Local THz Time Domain Spectroscopy of Duplex ... - ACS Publications

J. Phys. Chem. B 2009, 113, 15619–15628

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Local THz Time Domain Spectroscopy of Duplex DNA via Fluorescence of an Embedded Probe Andre´ Dallmann, Matthias Pfaffe, Clemens Mu¨gge, Rainer Mahrwald, Sergey A. Kovalenko, and Nikolaus P. Ernsting* Department of Chemistry, Humboldt UniVersity of Berlin, Germany ReceiVed: June 28, 2009

We demonstrate that THz vibrational activity of a biopolymer can be measured locally, on the effective length scale for polar solvation, with an embedded molecular probe. For this purpose, the polarity probe 2-hydroxy-7-nitrofluorene was linked into a 13mer DNA duplex opposite an abasic site. The NMR solution structure shows that the fluorene moiety occupies a well-defined position in place of a base pair but can flip around the long axis on a millisecond time scale. Femtosecond optical pump-probe experiments are used to measure the time-resolved Stokes shift of emission from the probe. The dynamic shifts for solution in H2O and D2O are quantified. Their difference is much larger than that expected for free water, implying that only bound water is observed. A weak 26 cm-1 spectral oscillation of the emission band is observed, which is not present when the probe is free in solution and is therefore caused by the supramolecular structure (DNA and hydration water). 1. Introduction Vibrational modes of biological molecules are important for their function, and many have frequencies below 200 cm-1 or 6 THz. Examples are the primary event of vision (60 cm-1),1 oxygen acceptance of hemoglobin (39 cm-1),2 chemical reactions in myoglobin (51 cm-1),3 and conformational change of bacteriorhodopsin (115 cm-1).4 For DNA transcription, the double helix must be opened to expose the coding bases to chemical reactions. Thermal melting of double-stranded (ds) polynucleotides is similar because it starts with a “denaturation bubble”;5 the latter is reached through collective modes between 60 and 140 cm-1, which compress and stretch the interbase H-bonds.6 We seek to observe such motion of dsDNA (to which the remainder applies exclusively) under physiological conditions by the use of molecular probes and here report initial results. Low-frequency motion which involves the helical base-base coordinates is strongly influenced by external forces. Water couples directly in the spine-of-hydration along the minor groove7,8 and, from the hydration sheath and further shells,9 contributes solvation energy to the configuration of DNA base pairs and sugar-phosphodiester backbones. Na+ or K+ counterions are condensed into the spine-of-hydration8,10,11 and distributed in an ion cloud.12,13 However, solvation by water and ions is not only essential for the structure and stability of biomolecules, it also affects the dynamics of supramolecular motion.14 DNA modes become mixed with those of hydration water, and the resulting oscillators may also be strongly damped by fluctuations in the second shell. Both effects lead to broadening, which renders spectroscopic resonances difficult to detect in the low-frequency region. Even though, the existence of low-frequency phonon modes in homopolymeric dsDNA was proved by neutron15 and Brillouin16 scattering of films and fibers. Raman scattering was used to characterize the spectral density of modes which may be considered acoustic.17-19 For some * To whom correspondence should be addressed. E-mail:[email protected].

samples, a peak was observed at 25 cm-1, and a broad band was observed near 85 cm-1, which were assigned to backbone motion and H-bond stretching in complementary base pairs, respectively.20 Far-infrared (FIR) absorption interrogates optical modes which modulate charge distributions; it is therefore dominated by the strong absorbance of hydration and bulk water.21 Measurements in the range of 0.1-3 THz (3-100 cm-1) are improved by time domain spectroscopy (TDS), where the electromagnetic field is generated by switching on a macroscopic Hertzian dipole. A molecular probe functions like a THz light source when its charge distribution is suddenly altered by femtosecond optical excitation S1 r S0. The electric field around the probe is changed instantly and acts on nearby groups with partial charges. Most of these change their nuclear position in an overdamped fashion, but some may oscillate briefly. Altogether, a reaction field R(t) is created which is reported by the polar probe molecule, through an emission frequency which depends on R(t).22 The probe molecule is therefore not only a light source but also a detector. In many ways, the experiment resembles one in which dielectric relaxation is measured locally, the timeresolved Stokes shift (TRSS) indicating the state of polarization. By forming the time derivative, one obtains a response function which is related to the local THz absorption spectrum, and in this way the low-frequency vibrational structure of biomolecules can be accessed. Until now, however, TRSS experiments with biomolecules could only resolve the overdamped, diffusive part of the spectral relaxation. Corresponding results for dsDNA23-27 have been interpreted in terms of solvation by water and ions, with the help of molecular dynamics (MD) simulations.28-35 The extent of the contribution from nucleotides is still controversial. Experiments are clearly needed to test the idea that solvation in complex environments includes the motion of components other than water. What is special in the present work is the ability to see oscillatory motion, that is, a small recurrence in R(t) which is associated with the supramolecular structure, including hydration water, around the probe.

