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2008, 112, 11194–11197 Published on Web 08/15/2008
Ultrafast Vibrational Dynamics of Adenine-Thymine Base Pairs in DNA Oligomers Jason R. Dwyer,‡ Łukasz Szyc, Erik T. J. Nibbering, and Thomas Elsaesser* Max-Born-Institut fu¨r Nichtlineare Optik und Kurzzeitspektroskopie, Max-Born-Strasse 2 A, D-12489 Berlin, Germany ReceiVed: June 19, 2008
N-H stretching excitations of DNA oligomers containing 23 alternating adenine-thymine base pairs are studied in femtosecond two-color pump-probe experiments. For a DNA film in a zero relative humidity atmosphere, transient vibrational spectra and their time evolution up to 10 ps demonstrate negligible spectral diffusion and allow for discerning different N-H stretching bands and the O-H stretching absorption of residual water molecules. Lifetimes on the order of 0.5 ps are found for both N-H and O-H stretching modes. The time-dependent pump-probe anisotropies of the different N-H excitations point to a pronounced coupling among them, whereas the O-H stretching anisotropy remains essentially constant. Introduction Hydrogen bonds play a key role for the structure of deoxyribonucleic acid (DNA) and its interaction with an aqueous environment. Intermolecular hydrogen bonds define the planar Watson-Crick geometry of adenine-thymine (A-T, Figure 1a) and guanine-cytosine base pairs in the double helix.1 Hydrogen bonding also mediates the interaction of water molecules with specific binding sites at the base pairs and the oligomer backbone, for example, with phosphate groups.2 Vibrational spectroscopy has been applied to identify particular functional groups and to characterize different helix structures of DNA oligomers in their native aqueous environment. The linear vibrational spectra of DNA are highly congested, both in the range of fingerprint vibrations and at frequencies between 3000 and 3600 cm-1 where different N-H and O-H stretching excitations occur. As a result of such complexity, a reliable separation of N-H and O-H stretching bands in condensedphase DNA has not been possible so far. Some authors have assigned the entire vibrational absorption above 3000 cm-1 to water.2 The study of isolated base pairs in the gas phase provides independent insight into vibrational properties. Infrared3,4 and double resonance laser spectroscopies5,6 have established characteristic frequencies for the N-H stretching vibrations of individual bases and hydrogen-bonded base pairs. A recent study of A-T pairs has revealed frequency positions of 3530 and 3326 cm-1 for the asymmetric and symmetric stretching vibration of the hydrogen-bonded NH2 group of adenine and 3295 cm-1 for the stretching vibration of the hydrogen-bonded NH group of thymine.6 The predominant A-T geometries in this gas-phase study are different from those of the Watson-Crick pair but display a similar strength of the intermolecular hydrogen bonds. * To whom correspondence should be addressed. Phone: +49 30 6392 1400. Fax +49 30 6392 1409. E-mail:
[email protected]. ‡ Present address: Department of Physics and Astronomy, University of British Columbia, Vancouver, B.C., Canada.
10.1021/jp8054119 CCC: $40.75
Figure 1. (a) Molecular structure of the adenine-thymine base pair in Watson-Crick geometry. (b) Sequence of bases in the DNA oligomers. (c) DNA film on a Si3N4 window as prepared by the method described in the text. The optical spot size on the film was 150 µm.
