Ultrafast Two-Dimensional Infrared Spectroscopy of Guanine

Article pubs.acs.org/JPCB

Ultrafast Two-Dimensional Infrared Spectroscopy of Guanine− Cytosine Base Pairs in DNA Oligomers Christian Greve and Thomas Elsaesser*

J. Phys. Chem. B 2013.117:14009-14017. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 11/08/18. For personal use only.

Max-Born-Institut für Nichtlineare Optik und Kurzzeitspektroskopie, D-12489 Berlin, Germany ABSTRACT: NH and OH stretching excitations of hydrated double-stranded DNA oligomers containing guanine−cytosine (GC) base pairs in a Watson−Crick geometry are studied by two-dimensional (2D) infrared spectroscopy. The 2D spectra measured at a low hydration level (∼4 water molecules/base pair) are dominated by NH stretch contributions from the NH2 groups of G and C and the NH group of G. Partially hydrated NH 2 groups display red-shifted NH stretch frequencies and a mixing of the wave functions of the two local NH oscillators via the mechanical vibrational coupling. The NH stretch lifetimes are of the order of 200−300 fs. Weak couplings exist between NH stretch oscillators within a base pair, while interactions between neighboring GC pairs in the double helix are negligible. The absence of spectral diffusion on a 1 ps time scale suggests a relatively rigid structure of the hydrogen bonds between DNA and residual water molecules. 2D spectra recorded with fully hydrated DNA oligomers exhibit NH and OH stretch contributions with a weak influence of water fluctuations on the NH stretch lineshapes. The femtosecond spectral diffusion of OH stretch excitations is slower than that in bulk H2O and originates from structural fluctuations of the water shell and the formation of a vibrationally hot ground state by vibrational relaxation. We compare our findings with measurements on hydrated adenine−thymine DNA oligomers and anhydrous GC base pairs in solution.

1. INTRODUCTION The pairing of nucleic bases by intermolecular hydrogen bonds is a key structural feature of the double-helical structure of deoxyribonucleic acid (DNA).1,2 In the so-called Watson− Crick geometry, guanine (G) and cytosine (C) form an essentially planar pair geometry with one NH···N and two NH···O hydrogen bonds (Figure 1a). The overall binding energy of GC base pairs of ΔHGC ≈ −88 kJ/mol or 0.91 eV/ pair is substantially higher than the binding energy ΔHAT ≈ −50 kJ/mol of adenine−thymine (AT) base pairs which contain two hydrogen bonds only.3 The lengths and strengths of the individual hydrogen bonds in the GC pair in a helix are slightly different from isolated GC pairs. X-ray diffraction studies have given a length of 0.286 nm for the strongest hydrogen bond between the guanine NH2 group and the cytosine CO group [G(NH2)···C(CO)], of 2.95 nm for the G(NH)···C(N3) bond, and of 0.291 nm for the G(CO)··· C(NH2) bond.4 Theoretical calculations of the geometries of isolated GC pairs have predicted a different pattern of hydrogen bond lengths with the G(CO)···C(NH2) bond being shortest, i.e., strongest.5−7 This discrepancy has been resolved by calculating the GC pair geometry in an environment of sodium counterions and water molecules, both changing the local electrostatic and orbital interactions.6 This treatment gives hydrogen bond lengths in good agreement with the experimental results of ref 4. While equilibrium structures of DNA have been studied in great detail, the knowledge about fluctuations and dynamics of © 2013 American Chemical Society

DNA structure on the sub-picosecond time scale of molecular motions is still limited. Moreover, the couplings and transport of electronic or vibrational excitations in DNA are far from being understood at the molecular level. Such phenomena are governed by microscopic interactions between bases and/or groups in the DNA backbone as well as between DNA and itsin most casesaqueous environment. Vibrational excitations of individual functional groups are highly sensitive probes of such dynamics which can be resolved in time by ultrafast nonlinear infrared spectroscopy.8−10 In particular, vibrations of NH and CO groups probe hydrogen bond dynamics within the base pairs and areon topinfluenced by interactions with water molecules in the hydrating water shell around the DNA helix. Femtosecond infrared spectroscopy has been applied to study both base pairs dissolved at low concentration in a liquid environment and in tailored model geometries including double-stranded DNA oligomers.11−20 NH stretch excitations of GC and AT base pairs in solution11−13 have been studied by two-dimensional (2D) infrared spectroscopy,8,21−23 providing the transition frequencies and the couplings of the different NH stretch oscillators. For both GC and AT base pairs in solution, a local mode picture was appropriate to describe the NH stretch manifold. For GC pairs, three oscillators are part of a hydrogen bond, Received: August 17, 2013 Revised: October 11, 2013 Published: October 15, 2013 14009

