize cross sections of (||^^) 2D spectra for population times between 0 and 700 fs. Cuts along the diagonal (Figure 3a) consist of broad components elongated along ν1 = ν3 and the 1263

dx.doi.org/10.1021/jp1090697 |J. Phys. Chem. B 2011, 115, 1262–1267

The Journal of Physical Chemistry B

ARTICLE

Figure 2. Two-dimensional vibrational spectra of DNA oligomers at 0% RH for (a) parallel linear polarizations of the 4 pulses (||||), and (b) perpendicular polarization of pulses 3 and 4 (||^^). The absorptive 2D signal is plotted as a function of the excitation frequency ν1 and the detection frequency ν3 for different population times T. The positive signals (yellow-red part) are due to the v = 0 to 1 transitions of the different NH stretching oscillators, and the negative signals (green-blue part) to the v = 1 to 2 transitions. Each spectrum is normalized to the respective maximum positive signal and contour lines correspond to 10% changes in amplitude. The thick solid lines in (a) represent the center lines (CL) of the different peaks.

Figure 3. Cross sections through the 2D spectra recorded at different population times T with the (||^^) polarization scheme. (a) Cross sections along the diagonal ν1 = ν3 (solid lines in Figure 2b). (b) Cross sections along an axis enclosing an angle of 63.4 with the detection axis (dashed line in Figure 2b). (c) Cross sections along the detection axis for an excitation frequency νex = 3345 cm-1 (dash-dotted line in Figure 2b).

additional narrower peak close to 3200 cm-1. As shown in Figure 3b, the latter peak is well pronounced in the cross sections along the dashed line in Figure 2b (upper right panel). The cross sections for a fixed excitation frequency ν1 = 3345 cm-1 (Figure 3c, cf. dash-dotted line in Figure 2b) display the negative signal due to the v = 1 to 2 transitions at small ν3, the positive cross peak P3 at ν3 = 3200 cm-1 and the upper diagonal peak P2

Figure 4. Normalized spectrally integrated intensity of the (a) diagonal peaks P1 (ν3 = 3200 cm-1) and P2 (ν3 = 3350 cm-1) and (b) the cross peaks P3 (ν3 = 3200 cm-1) and P4 (ν3 = 3350 cm-1) as a function of population T. The intensities were extracted from the 2D spectra recorded with the (||^^) polarization scheme. (c) Intensity ratio P3/ P2 as a function of population time T. The rise of the relative intensity of P3 for T g 75 fs gives evidence of vibrational energy transfer (solid line is guide to the eye).

at ν3 = 3350 cm-1. The negative signal and the positive cross peak show a different time evolution. With increasing population time T, the strength of the cross peak P3 grows substantially relative to that of the diagonal peaks P1 and P2. In Figure 4a,b, we plot the spectrally integrated intensities of the diagonal and cross peaks as a function of population time T. The two diagonal components P1 and P2 show a decay within the first 500 fs, followed by a slower decrease (not shown). This behavior is close to the kinetics of vibrational populations observed in femto1264

dx.doi.org/10.1021/jp1090697 |J. Phys. Chem. B 2011, 115, 1262–1267

The Journal of Physical Chemistry B second pump-probe experiments and due to the decay of the v = 1 states and the repopulation of the v = 0 states of the oscillators.13,14 The kinetics of the two cross peaks P3 and P4 differ substantially (Figure 4b). P3 shows a delayed rise and a partial decay by a factor of 2 within the first 700 fs, slower than any of the other peaks. The intensity ratio of P3 relative to the diagonal peak P2 increases strongly with T (Figure 4c). In an independent series of experiments, we measured 2D spectra of DNA oligomers containing 23 nonalternating A-T pairs, i.e., vertical “stacks” of adenine and thymine moieties linked to the respective backbone strand. Within the experimental accuracy, such 2D spectra agree with the results presented in Figure 2.