Changes of vibrational spectra upon DNA oligomer formation and/or dissolution in an aqueous condensed phase have remained unresolved. While such work has addressed the time-averaged equilibrium properties of DNA and/or individual base pairs, understanding structural fluctuations and the elementary steps of conformational changes on the molecular level requires insight into ultrafast vibrational dynamics.7 So far, there exist only a few studies of the ultrafast vibrational dynamics of DNA, mainly in the fingerprint range.8-10 In this Letter, we address hydrogenbonding vibrational dynamics and couplings of A-T base pairs. To work with a well-defined molecular configuration, we study DNA oligomers containing 23 alternating A-T pairs in Watson-Crick geometry. These oligomers are complexed with a cationic surfactant, cast into thin films, and held in an atmosphere of well-defined relative humidity (r.h.) that determines the film water concentration. As a benchmark case, we concentrate on DNA at 0% r.h., corresponding to approximately 2 water molecules per base pair. Transient infrared spectra measured by femtosecond vibrational spectroscopy display 2008 American Chemical Society
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J. Phys. Chem. B, Vol. 112, No. 36, 2008 11195
different N-H and O-H stretching bands between 3000 and 3600 cm-1 with negligible spectral diffusion in a 10 ps time range. The pump-probe anisotropy displays an ultrafast partial decay for coupled N-H stretching excitations and a nearly constant value of 0.4 for the high-frequency O-H stretching mode. Experimental Section Preparation of DNA-Surfactant Complex Films. High optical quality films of artificial 23 base pair DNA oligomer duplexes were prepared by replacing the sodium counterions with surfactant molecules. The duplexes were composed of 5′T(TA)10-TT-3′ and its complement (Figure 1b), purchased from Thermo Scientific (HPLC/desalted). The DNA was dissolved in sterilized water. The surfactant cetyltrimethylammonium chloride (CTMA chloride) was also dissolved in sterilized water and was prepared to yield a 1:1 ratio between the number of CTMA cations and the number of phosphate groups in the DNA duplex.11,12 The DNA/CTMA complex formed spontaneously upon dropwise addition of the CTMA chloride solution to the DNA. The complex was isolated by repeatedly centrifuging, drawing off the supernatant, and rinsing with water. The DNA/ CTMA complex was then dissolved in ethanol and dried overnight at room temperature. The dry DNA:/CTMA complex was dissolved in tert-butanol, drop-cast onto 500 nm thick silicon nitride membranes (Silson Ltd., Northampton, U.K.), and allowed to dry in air and then under a continuous nitrogen flow (Figure 1c). A custom stainless steel humidity cell was constructed to control the water content of the film. A channel connected the sample chamber to a reservoir where various agents could be placed to control the sample chamber r.h. The sample on the Si3N4 membrane was sealed into the humidity cell with P2O5 in the reservoir to maintain 0% r.h. inside of the chamber. X-ray diffraction analysis has shown that changes of r.h. drive macroscopic changes in the helix conformation of DNA without surfactant counterions13 and that similar changes occur in DNA/surfactant complexes.14-16 DNA oligomers with alternating A-T pairs exist in the D conformation at a r.h. between 40 and 70% and in the B conformation at high humidity levels.13 X-ray diffraction from thin films at 0% r.h. suggests a well-defined helix structure without, however, an in-depth structural analysis.15 Femtosecond Pump-Probe Experiments. Independently tunable pump and probe pulses were generated in two parametric frequency converters driven by amplified pulses from a Ti: sapphire laser (repetition rate, 1 kHz). The energy of the pump pulses was 2 µJ, and the temporal width of the cross-correlation with the probe pulses was 150 fs. In the following, we present results for three different spectral positions of the pump pulses (Figure 2a). Experiments were performed with parallel and perpendicular linear polarizations of the pump and probe and under magic angle conditions. After interaction with the DNA sample, the probe pulses were dispersed in a monochromator and detected with a 16 element HgCdTe detector array (resolution 8 cm-1). Nonlinear pump-probe signals from the Si3N4 membranes are negligible, as was confirmed in measurements with uncoated membranes. Results In Figure 2a, the linear absorption spectrum of the DNA films between 3000 and 3600 cm-1 (thick solid line) is shown together with the spectral profiles of the pump pulses. The absorption maximum at around 3050 cm-1 is due to C-H stretching
Figure 2. (a) Infrared absorption spectrum of a DNA film consisting of DNA oligomers with 23 A-T base pairs in the range of the N-H and O-H stretching absorption for 0% relative humidity (thick solid line). Thin solid lines: Spectral profiles of femtosecond pump pulses for three center frequencies. (b) Transient vibrational spectra measured with pump pulses centered at Eex ) 3150 cm-1. The absorbance change ∆A ) -log(T/T0) is plotted as a function of (probe) frequency for delay times of 60, 100, 200, and 500 fs and 1 and 2.5 ps (colored symbols, T0, T: sample transmission before and after excitation). The transient spectra display distinct components and negligible spectral diffusion. (c,d) Transient spectra measured with pump pulses centered at Eex ) 3250 and 3550 cm-1 for the same time delays as those in (b).