dx.doi.org/10.1021/jp408229k | J. Phys. Chem. B 2013, 117, 14009−14017

The Journal of Physical Chemistry B

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So far, couplings between NH stretching oscillators of GC pairs in double-stranded DNA and the interaction of such oligomers with their hydration shells have remained unaddressed. Changes of the intermolecular hydrogen bond strengths compared to isolated GC pairs and the interplay with OH stretch excitations of water molecules in the environment should be reflected in the 2D spectra of such modes. In particular, structural fluctuations of the water shell represent a basic mechanism from which spectral diffusion of vibrational transitions arises. In this article, we present a femtosecond 2D infrared study of NH stretch excitations of GC base pairs in double-stranded DNA oligomers. A wide range of hydration levels are covered andat high hydration levelalso the OH stretch contributions of water to the 2D spectra are addressed. In analyzing the experimental results, 2D spectra of GC base pairs in solution and of DNA oligomers containing AT base pairs serve as a benchmark. The article is organized as follows. In section 2, we describe the preparation of the DNA samples and the methods applied in the femtosecond 2D infrared experiments. Results are presented in section 3, starting with the linear vibrational spectra in subsection 3.1 and followed by the 2D results in subsection 3.2. The experimental results are discussed in section 4, and conclusions are given in section 5.

2. EXPERIMENTAL TECHNIQUES The DNA samples consist of artificial double-stranded oligomers containing 23 GC pairs in the sequence which is schematically shown in Figure 1b (supplier Thermo Scientific, HPLC, desalted). The individual GC pairs are in the Watson− Crick geometry shown in Figure 1a. Thin film samples of high optical quality were prepared on Si3N4 substrates by replacing the sodium counterions of the oligomers with cetyltrimethylammonium chloride (CTMA), a surfactant forming complexes with DNA. The ∼20 μm thick films were generated with a similar procedure as described in detail in refs 17, 24, and 25, however, with partly different solvents. For an intermediate drying process, the DNA/CTMA complex was dissolved in 2butanol instead of ethanol, and for the following final step of casting the film, the dry DNA/CTMA complexes were dissolved in 2-methyl-1-propanol instead of tert-butanol. The film samples contain DNA in a concentration of approximately 10−2 M. To control the water content of the samples in a defined way, the DNA films were integrated into a home-built humidity cell which was connected to a reservoir containing different agents for controlling the relative humidity (R.H.) in the cell volume and the film sample.17,26 Here, we focus on data sets recorded at 0% R.H., roughly corresponding to a water concentration of up to 4 water molecules per base pair (cf. section 3.1), and at 92% R.H. with more than 20 water molecules per base pair. In the latter case, the DNA oligomers are embedded in a complete first hydration shell. In a separate experiment, the linear infrared absorption spectrum was measured with DNA films dried in a vacuum (pressure 50 mbar) for a period of 3 days to reduce the residual water content. Systematic studies show that the hydration level of the vacuum-dried films can be changed in a fully reversible way between the lowest and highest (92% R.H.) water concentration. 2D infrared spectra were derived from femtosecond heterodyne detected three-pulse photon echoes.19−21,23 A parametric infrared source driven by a regeneratively amplified Ti:sapphire laser system (repetition rate 1 kHz) provides pulses

Figure 1. (a) Molecular structure of the guanine−cytosine base pair in Watson−Crick geometry together with sugar and phosphate units. (b) Base pair sequence of the double-stranded DNA oligomers. (c) Linear infrared absorption spectra of the DNA thin film sample for different levels of hydration (solid lines, R.H.: relative humidity). The dashed line represents the infrared spectrum of guanosine−cytidine base pairs in chloroform solution (concentration 50 mM, taken from ref 11). The liquid phase spectrum was normalized to the spectrum of the vacuumdried sample at 3150 cm−1 where CH and OH stretch contributions are negligible, in order to compensate for notable differences in sample thickness and concentration. NH stretch bands are located between 2700 and 3600 cm−1 and overlap partially with the OH stretch absorption of H2O which is most pronounced at 92% R.H. The asymmetric phosphate stretch [(PO2)−] vibration undergoes a characteristic red-shift upon hydration which is shown in detail in the inset.

νG(NH2)b, νG(NH), and νC(NH2)b, and there are two free NH stretch oscillators of the G and C NH2 groups, νG(NH2)f and νC(NH2)f (b, f: bound, free).11,12 This vibrational pattern is static on the time scale of the 2D experiments, demonstrating the absence of a breaking and reformation of interbase hydrogen bonds. The free NH stretch modes display a v = 1 lifetime of the order of 3 ps, while the stretch modes of the hydrogen bonded NH oscillators show a much shorter lifetime between 200 and 400 fs. Vibrational couplings between the NH stretch oscillators are of the order of 5 cm−1 and lead to an energy transfer from νG(NH2)b to νG(NH) on a 200 fs time scale. Coupling strengths of 5−10 cm−1 have been derived from 2D infrared spectra of the GC CO stretch modes in oligonucleotides and have been interpreted in terms of intraand interstrand interactions.15,16 14010