4. DISCUSSION We first discuss the vibrational assignments of the different diagonal peaks. In the A-T base pairs (inset of Figure 1b), two NH stretching oscillators are located on the adenine NH2 group and one on the thymine NH group. For isolated adenine molecules in the gas phase, the NH2 vibrations have been classified in terms of a symmetric and an asymmetric NH2 stretching mode with a measured frequency splitting ΔνNH = νAS - νS ≈ 3569 3451 ≈ 120 cm-1 (refs 28 and 29). This splitting corresponds to a coupling of ΔνNH/2 = 60 cm-1 between the two local N-H oscillators, each having a frequency of approximately ν0 = 3510 cm-1. In between the two NH2 bands, the stretching band of the single N9H group of free adenine is located at 3509 cm-1. Upon formation of the A-T pair, the hydrogen bond to the thymine CO group introduces a symmetry distortion compared to a free NH2 group. As a result, it is a priori unclear if the NH2 modes are described appropriately by symmetric and asymmetric NH2 modes or by individual stretching modes of the two local oscillators, one hydrogen bonded and the other free. Gas-phase spectra of isolated A-T pairs28 display two stretching bands of the adenine NH2 group at 3326 and 3530 cm-1 (ΔνNH ≈ 200 cm-1) and the stretching band of the free N9H group at 3507 cm-1. This spectral pattern is very similar to that of isolated adenine and the stretching frequency of 3530 cm-1 is higher than ν0, the frequency an uncoupled local NH oscillator would display. Moreover, the spectral downshifts of the NH bands caused by hydrogen bonding in the A-T pairs are of the same order of magnitude as ΔνNH and theoretical calculations suggest couplings between the adenine and thymine NH stretching oscillators much smaller than ΔνNH/2.30 We conclude that a description of adenine NH2 vibrations in terms of symmetric and asymmetric stretching modes represents a reasonable approximation also in the base pair. Isolated thymine molecules in the gas phase display stretching frequencies of 3435 ( 2 cm-1 (N3H) and 3482 ( 2 cm-1 (N1H) group.28,31 Upon formation of hydrogen-bonded A-T pairs, the frequency of the hydrogen-bonded NH group shifts down to 3295 cm-1, a value close to the frequency of the symmetric NH2 stretching vibration of adenine in the base pair (3326 cm-1).28 In the 2D spectra, we assign-in agreement with the gas-phase spectra and our previous pump-probe experiments on the same DNA oligomers13,14-the upper diagonal peak P2 (at ν1 = ν3 = 3350 cm-1) to the asymmetric NH2 stretch. Around ν1 = ν3 = 3200 cm-1 (P1), the 2D spectra and cross sections display two different features, (i) a broad band elongated along the diagonal and (ii) a substantially narrower peak which is tilted toward the ν1 axis (cf. Figures 2 and 3a,b). The 2D spectra and the cuts along the diagonal (Figure 3a) suggest a similar spectral profile and

ARTICLE

width of the broad component (i) and the upper diagonal peak P2. We, thus, assign component (i) to the symmetric NH2 stretch. Based on the experimental gas phase spectra, one expects the thymine NH stretching frequency to lie in the same range as the symmetric NH2 stretch. We, therefore, assign component (ii) around 3200 cm-1 to the thymine NH stretching mode. For excitation pulses centered at 3250 cm-1 and the very low water concentration in the DNA sample at 0% RH, the OH stretching vibrations of the residual water molecules around 3500 cm-1 (ref 13) make a negligible contribution to the 2D spectra. The shape of the adenine NH2 stretching bands reflects a substantial inhomogeneous broadening which remains unchanged up to T ≈ 1 ps; i.e., there is no measurable spectral diffusion on the subpicosecond time scale. At the very low hydration level of 0% RH which corresponds to one water molecule per phosphate group in the DNA backbone, fluctuating forces and, thus, spectral diffusion originating from water motions play a minor role. The constant pump-probe anisotropy of water OH stretch excitations observed under such conditions points to a rather rigid attachment of water molecules to the phosphate groups and a minor role of hydrogen bonds between base pair groups and water.13 Instead, we assign the “quasi-static” inhomogeneity mainly to structural disorder in the DNA helix and the attached CTMA counterions which is mapped onto the vibrational frequencies of the NH2 groups with one free (nonhydrogen-bonded) NH unit. With spectral diffusion playing a minor role, one expects a correlated broadening of the symmetric and asymmetric NH2 stretching modes of a particular NH2 group. In contrast, the NH group of thymine is part of a welldefined hydrogen bond inside the base pair and, thus, should be less sensitive to structural inhomogeneity. This picture is in line with the more homogeneous line shape of the thymine NH stretching band. The 2D spectra clearly display two cross peaks, P3 at (3350,3200) cm-1 and P4 at (3200,3350) cm-1. The cross peaks indicate an anharmonic coupling between the asymmetric NH2 oscillators and the modes around 3200 cm-1. As a function of population time T, the intensities of P4 and P3 behave differently (Figure 4b): P4 decays on a similar time scale as the diagonal peaks P1 and P2 (Figure 4a) whereas P3 displays a delayed rise and a slower decay. In particular, the P3/P2 intensity ratio (Figure 4c) increases strongly as a function of T. In general, a cross peak caused by the off-diagonal anharmonic coupling of two oscillators should display a dispersive line shape.32 This line shape consists of a positive component at ν01, the frequency of the v = 0 to 1 transition, which is due to the depletion of the common ground state and a negative component at ν0 01 6¼ ν01, due to the shift of the v = 0 to 1 absorption when the other coupled oscillator is excited. The sign and the absolute value of the spectral shift are determined by the offdiagonal anharmonicity. To clearly detect such dispersive line shapes in a 2D experiment, the spectral widths of the original and the anharmonically shifted transition need to be smaller than the spectral shift, i.e., the off-diagonal anharmonic coupling. In the opposite limit of line shapes broad compared to the spectral shift, the compensation of the spectrally overlapping positive and negative components reduces the amplitude of the dispersive feature strongly. The 2D spectra in Figure 2 display diagonal and cross peaks of a spectral width (fwhm) between approximately 70 and 150 cm-1, resulting in an overlap of their spectral wings. In the range of the cross peak P4, one observes an overall positive signal 1265