vibrations. The different maxima and shoulders above 3100 cm-1 reflect both N-H stretching bands of A-T pairs and the O-H stretching absorption of water without a clear assignment available so far. In Figure 2b-d, we present transient spectra for the different pumping conditions (cf. Figure 2a). The change of absorbance ∆A is plotted as a function of the probe frequency for pump-probe delays between 60 fs and 2.5 ps (colored symbols, parallel polarization of pump and probe). At low probe frequencies, the transient spectra display a spectrally broad enhanced absorption (∆A > 0) that originates from the V ) 1 f 2 transitions of the excited oscillators. Such enhanced absorption decays on a time scale of approximately 1 ps, reflecting the depopulation of the excited V ) 1 states. At higher probe frequencies, one observes a decrease of absorption (∆A < 0) with a spectral envelope depending on the pump frequency. For excitation at Eex)3150 and 3250 cm-1, a strong component centered at around 3200 cm-1 is complemented by weaker contributions at around 3350 and 3480 cm-1.17 The latter contribution dominates when exciting at Eex ) 3550 cm-1. The relative strength of the different components is roughly given by the overlap of the respective pump spectrum with the linear absorption spectrum. It is important to note that the nonlinear spectra display negligible spectral diffusion up to delay times of ∼10 ps, in sharp contrast to disordered fluctuating hydrogenbonded systems such as bulk liquid water.18 Pump-probe transients measured with pump pulses centered at Eex ) 3250 cm-1 and different probe frequencies are
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Figure 4. (a) Pump-probe transients measured with Eex ) 3250 cm-1 and Epr ) 3335 cm-1 for parallel (solid circles), perpendicular (solid squares), and magic angle (open diamonds) linear polarizations of pump and probe pulses. Dashed line: Calculated magic angle signal as in Figure 3. (b) Anisotropy decay (solid line). The dashed line at r(t) ) 0.16 marks the average value reached for delay time t > 1500 fs. Figure 3. (a,b) Pump-probe transients measured at probe photon energies of Epr ) 3075 and 3205 cm-1 after excitation with pump pulses centered at Eex ) 3250 cm-1. The change of absorbance ∆A is plotted as a function of pump-probe delay for parallel (solid circles) and perpendicular (solid squares) linear polarization of pump and probe pulses, as well as for polarizations at the magic angle (open diamonds). The dashed line in (b) gives the magic angle signal ∆Ama ) (∆Apa + 2∆App)/3 calculated from the transients for parallel (∆Apa) and perpendicular (∆App) polarization. (c) Magic angle transient measured with Eex ) 3150 cm-1 and Epr ) 3205 cm-1. (d) Time-dependent anisotropy r(t))(∆Apa - ∆App)/(∆Apa + 2∆App) derived from the data in (b) (solid line) and from measurements with Eex ) 3150 cm-1 and Epr ) 3205 cm-1 (dashed line).
presented in Figures 3 and 4. The transient V ) 1 f 2 absorption at Epr ) 3075 cm-1 (Figure 3a) decays with a time constant of ∼0.5 ps, similar to the initial recovery of the magic angle bleaching signal at Epr ) 3205 cm-1 (open diamonds), that is, at the maximum of the strong bleaching feature in Figure 2b,c. The decrease of absorption is caused by bleaching due to the pump-induced depopulation of the V ) 0 state and by stimulated emission on the V ) 1 f 0 transition. The 0.5 ps kinetics reflect the decay of the V ) 1 population back to a ground state in which the oscillators are in their V ) 0 states and the vibrational excess energy is contained in other anharmonically coupled vibrations. The reshaped absorption band of the hot ground state gives rise to the longer-lived residual bleaching found in Figure 3b,c, a behavior similar to that in other hydrogen-bonded systems.7 The measured magic angle transient (Figure 3b) in which ∆A is not affected by a potential rotational relaxation of the excited molecules agrees very well with the magic angle transient (dashed line) calculated from the data taken with parallel (solid circles) and perpendicular (solid squares) polarizations of the pump and probe. The magic angle data recorded with the excitation centered at Eex ) 3150 cm-1 (Figure 3c) show a similar time evolution. The time-dependent anisotropy r(t) )(∆Apa - ∆App)/(∆Apa + 2∆App) (solid line in Figure 3d) derived from the transients in Figure 3b exhibits an initial femtosecond decay from its initial value of 0.4 to a residual value of r ≈ 0.18, which is constant at later picosecond delay
Figure 5. (a,b) Same as Figure 4a,b for Eex ) 3550 cm-1 and Epr ) 3505 cm-1. Note the constant anisotropy of 0.4.