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of up to 7 μJ energy tunable from 2800 to 4000 cm−1 (spectral pulse width >250 cm−1) with a pulse duration of 55 ± 5 fs. Two phase-locked pulse pairs are generated by reflection from a diffractive optical element. The first pulse pair generates a coherence and a population grating in the DNA sample for different values of the coherence time τ, the delay between pulses 1 and 2. The photon echo signal is generated by pulse 3 interacting with the sample after the population time T (relative to pulse 2) and is heterodyned with pulse 4 serving as a local oscillator. The interferogram is detected as a function of the detection frequency ν3 with the help of a monochromator and an infrared array detector (16 elements, spectral resolution 8 cm−1). The excitation frequency coordinate ν1 is generated by Fourier transforming data recorded for different values of the coherence time τ. Details of the photon echo setup and the procedures applied for data analysis have been presented in refs 19, 20, and 27. In the following, we present 2D spectra measured with pulses which were centered at 3260 and 3400 cm−1. The data were collected with parallel linear polarizations of pulses 1 and 2 and perpendicular polarizations for pulses 3 and 4 (∥⊥⊥). The fraction of excited NH or OH stretching oscillators was below 1%. All measurements were performed at ambient temperature (T = 295 K).

stretch band at 0% R.H. is red-shifted by 4−5 cm−1. From the red-shift data of refs 29 and 30 and the gravimetric calibration of water content in ref 31, one estimates a residual concentration of approximately 4 water molecules per base pair at 0% R.H. 3.2. Two-Dimensional Infrared Spectra of NH and OH Stretch Excitations. Two-dimensional infrared spectra were measured with femtosecond infrared pulses at peak spectral positions of νmax = 3260 and 3400 cm−1. At the first spectral position, the full range of v = 0 to 1 and part of the v = 1 to 2 NH stretch spectra are covered. Pulses centered at νmax = 3400 cm−1 allow for extending the 2D spectra up to a frequency of ∼3600 cm−1 and show a predominant overlap with the water OH stretch band at high hydration level. Spectra recorded at a hydration level of 0% R.H. are summarized in Figure 2 for population times T between 0 and 1000 fs. The absorptive 2D signal, the real part of the sum of the rephasing and nonrephasing signal,32 is plotted for different population times T as a function of the detection frequency ν3 (abscissa) and the excitation frequency ν1 (ordinate). For excitation centered at νmax = 3260 cm−1 (upper part of Figure 2), the positive signal due to the v = 0 to 1 transitions of the different NH stretch oscillators (yellow-red contours) consists of two pronounced diagonal peaks around 3170 and 3320 cm−1 which are clearly distinguished in the frequency cuts along the diagonal ν1 = ν3 presented in Figure 3a. The maximum intensities of the two peaks are similar, and the high-frequency tail of the lower peak overlaps significantly with the low-frequency tail of the upper peak. Both diagonal peaks are elongated along the diagonal, revealing a pronounced inhomogeneous broadening that will be discussed in section 4.1. The solid line in Figure 3a shows a diagonal cross section of the 2D spectrum of the guanosine− cytidine pairs in solution (population time T = 400 fs) from ref 12. Here, the peak at 3150 cm−1 is much stronger than the signal between 3250 and 3400 cm−1, and an additional peak due to free NH stretch oscillators occurs at 3490 cm−1. In addition, the 2D spectra of Figure 2 exhibit two cross peaks at (ν1, ν3) = (3320, 3170) cm−1 and (ν1, ν3) = (3170, 3320) cm−1. As a function of T, the overall peak pattern is preserved, pointing to minor spectral diffusion on a frequency scale set by the spectral width of the individual peaks. In Figure 4a, the intensity of the peaks numbered 1−5 in Figure 2 is plotted as a function of population time T. The transients of peaks 1−4 show an initial decay with time constants of approximately 250 fs (peak 1), 210 fs (peak 2), 500 fs (peak 3), and 200 fs (peak 4). The time constants were derived from the numerical fits including a monoexponential decay and a constant component (solid lines in Figure 4a). The negative signal observed at low detection frequencies (blue contours) is caused by v = 1 to 2 transitions of the NH stretch oscillators. A separation into contributions from the individual NH stretch oscillators is impossible because of the broad line shape with overlapping individual components. The negative signal decays with a time constant of 200 fs, representing the average v = 1 lifetime of the NH stretch oscillators. A time trace recorded at the maximum of the negative peak 5 is shown in Figure 4a (black triangles). It should be noted that peak 5 decays almost completely with the first 1000 fs, whereas a significant residual intensity is found for the positive peaks 1−4. At population times T = 750 and 1000 fs, the 2D spectra exhibit a weak negative signal at high detection frequencies which is marked as peak 6 in the T = 1000 fs panel in the upper part of Figure 2. This signal points to