dx.doi.org/10.1021/jp1090697 |J. Phys. Chem. B 2011, 115, 1262–1267

The Journal of Physical Chemistry B with a slight intensity modulation toward smaller detection frequencies. We attribute this behavior to the limited amplitude of the dispersive feature which is caused by the partial compensation of the spectrally broad positive and negative P4 components, and to a positive background originating from the very broad diagonal peaks. We conclude that the off-diagonal anharmonic coupling between the modes at 3200 and 3350 cm-1 is substantially smaller than the spectral width of P4 of 70 cm-1. A different behavior is found in the range of the upper cross peak P3. The cuts plotted in Figure 3c demonstrate that the positive P3 component is superimposed on the initially (T = 0 fs) predominant, spectrally broad negative contribution due to the v = 1 to 2 absorption which undergoes a rapid reshaping and partial decay during the temporal pulse overlap up to T ≈ 75 fs. Due to the spectral overlap with the v = 1 to 2 absorption, the positive P3 amplitude reaches its maximum only at T ≈ 100 fs, followed by a decay which is substantially slower than that of P1, P2, and P4. It is important to note that for T g 75 fs the positive P3 peak strongly dominates over the residual negative signal and that the P3 amplitude increases relative to that of the diagonal peak P2. Such features are characteristic for an energy transfer process from the asymmetric NH2 stretching oscillator at ν01 = 3350 cm-1 to the ν01 = 3200 cm-1 oscillators in which the upper oscillators are deactivated and the (unshifted) v = 0 to 1 transition of the lower oscillators excited. The energy difference between the two excitations is accepted by the vibrational manifold of DNA. This downhill energy transfer enhances the positive P3 component relative to P2 whereas a negative spectrally shifted P3 component decays in parallel to the deactivation of the upper oscillators. The energy transfer process results in an additional channel of the v = 1 population decay of the upper oscillators and enhances the v = 1 population of the lower oscillators. As a result, the P3 decay (Figure 4b) is slowed down compared to the P1 decay (Figure 4a) and the intensity ratio of the two positive peaks P3 and P2 increases as a function of population time T (Figure 4c). The inverse energy transfer, an uphill process from the modes at ν01 = 3200 cm-1 to the asymmetric NH2 stretching mode at ν01 = 3350 cm-1 is less efficient because of the energy mismatch. It would, however, enhance the intensity of the positive cross peak P4. Such behavior is absent in the 2D data; i.e., the rate of the uphill transfer is substantially smaller than the population decay rate of the asymmetric NH2 stretching oscillator. The time evolution of the intensity ratio P3/P2 for T < 75 fs (Figure 4c) is strongly affected by the spectrally overlapping, rapidly decaying v = 1 to 2 absorption (cf. Figure 3c). In contrast, the rise of the intensity ratio for T g 75 fs mainly reflects the kinetics of energy transfer occurring on a ∼500 fs time scale. Taking the latter as a measure for the incoherent energy transfer time, a standard Fermi golden rule approach gives an absolute value of the coupling strength between the two oscillators of the order of 5 cm.1. Couplings of the same order of magnitude between vibrational transition dipoles in base pairs have been found in ref 12 for fingerprint modes and in ref 14 for NH stretching modes. It should be noted that such couplings are much smaller than the spectral widths of the different peaks in the 2D spectra and, thus, cannot be derived by a line-shape analysis. In the DNA oligomers with alternating A-T pairs studied here, energy transfer by dipole-dipole coupling favors a transfer from the asymmetric NH2 stretching mode of adenine to the NH mode of thymine in the same base pair because of their comparably small separation of approximately 0.35 nm and the ∼30 angle between their transition dipoles, both being determined by the base pair geometry.2,28-31 The more or less vertical center