times. A similar behavior is found with Eex ) 3150 cm-1 and Epr ) 3205 cm-1 (dashed line). Different time evolutions of the absorbance change ∆A and the anisotropy are found on the second pronounced bleaching feature in the transient spectra (Figure 2b,c), for example, at Epr ) 3335 cm-1 (Figure 4). Here, ∆A shows a fast partial decay within the first 200 fs (Figure 4a), followed by a ∼0.5 ps component and weak residual bleaching. The anisotropy (Figure 4b) decays to a residual value of r ≈ 0.16 on a 1 ps time scale, somewhat slower than the 300 fs time scale in Figure 3d. After excitation at high frequencies (Eex ) 3550 cm-1), one finds a population decay on a 1 ps time scale (Figure 5a) with a constant anisotropy r(t) ) 0.4 (Figure 5b). Discussion The spectrally and temporally resolved pump-probe data clearly demonstrate the occurrence of different fundamental (V
Letters ) 0 f 1) vibrational transitions in the N-H/O-H stretching range (Figure 2), displaying similar V ) 1 decay times on the order of 0.5 ps (Figures 3-5). The observation of such individual bands is facilitated by their limited spectral diffusion, being less than the individual line widths. In general, spectral diffusion arises from a modulation of the transition frequency by structural fluctuations of the hydrogen bond geometries or by fluctuating forces exerted by the environment. In our case, the well-defined hydrogen bond geometries are essentially timeindependent as the DNA backbone sets the steric boundary conditions for the A-T pair geometry. Nevertheless, the DNA helix structure and its polar groups, for example, the phosphate groups, undergo fluctuating thermal motions which result in a fluctuating long-range Coulomb interaction with the vibrational transition dipoles. The minor spectral diffusion found here points to a low frequency of the fluctuating motionssas supported by the sub-20 cm-1 frequency of DNA backbone motions19sand to a limited interaction strength. The latter may arise from the substantial distance between the polar groups on the outside and the A-T pairs in the inner part of the DNA structure. The assignment of the different transitions to particular N-H and/or O-H modes is not straightforward. Following the frequency pattern derived from the gas-phase spectra of isolated A-T dimers, one expects contributions from the asymmetric and symmetric stretching modes of the adenine NH2 group and the stretching mode of the hydrogen-bonded N-H group of thymine, the latter at a similar frequency as the symmetric NH2 vibration.6 We thus assign the spectral component at 3350 cm-1 to the asymmetric NH2 stretching mode and the component with maximum at 3200 cm-1 to a superposition of the symmetric NH2 stretching mode of adenine and the N-H stretching mode of thymine. The anisotropy r(t) at 3205 and 3335 cm-1 shows a subpicosecond partial decay to a constant value (Figures 3 and 4). In view of the steric boundary conditions set by the DNA, rotation of functional groups and/or the base pairs is negligible on this time scale. Instead, the changes of r(t) are due to a coupling of the different N-H stretching oscillators. At 3205 cm-1, the initial r(0) ) 0.4 suggests the preferential excitation of one oscillator per base pair from which excitation is transferred to the other oscillators with a different direction of their transition dipoles. After ∼300 fs, the distribution of excited dipoles gives rise to a constant r(t) ≈ 0.18. The coupling of the asymmetric NH2 mode (3335 cm-1) is evident from the smaller initial value r(0) ≈ 0.3 and the subsequent decay to r(t) ≈ 0.16. Future studies of the two-dimensional N-H stretching spectra will give more detailed information on the underlying coupling mechanisms. The broad band at around 3550 cm-1 is assigned to the O-H stretching mode of the residual water molecules. At 0% r.h., the ∼2 water molecules per base pair are expected to be located at the ionic phosphate groups of the DNA backbone. Similar to water molecules interacting with the ionic heads of reverse micelles,20,21 we observe a blue-shifted O-H stretching band