3. RESULTS 3.1. Linear Vibrational Spectra. Linear infrared absorption spectra of the DNA oligomers are shown in Figure 1c for different levels of hydration (solid lines). The NH stretch absorption extends from approximately 2700 to 3600 cm−1. It is superimposed by CH stretch bands around 3000 cm−1 and by the water OH stretch band which displays a maximum at 3400 cm−1 and is most pronounced for full hydration of DNA (92% R.H.). The black solid line represents the spectrum of a DNA film sample that was dried in a vacuum to extract the residual water molecules from the sample. The infrared spectrum of guanosine−cytidine base pairs in solution (concentration 50 mM in chloroform) is shown as a dashed line.11,12 Here, a Watson−Crick pairing geometry is enforced by bulky side groups, and the base pairs represent the by-far dominating species in the solution, as has been discussed in refs 11, 12, and 28. The concentration of water molecules in the solution is negligibly small. The main components of this infrared spectrum are the comparably narrow stretching band of the free NH units on the NH2 groups of G and C at 3490 cm−1 and two bands due to the hydrogen bonded NH groups. The band with a maximum at 3150 cm−1 has been assigned to the νG(NH) and νC(NH2)b vibrations, while the band at 3300 cm−1 has been attributed to the νG(NH2)b mode. A more detailed analysis has shown that Fermi resonances with combination and overtones of fingerprint vibrations affect the overall line shape of the NH stretch absorption substantially.12 In particular, the low frequency tail below 3000 cm−1 originates from this mechanism. It is important to note that the GC pairs in the DNA oligomer display a much less pronounced band of free NH groups around 3490 cm−1 than guanosine−cytidine pairs in solution. A change of hydration level is connected with characteristic frequency shifts of the asymmetric (PO2)− stretch vibration of the phosphate groups in the DNA backbone which represent major hydration sites.29,30 The inset of Figure 1c shows this behavior for the present DNA oligomers. Compared to the vacuum-dried sample, the maximum of the asymmetric (PO2)− 14011



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Figure 3. (a, b) Spectral cuts through the 2D spectra in Figure 2 along the diagonal ν1 = ν3 for different population times T and the two center frequencies of the pulses (symbols connected by lines to guide the eye). The solid line represents a diagonal cross section of the 2D spectrum of guanosine−cytidine pairs in solution measured for T = 400 fs under similar experimental conditions.12 (c, d) Same for the 2D spectra of Figure 5.

Figure 2. Top row: Linear infrared absorption of the DNA sample at 0% R.H. (solid lines) and intensity spectra of the femtosecond pulses centered at νmax = 3260 and 3400 cm−1 (dashed lines). Second and third row: 2D infrared spectra of the DNA sample at 0% R.H. measured with femtosecond pulses centered at νmax = 3260 cm−1. The absorptive 2D signal, i.e., the real part of the sum of the rephasing and non-rephasing signal, is plotted for different population times T as a function of the detection frequency ν3 (abscissa) and the excitation frequency ν1 (ordinate). Yellow-red features represent positive signals related to v = 0 to 1 excitations of NH stretch oscillators. Blue features of negative sign are caused by v = 1 to 2 transitions at low detection frequencies and by a vibrationally hot ground state at high detection frequencies. The intensity change between neighboring contour lines is 10%. Fourth and fifth row: 2D infrared spectra recorded with pulses centered at νmax = 3400 cm−1. The white lines represent center lines, connecting the maxima of spectral slices at fixed detection frequency ν3, i.e., parallel to the ν1 axis.

and 3480 cm−1 (solid squares). The open symbols represent the time evolution after subtraction of the residual intensity value at T = 1000 fs. Within the experimental accuracy, such two transients display the same kinetics, a decay with a time constant of the order of 250 fs. Again, this behavior is in contrast to guanosine−cytidine base pairs in solution where the intensity of the diagonal peak of the free NH stretch vibrations at 3490 cm−1 (cf. Figure 3a) follows a 2.9 ps decay (dashed line in Figure 4b). To assess spectral diffusion in the 2D spectra, we analyzed the center lines (CLs) of the spectra.33 For deriving the CLs, the maximum intensity of spectral slices for a fixed detection frequency ν3 was determined and their spectral positions (ν1, ν3) in different slices were connected. The thick white lines in the lower part of Figure 2 represent a linear fit of the center lines. In Figure 6, the slope of such lines (CLS: center line slope) is plotted versus the population time T (open symbols). The CLS as a function of T reflects the frequency fluctuation correlation function of the oscillators.33 Within the experimental accuracy, there is no significant change of the slope from its initial value around 0.75. We conclude that spectral diffusion is minor on this time scale.

a vibrationally hot ground state of the system, as will be discussed in section 4.1. The 2D spectra recorded with infrared pulses centered at νmax = 3400 cm−1 (lower part of Figure 2) display a similar peak pattern with an enhanced signal intensity at high excitation/ detection frequencies. There are no distinct diagonal or cross peaks in the range around ν1 = ν3 = 3480 cm−1 where the v = 0 to 1 transitions of free NH stretch oscillators would occur. This fact is also evident from the diagonal cuts shown in Figure 3b and in contrast to the 2D spectra of guanosine−cytidine base pairs in solution which display well-separated diagonal and cross peaks due to free NH stretch vibrations.11,12 In Figure 4b, we compare the change of the 2D signal intensity with T at the diagonal frequency positions ν1 = ν3 = 3300 cm−1 (solid circles) 14012



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Figure 4. (a) Intensity of peaks 1−5 in the 2D spectra of Figure 2 (second and third row, νmax = 3260 cm−1) as a function of population time T (symbols). The solid lines represent numerical fits to the decay kinetics. (b) Normalized intensity of the 2D signal at ν1 = ν3 = 3300 and 3400 cm−1 (solid symbols) as a function of population time T derived from the 2D spectra of Figure 2 (fourth and fifth row, νmax = 3400 cm−1, solid lines are to guide the eye). The open symbols represent the time evolution after subtracting the signal at 1000 fs from the measured values. Dashed line: 2.9 ps decay.