ARTICLE

line of the cross peak P3 (cf. Figure 2a) and the spectral width of P3 close to that of the thymine NH stretching component of the diagonal peak P1 supports this picture. A comment should be made on an alternative coupling scheme in which a predominant anharmonic interaction occurs between the symmetric and the asymmetric NH2 stretching modes. Under the broadband excitation conditions applied here and in our previous pump-probe experiments on the same DNA system,13-15 such a coupling would give rise to quantum beats33 with a frequency/period of approximately 150 cm-1/220 fs as determined by the frequency separation of the two modes. Such quantum beats are absent in the large set of spectrally resolved pump-probe data which were reported in refs 13-15 for hydration levels of 0% RH (present case) and 92% RH, and in the spectrally resolved pump-probe traces which we derived from the 2D spectra by integrating along the ν1 axis (not shown). Moreover, for a significant coupling between and/or energy transfer from the asymmetric to the symmetric NH2 stretching mode, the spectral envelope of P3 should display a pronounced inhomogeneous broadening and a width comparable to that of P2 and the broad component of P1, signatures which are absent in the 2D spectra. We, thus, conclude that the coupling between the two NH2 stretching modes is-if existing at all-substantially smaller than the coupling between the asymmetric NH2 stretch and the NH stretching mode of thymine. It should be noted that the 2D spectra of DNA oligomers with 23 stacked nonalterating A-T pairs display no major differences to the spectra presented here, although the interbase pair couplings are different in the two types of oligomers. This fact suggests that the strength of the cross peaks in the 2D spectra is mainly determined by intrabase pair coupling. A more detailed quantitative analysis of the 2D spectra requires in-depth calculations of vibrational transition frequencies and anharmonic couplings in the highly complex DNA structure. Beyond local interactions, the long-range Coulomb forces generated by the ionic phosphate groups in the DNA backbone and by the CTMA counterions need to be included for a realistic treatment. Such work is beyond the scope of this experimental study.

5. CONCLUSIONS In conclusion, we have separated and characterized the different NH stretching excitations of adenine-thymine base pairs in doublestranded DNA oligomers at low hydration level. Two-dimensional infrared correlation spectroscopy allows for an assignment of the different NH stretching bands which are highly congested in the linear infrared spectra, and for an analysis of their different line shapes. Spectral diffusion plays a minor role in the 2D spectra recorded up to population times of 1 ps. The time-independent inhomogeneous broadening of the NH2 stretching bands of adenine points to structural disorder in the DNA oligomers while the NH stretching band of thymine is close to homogeneously broadened. The cross peaks in the 2D spectra give evidence of anharmonic couplings between the NH oscillators and of vibrational energy transfer. The results presented here can serve as a benchmark for studies of fully hydrated DNA, addressing the interaction between DNA and its water shell. Such work is presently underway. ’ AUTHOR INFORMATION Corresponding Author