J. Phys. Chem. B, Vol. 112, No. 36, 2008 11197 that peaks at around 3500 cm-1 and an O-H stretching lifetime of ∼0.5 ps that is substantially longer than that in bulk H2O.18 The constant anisotropy of r(t) ) 0.4 suggests that the O-H stretching vibrations are localized on a single O-H group of the water molecules22 and that excitation transfer to other vibrations and/or reorientation processes play a minor role. Such conclusions are supported by results for DNA films at higher hydration levels.23 In summary, femtosecond vibrational spectroscopy allows for discerning different N-H stretching bands of alternating adenine-thymine base pairs in DNA oligomers. Transient pump-probe spectra display negligible spectral diffusion in this macromolecular environment and similar lifetimes of ∼0.5 ps for the different N-H excitations. The time-dependent pumpprobe anisotropies of the different N-H excitations point to a pronounced coupling among them, whereas the anisotropy of the O-H stretching band of residual water molecules occurring at higher frequency remains essentially constant. Acknowledgment. This work has been supported by the Deutsche Forschungsgemeinschaft (SFB 450). J.R.D was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. References and Notes (1) Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 737. (2) Ouali, M.; Gousset, H.; Geinguenaud, F.; Liquier, J.; Gabarro-Arpa, J.; Le Bret, M.; Taillandier, E. Nucleic Acids Res. 1997, 25, 4816. (3) Dong, F.; Miller, R. E. Science 2002, 298, 1227. (4) Choi, M. Y.; Miller, R. E. J. Phys. Chem. A 2007, 111, 2475. (5) Nir, E.; Kleinermanns, K.; de Vries, M. S. Nature 2000, 408, 949. (6) Pluetzer, C.; Huenig, I.; Kleinermanns, K.; Nir, E.; de Vries, M. S. ChemPhysChem 2003, 4, 838. (7) Nibbering, E. T. J.; Elsaesser, T. Chem. ReV. 2004, 104, 1887. (8) Krummel, A. T.; Mukherjee, P.; Zanni, M. T. J. Phys. Chem. B 2003, 107, 9165. (9) Krummel, A. T.; Zanni, M. T. J. Phys. Chem. B 2006, 110, 13991. (10) Woutersen, S.; Cristalli, G. J. Chem. Phys. 2004, 121, 5381. (11) Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679. (12) Wang, L.; Yoshida, J.; Ogata, N. Chem. Mater. 2001, 13, 1273. (13) Mahendrasingam, A.; Rhodes, N. J.; Goodwin, D. C.; Nave, C.; Pigram, W. J.; Fuller, W.; Brahms, J.; Vergne, J. Nature 1983, 301, 535. (14) Yang, C. Y.; Yang, W. J.; Moses, D.; Morse, D.; Heeger, A. J. Synth. Met. 2003, 137, 1459. (15) Yang, C. Y.; Moses, D.; Heeger, A. J. AdV. Mater. 2003, 15, 1364. (16) Wang, L.; Yoshida, J.; Ogata, N.; Sasaki, S.; Kajiyama, T. Chem. Mater. 2001, 13, 1273. (17) The positive signal ∆A > 0 found at a delay time of 60 fs with Eex ) 3250 cm-1 (Figure 2c) is due to cross-phase modulation during the temporal overlap of pump and probe pulses. (18) 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. (19) Urabe, H.; Hayashi, H.; Tominaga, Y.; Nishimura, Y.; Kubota, K.; Tsuboi, M. J. Chem. Phys. 1985, 82, 531. (20) Cringus, D.; Bakulin, A.; Lindner, J.; Voehringer, P.; Pshenichnikov, M. S.; Wiersma, D. A. J. Phys. Chem. B 2007, 111, 14193. (21) Tan, H. S.; Piletic, I. R.; Riter, R. E.; Levinger, N. E.; Fayer, M. D. Phys. ReV. Lett. 2005, 94, 057405. (22) Volkov, V. V.; Palmer, D. J.; Righini, R. Phys. ReV. Lett. 2007, 99, 078302. (23) Szyc, Ł.; Unpublished results.
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