Figure 5. 2D infrared spectra of the DNA oligomers at a hydration level of 92% R.H. The data are plotted in the same way as the 2D spectra in Figure 2. The white thick lines in the lower panels represent the center lines from which the slopes plotted in Figure 6 (solid symbols) were derived.

A set of 2D spectra measured with the DNA sample at a high hydration level of 92% R.H. is shown in Figure 5. The NH stretch peak pattern observed at early population times T with pulses centered at νmax = 3260 cm−1 (upper part of Figure 5 and diagonal cuts in Figure 3c) is very similar to the spectra recorded at 0% R.H. (upper part of Figure 2). With increasing T, however, a reshaping toward an overall round shape with a maximum around ν1 = ν3 = 3400 cm−1 occurs and a stronger negative signal develops at high detection frequencies ν3. Such changes are hallmarks of spectral diffusion of OH stretch excitations of the water shell around the DNA oligomers and the formation of a vibrationally hot ground state with a blueshifted OH stretch absorption. The 2D spectra measured with pulses centered at νmax = 3400 cm−1 are dominated by the OH stretch contribution from the water shell and exhibit its spectral diffusion most clearly. Diagonal cuts of such spectra are shown in Figure 3d. The white lines in the lower part of Figure 5 are CLs, the slopes of which are plotted as a function of population time T in Figure 6 (solid symbols). The CLS decreases from a value of 0.9 at T = 0 fs to ∼0.3 within the first 400 fs and levels off at a later T. The initial decay is slower than the sub-100 fs decay observed in bulk H2O but demonstrates significant spectral diffusion on a sub-picosecond time scale.

Figure 6. Center line slopes of the 2D spectra recorded at 0 and 92% R.H. with pulses centered at νmax = 3400 cm−1 (cf. white lines in Figures 2 and 5) as a function of population time T. The center lines were derived from spectral cuts through the 2D spectra taken at a fixed detection frequency ν3. For the data measured at 92% R.H. the solid line shows a monoexponential fit curve with a 250 fs monoexponential decay and a constant component.

4. DISCUSSION 4.1. GC Oligomers at Low Hydration Level. The double helix structure of DNA undergoes characteristic modifications 14013



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upon changes of the hydration level. X-ray studies of fibers of GC double helices suggest a predominant B- and A-form at hydration levels higher and lower than 90% R.H., respectively.34,35 X-ray scattering data have also been reported for thin films of calf thymus and salmon testes DNA which were prepared in the same fashion as our present samples.24,25 Such DNA helices, which contain approximately 45 mol % GC pairs, adopt the B-form under full hydration conditions (R.H. ≥ 90%) and transform into the A-form or a helix geometry with an edge-stacking of base pairs at low hydration level. In all such geometries, the GC base pairing in Watson−Crick geometry is preserved. The linear infrared spectra of the GC oligomers after vacuum drying and at 0% R.H. (Figure 1c) display a similar pattern of NH stretch bands. The spectral positions of the maxima are close to the spectrum of the guanosine−cytidine pairs in chloroform solution with a negligible water content (dashed line in Figure 1c). In the solution-phase system which serves as a benchmark for interpreting the linear and 2D spectra of the oligomers, vibrational assignments were made on the basis of the linear infrared spectrum, the 2D spectra, and time-resolved pump−probe data.11,12 A description in terms of local NH stretch modes was found to be a good approximation, although Fermi resonances occur of the NH stretch modes with overand combination tones of fingerprint modes and affect the overall line shapes. In the local mode picture, the band with maximum at 3150 cm−1 was assigned to the superposition of νG(NH) and νC(NH2)b, the much weaker band at 3300 cm−1 to νG(NH2)b, and the band at 3490 cm−1 to the free NH stretch bands νG(NH2)f and νC(NH2)f.11,12 The occurrence of a distinct stretch band of free NH groups demonstrates an effective “decoupling” of the two NH oscillators on each of the NH 2 groups. The frequency red-shifts of ∼200 cm −1 (νG(NH2)b) and 345 cm−1 (νC(NH2)b) with respect to νG,C(NH2)f are caused by hydrogen bonding and larger than the frequency splitting of 2V ≈ 110 cm−1 (V: mechanical coupling) expected for symmetric and asymmetric NH2 stretch modes of mechanically coupled NH oscillators. The decoupled oscillators display markedly different relaxation times of 210 fs for the hydrogen bonded and 2.9 ps for the free NH stretch vibrators. For an analysis of the GC oligomer spectra, we recall the following facts: (i) the hydrogen bond strengths of isolated GC pairs and GC pairs in DNA helices are different, resulting in different spectral red-shifts of the NH stretch oscillators being part of interbase hydrogen bonds;4,6 and (ii) up to 4 H2O molecules per base pair are present in the DNA film samples even at 0% R.H. For GC pairs in DNA, both experiment and theory give the highest strength for the G(NH2)···C(CO) hydrogen bond, followed by the G(CO)···C(NH2) bond and finally the G(NH)···C(N) bond.4,6 In the local mode picture, one, thus, would assign the NH stretch band with maximum at 3150 cm−1, i.e., with the strongest red-shift compared to a nonhydrogen-bonded NH stretch oscillator, to a superposition of νG(NH2)b and νC(NH2)b and the band at 3300 cm−1 to νG(NH). However, there is no clear evidence for a decoupling of the NH oscillators on the NH2 groups in the GC oligomers, given the lack of a pronounced separate stretching feature of free NH groups in the linear and 2D infrared spectra (cf. diagonal cross sections in Figure 3a) at 0% R.H. as well as the very similar decay times of the diagonal NH stretch signals in the 2D spectra at 3150, 3300, and 3480 cm−1 (cf. Figure 4).