*Phone: þ49 30 63921400. Fax: þ49 30 63921409. E-mail: [email protected]. 1266

dx.doi.org/10.1021/jp1090697 |J. Phys. Chem. B 2011, 115, 1262–1267

The Journal of Physical Chemistry B

ARTICLE

’ ACKNOWLEDGMENT We thank Nils Huse, Berkeley, for his help in the early phase of our 2D study. Valuable discussions with Erik T. J. Nibbering and Henk Fidder are gratefully acknowledged. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 247051. ’ REFERENCES (1) Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 737. (2) Saenger, W. Principles of Nucleic Acid Structure; Springer: New York, 1984. (3) Dong, F.; Miller, R. E. Science 2002, 298, 1227. (4) Tsuboi, M. J. Am. Chem. Soc. 1957, 79, 1351. (5) Falk, M.; Hartman, K. A.; Lord, R. C. J. Am. Chem. Soc. 1963, 85, 387. (6) Prescott, B.; Steinmetz, W.; Thomas, G. J., Jr. Biopolymers 1984, 23, 235. (7) Letellier, R.; Ghomi, M.; Taillandier, E. J. Biomol. Struct. Dyn. 1986, 3, 671. (8) Fayer, M. D., Ed. Ultrafast infrared and Raman spectroscopy; Dekker: New York, 2001. (9) Nibbering, E. T. J.; Elsaesser, T. Chem. Rev. 2004, 104, 1887. (10) For a recent overview : Mukamel, S., Tanimura, Y., Hamm, P., Eds. Acc. Chem. Res. 2009, 42, 1207-1469; Issue 9: Special Issue on Coherent multidimensional optical spectroscopy. (11) Krummel, A. T.; Mukherjee, P.; Zanni, M. T. J. Phys. Chem. B 2003, 107, 9165. (12) Krummel, A. T.; Zanni, M. T. J. Phys. Chem. B 2006, 110, 13991. (13) Dwyer, J. R; Szyc, y.; Nibbering, E. T. J.; Elsaesser, T. J. Phys. Chem. B 2008, 112, 11194. (14) Szyc, y.; Dwyer, J. R.; Nibbering, E. T. J.; Elsaesser, T. Chem. Phys. 2009, 357, 36. (15) Szyc, y.; Yang, M.; Nibbering, E. T. J.; Elsaesser, T. Angew. Chem., Int. Ed. 2010, 49, 3598. (16) Szyc, y.; Yang, M.; Elsaesser, T. J. Phys. Chem. B 2010, 114, 7951. (17) Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679. (18) Yang, C.; Moses, D.; Heeger, A. J. Adv. Mater. 2003, 15, 1364. (19) Falk, M.; Hartman, K. A.; Lord, R. C. J. Am. Chem. Soc. 1962, 84, 3843. (20) Asplund, M. C.; Zanni, M. T.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8219. (21) Mukamel, S. Annu. Rev. Phys. Chem. 2000, 51, 691. (22) Jonas, D. M. Annu. Rev. Phys. Chem. 2003, 54, 425. (23) Khalil, M; Demird€oven, N.; Tokmakoff, A. J. Phys. Chem. A 2003, 107, 5258. (24) Cowan, M. L.; Ogilvie, J. P.; Miller, R. J. D. Chem. Phys. Lett. 2004, 386, 184. (25) Cowan, M. L.; Bruner, B. D.; Huse, N.; Dwyer, J. R.; Chugh, B.; Nibbering, E. T. J.; Elsaesser, T.; Miller, R. J. D. Nature 2005, 434, 199. (26) Kraemer, D.; Cowan, M. L.; Paarmann, A.; Huse, N.; Nibbering, E. T. J.; Elsaesser, T.; Miller, R. J. D. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 437. (27) Kwak, K.; Rosenfeld, D. E.; Fayer, M. D. J. Chem. Phys. 2008, 128, 204505. (28) Pluetzer, C.; Huenig, I.; Kleinermanns, K.; Nir, E.; de Vries, M. S. ChemPhysChem 2003, 107, 9165. (29) Choi, M. Y.; Dong, F.; Han, S. W.; Miller, R. E. J. Phys. Chem. A 2008, 112, 7185. (30) Wang, G. X.; Ma, X. Y.; Wang, J. P. Chin. J. Chem. Phys. 2009, 22, 563. (31) Colarusso, P.; Zhang, K. Q.; Guo, B.; Bernath, P. F. Chem. Phys. Lett. 1997, 269, 39. (32) Woutersen, S.; Hamm, P. J. Phys.: Condens. Matter 2002, 14, R1035. (33) Khalil, M.; Demird€oven, N.; Tokmakoff, A. J. Chem. Phys. 2004, 121, 362.

1267

dx.doi.org/10.1021/jp1090697 |J. Phys. Chem. B 2011, 115, 1262–1267