The absence of a distinct free NH stretch band in the GC oligomer spectra points to local hydrogen bonds of the “free” NH units on the two NH2 groups with water molecules which are present in the environment even at the low hydration level of 0% R.H. Such interactions are expected to red-shift the NH stretch frequency and bring it closer to that of NH groups being part of the Watson−Crick pairing. X-ray diffraction studies of A-form DNA helices have shown that the NH2 groups of G (minor groove of the helix) and C (major groove of the helix) are prominent hydration sites of the bases where hydrogen bonds are formed between the NH units and the water oxygens.36 Though the majority of water molecules present interact with the phosphate groups of the backbone at low hydration level, a certain fraction of them is spread out over the DNA surface and attached to NH2 groups of the bases. This mechanism introduces structural disorder and results in a spreading of NH stretch frequencies from 3490 cm−1 toward lower frequencies, i.e., an inhomogeneous broadening. This behavior is obvious from the substantially larger line width of the 3300 cm−1 peak in the infrared absorption spectra of the GC oligomers (solid lines in Figure 1c) compared to the solution-phase guanosine−cytidine spectrum (dashed line). Correspondingly, the diagonal cuts of the oligomer 2D spectra display a larger spectral width than the diagonal cut of the guanosine−cytidine 2D spectra in solution (cf. Figure 3a). Under such conditions, the local mode picture for the NH stretch vibrations represents an approximation valid just for a subset of local unhydrated geometries in the GC oligomers. For NH2 units hydrogen bonded to water molecules, one expects a smaller frequency separation of the two local NH oscillators and, induced by the mechanical coupling V, a stronger mixing of the wave functions of the two oscillators. In second-order perturbation theory, the total v = 1 wave functions of the coupled oscillators 1 and 2 are given by β β + φ2 sin 2 2 β β ψ2 = −φ1 sin + φ2 cos 2 2 ⎡ 2V ⎤ β = arctan⎢ ⎥ ⎣ E1 − E2 ⎦ ψ1 = φ1 cos

where φ1,2 are the v = 1 wave functions of the local NH stretch oscillators and E1,2 the energy of their v = 1 states.37 While the exact values of E1,2 are unknown for the GC oligomers, the energy difference (E1 − E2) is of the order of 100−200 cm−1 and V ≈ 55 cm−1, as derived from infrared spectra of the G and C monomers.11 This results in values of β = 28.8−47.7° and a ratio of tan(β/2) = sin(β/2)/cos(β/2) = 0.25 − 0.44. Thus, a significant mixing of the local oscillator wave functions occurs which is, however, less pronounced than for symmetric and asymmetric NH2 stretching modes with tan(β/2) = 1, i.e., identical amplitudes of the two local oscillator wave functions. This analysis shows that the NH stretch mode character in the GC oligomers is in between the limiting cases of local NH stretch vibrations and NH2 stretch vibrations. We now discuss the pattern of NH stretch peaks in the 2D spectra at 0% R.H. The diagonal peaks of such spectra display a substantial inhomogeneous broadening and overlap of different spectral features (cf. Figure 3a,b). There are no indications of a pronounced spectral diffusion up to our longest population time of T = 1000 fs, a fact also evident from the CLS plotted in 14014



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directly. This behavior is caused by the randomization of excess energy which is released in the v = 1 population decay and the concomitant formation of a vibrationally hot ground state of the oligomers and the residual water shell. In the hot ground state, the NH and OH stretch lineshapes are different from the initial ones, leading to a residual positive 2D signal over the range of the NH and OH v = 0 to 1 transitions. Moreover, there is a negative 2D signal (peak 6 in Figure 2) which is weakly blue-shifted and elongated along the excitation frequency axis, i.e., of homogeneous line shape. This component is dominated by OH stretch oscillators with weakened hydrogen bonds and, thus, higher v = 0 to 1 transition frequencies in the hot ground state.20 It should be noted that this signal is much more pronounced at high hydration levels (cf. Figure 5), due to the much stronger OH stretch component in the 2D spectra.18,20 The intensity of the cross peaks 3 and 4 in Figure 2 evolves differently in time. While peak 4 shows an initial decay similar to the diagonal peaks, peak 3 decays substantially slower. This observation points to a transfer of vibrational energy from the NH stretch oscillator at 3300 cm−1 to the NH stretch oscillators at lower frequency. A similar transfer has been observed in the guanosine−cytidine base pairs in solution where, however, the intensity ratio of peak 3 vs peak 2 showed an increase with population time. Such a clear independent signature of energy transfer is absent in the present data set. 4.2. Fully Hydrated GC Oligomers. In the 2D spectra recorded at 92% R.H. (Figure 5), the intensity of the upper diagonal peak is strongly enhanced compared to the lower one and the maximum of the upper peak is shifted to higher frequencies compared to the 0% R.H. spectra. Both changes originate from the much stronger contribution of OH stretch excitations of the hydrating water shell around the GC oligomers. The diagonal cuts in Figures 3c show an asymmetric profile of the upper peak consisting of a broad NH stretch component around 3300 cm−1 and an OH stretch component with a maximum around 3390 cm−1. The upper diagonal peak of the 2D spectra measured with pulses centered at 3260 cm−1 (upper panels in Figure 5) exhibits a “bifurcated” line shape which consists of an inhomogeneously broadened NH stretch component elongated along the diagonal and an OH stretch component which is more parallel to the ν1 axis, i.e., closer to homogeneously broadened. At longer T, both components overlap with the enhanced absorption of the hot water ground state occurring as a homogeneously broadened feature at high detection frequencies ν3. Such findings suggest that the NH and OH stretch contributions are essentially additive with an NH stretch line width comparable to the 2D spectra for 0% R.H. This conclusion is supported by the fact that the frequency positions and shapes of the lower diagonal and the off-diagonal peaks are very similar to the 0% R.H. 2D spectra (Figure 2). Obviously, the water shell makes a limited contribution to the dephasing/spectral diffusion of NH stretch excitations, a behavior we also observed with hydrated AT oligomers.20 The 2D spectra recorded with pulses centered at 3400 cm−1 (lower panels of Figure 5) are dominated by the upper diagonal peak and, in particular, the OH stretch contribution. The spectra show substantial changes of the line shape which becomes more round due to spectral diffusion of OH stretch excitations. To quantify the reshaping, we consider center lines in the lower part of this peak (white solid lines) and plot their slope (CLS) as a function of T in Figure 6 (solid symbols). There is a pronounced decrease of the CLS within the first 400

Figure 6. The slight reshaping of the diagonal cuts of Figure 3b at T = 750 and 1000 fs originates from the different kinetics of the underlying NH stretch component around 3300 cm−1 and the OH stretch contribution around 3400 cm−1. From the relative oscillator strength of NH stretch and OH stretch transitions of F = f(NH)/f(OH) ≈ 2.5 and the relative concentrations of high-frequency NH stretch oscillators and OH stretch oscillators C = c(NH)/c(OH)≈0.75, one estimates a ratio of the NH to the OH stretch 2D signals of R = F2C ≈ 4.5; i.e., the NH stretch signal clearly dominates at 0% R.H. The absence of spectral diffusion points to a static structural inhomogeneity in the weakly hydrated GC oligomers. In the short helices, one expects slight differences of base pair geometries in the center and at the two ends of the helix, resulting in different hydrogen bond strengths within the base pairs, and, thus, a distribution of NH stretch frequencies. The incomplete hydration results in a random distribution of water molecules hydrating the “free” NH units on the two NH2 groups and, thus, a distribution of transition frequencies. The calculations of ref 6 have clearly shown that both hydrogen bond strength and NH stretch frequencies are highly sensitive to small changes of the molecular geometries, including the location of and interaction with the ionic backbone and the counterions. The absence of spectral diffusion in the 2D spectra suggests a comparably rigid hydration geometry of the residual water molecules with a lifetime of water−DNA hydrogen bonds longer than 1 ps. Such behavior is in line with earlier studies of DNA oligomers containing adenine−thymine base pairs19 and with theoretical simulations.38,39 The cross peaks in the 2D spectra demonstrate a coupling of the different NH stretching oscillators, in particular between the νC(NH2)b/νG(NH2)b oscillators and the νG(NH) vibrator. This part of the peak pattern is very similar to that of guanosine−cytidine base pairs in solution for which coupling strengths of the order of 5 cm−1 have been invoked.11,12 We conclude that couplings between neighboring GC pairs in the double helix structure play a minor role. The time evolution of the intensity of the diagonal peaks (Figure 4a) shows an initial decay to a residual value, whereas the v = 1 to 2 signal decays completely within the first picosecond. From the fast decays, one derives v = 1 lifetimes of the different NH stretch oscillators between 200 and 300 fs, values which are close to the lifetimes of the hydrogen bonded NH stretch oscillators in the solution phase base pairs.11,12 Similar to other pair and dimer systems with hydrogen bonded NH groups, the pathway of population relaxation most probably involves overtones of NH bending modes and combination tones including other fingerprint modes.40−42 The transients of Figure 4b demonstrate that the diagonal 2D signals at 3300 and 3480 cm−1 display the same sub-picosecond decay component, a behavior in marked contrast to the guanosine−cytidine base pairs in solution. The latter show a decay time of 210 fs at 3300 cm−1 and a 2.9 ps lifetime of the free NH stretch oscillators around 3490 cm−1. We assign the fast decay in the GC oligomers at 3480 cm−1 to OH stretch excitations of the hydrating water and a contribution from hydrogen bonded NH stretch oscillators. Free NH units may contribute to kinetics beyond population times of 1 ps which have not been studied here and are partly masked by a longlived residual contribution discussed next. The residual intensity observed at long population times shows that the initial vibrational ground state is not repopulated 14015



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interactions between neighboring GC pairs in the double helix. At low hydration, the absence of spectral diffusion on a time scale up to 1 ps points to a relatively rigid structure of the hydrogen bonds between DNA and residual water molecules. The 2D spectra measured under conditions of full hydration (92% R.H.) reveal additive contributions of NH and OH stretch excitations with a weak influence of water fluctuations on the 2D lineshapes of NH stretch excitations. The dynamics of the water shell is manifested in the femtosecond spectral diffusion of OH stretch excitations which is somewhat slowed down compared to bulk H2O. The main mechanisms behind spectral diffusion are structural fluctuations of the water shell and the formation of a vibrationally hot ground state by vibrational relaxation. The overall behavior of GC base pairs in DNA found here is different from that of isolated base pairs in solution. The dynamics of the water shell as manifested in the OH stretch dynamics is similar to other systems such as hydrated DNA oligomers containing adenine−thymine base pairs and water nanopools in reverse micelles.

fs, followed by a leveling off which points to a slower or even constant CLS. The initial decay with a time constant of approximately 250 fs (solid line) is due to spectral diffusion which is somewhat slower than in bulk H2O27 but definitely in the femtosecond regime. There are two mechanisms contributing to spectral diffusion: first, resonant energy transfer between OH stretch oscillators on different water molecules and structural fluctuations, occurring mainly in the outer part of the hydration shell, result in frequency jumps of OH stretch oscillators. Compared to bulk H2O, intermolecular energy transfer is slowed down because of the lower water concentration which is of the order of 10 M and results in a larger distance between OH stretch oscillators, sensibly affecting the energy transfer rates.20,43 Structural fluctuations are substantially slowed down in the first water layer interacting with the DNA surface, due to the steric constraints in the minor DNA groove and strong hydrogen bonding with DNA groups, e.g., the phosphate groups in the backbone. As the experiment averages over all parts of the hydration shell, the overall fluctuations and spectral diffusion are slower than in bulk H2O. It should be noted that this interpretation is in line with recent molecular dynamics simulations of water at DNA surfaces, predicting a slowing down of the decay of the frequency fluctuation correlation function by a factor of 2−3 compared to bulk water.44 The second mechanism contributing to the apparent spectral diffusion is the formation of the hot water ground state. Weakening and/or breaking of water−water hydrogen bonds in the ground state heated via the release of excess energy in the decay of NH and OH stretch excitations results in a positive 2D signal (bleach) extending over the full range of the 2D peak shown in the lower panels of Figure 5 and a negative 2D signal (enhanced absorption) which becomes visible at high ν3. As the initial excitation is totally randomized in the relaxation process, the line shape of the heated ground state is homogeneous; i.e., the bleach component is round in the (ν1, ν3) plane and the enhanced absorption elongated along the ν1 axis. The hot ground state is formed on the femtosecond time scale of the population decay of the initially excited oscillators (cf. Figure 4), and thus, the formation of its OH stretch band contributes to the sub-picosecond reshaping observed in the 2D spectra. A very similar behavior has been found with hydrated AT oligomers20 and, most recently, in nanopools of water confined in reverse micelles.45

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 30 63921400. Fax: +49 30 63921409. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Ł. Szyc for help with the preparation of the GC oligomer film samples. 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.

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REFERENCES

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5. CONCLUSIONS In conclusion, we have studied the dynamics and couplings of NH and OH stretching excitations of hydrated DNA oligomers containing guanine−cytosine (GC) base pairs in a Watson− Crick geometry. The 2D infrared spectra measured at a low hydration level of 0% R.H. are dominated by NH stretch contributions from the NH2 groups of G and C and the NH group of G. In contrast to guanosine−cytidine pairs in waterfree solution, there is an incomplete decoupling of the two local NH oscillators on the NH2 groups and a wider distribution of NH stretch frequencies. This behavior is attributed to local hydrogen bonds between NH and water molecules, resulting in a red-shift of the NH stretch frequencies and a mixing of the wave functions of the two local oscillators via the mechanical vibrational coupling. The lifetimes of the NH stretch excitations are of the order of 200−300 fs, similar to guanosine−cytidine pairs in solutions. There are weak couplings between the NH stretch oscillators within a base pair but no significant 14016



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Ultrafast Two-Dimensional Infrared Spectroscopy of Guanine

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