10.1021/jp906037g CCC: $40.75  2009 American Chemical Society Published on Web 09/18/2009

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SCHEME 1: Nucleotide Numbering Scheme for the 13mer Duplexa

Figure 1. Absorption changes upon hybridization, when lowering the temperature from 85 to 25 °C (total single-strand concentration of 23.5 µM). The well-known hyperchromism of the UV absorbance (blue arrow) is accompanied by a 1190 cm-1 red shift of the HNF absorption band. a X refers to an abasic site, that is, a nucleobase is replaced by a H atom.

Molecular THz spectroscopy should reduce inhomogeneous broadening because the perturbing electric field and the reaction field are local. Only those modes which have an oscillator strength in the region at the right direction will interact. The obvious disadvantage is the need to embed a probe molecule inside of dsDNA as an artificial nucleobase. It should be mentioned that the probe has to be free of internal modes which are active below ∼300 cm-1 since they would mix with the macromolecular dynamics to be reported. For this reason the best-studied polarity probes, coumarins,36,37 are not eligible. Instead one must use chromophores which have been shown to report the FIR spectrum of pure liquids such as acetonitrile38,39 or water.40 Required is bio-organic development guided by optical femtosecond spectroscopy in the condensed phase. For a proof-of-principle, we synthesized 2-hydroxy-7-nitrofluorene (HNF) as a polarity probe for oligonucleotides. The chromophore was linked into a 13mer DNA duplex, and the opposite site was taken to be abasic (Scheme 1). The NMR solution structure shows that the fluorene moiety occupies a well-defined position in place of a base pair, even though it is connected by an R-glycosidic bond (rather than β-glycosidic as in natural DNA). However, it can flip around the long axis on a millisecond time scale, resulting in conformational averaging. Femtosecond optical pump-probe experiments were used to measure the time-resolved Stokes shift of emission from the probe. The diffusive dynamics of the time-resolved Stokes shift was quantified for solution in H2O and D2O. Their difference is much larger than that expected for free water, suggesting that only bound water is observed. A weak 26 cm-1 spectral oscillation of the emission band was observed, which was not present when the probe was free in solution; the THz oscillation is therefore caused by the supramolecular structure. A similar oscillation was noted recently in MD simulations.35 To which extent DNA reorganization is reported by the time-resolved Stokes shift seems to depend on the chemical system being studied. This is why the paper focuses equally on structure and femtosecond spectral dynamics. 2. Results 2.1. Hybridization and NMR structure. UV/vis absorption spectra of the DNA-HNF duplex are shown in Figure 1 as a function of temperature. The nucleobases absorb intensely below

Figure 2. Temperature dependence of the spectra in Figure 1. The amplitudes of the two UV components (blue and olive points, right scale, see SI) indicate a melting point of 64 °C (vertical line). The peak position of the HNF absorption band (red points, left scale) shows a melting point which is lower by 3 °C. A simultaneous fit was obtained with a modified zipper model (lines, see text).

300 nm, while the HNF chromophore is seen by a weak band at around 380 nm, corresponding to the S1 r S0 transition. At 85 °C, only single strands are present. As the temperature is lowered to 25 °C spectral changes reflect hybridization to the duplex. Contrast this with the behavior of the HNF absorption band, which shifts significantly to the red upon hybridization (stages are quantified in the Supporting Information, SI). The thermodynamics of duplex formation is examined first, to check whether the probe intercalates and how stable the local arrangement is. Spectral change with temperature is quantified by “melting curves” (Figure 2). The relative UV absorption amplitude decreases from 1 in single strands to 0.74 in the duplex (right scale, blue and olive points for two spectral components as described in the SI). A melting point TM of 64 °C is found for the total concentration cT ) 23.5 µM of single strands, and a hybridization enthalpy of -460 ( 30 kJ/mol41 is estimated from the dependence of TM on cT. The HNF peak position is shown as red points (left scale). With this measure the melting point is located 3 °C lower, indicating local melting

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Figure 3. NMR solution structures showing HNF in the center of the helix. Two orientations are equally populated. Left: the HNF methylene group points toward the major groove, “face-up” to the reader. Right: “face-down” to the minor groove.

or bubble formation around the chromophore. Several models are available to describe the microscopy of duplex melting and to extract thermodynamic parameters from the measurements.42 An essential feature of small oligomers is that near and above TM, they are paired predominantly in the middle of the sequence, while the ends tend to fray. This behavior is captured by the zipper model, which uses generic statistics and needs relatively few parameters. Bubble formation and unstacking of separated strand sections for T > TM were also treated recently.43 We link the UV absorbance change and shifts of the visible HNF absorption band in a similar manner (see SI) and, by fitting the two data sets simultaneously, obtain the corresponding curves in the figure. Comparison with normal dsDNA is instructive. For example, when HNF in Scheme 1 is replaced by a GC base pair, we measure TM ) 69.35 °C under the same conditions. It follows that HNF-DNA is less stable by 6.9 kJ/mol compared to the GC reference,44 equivalent to the lack of enthalpy from hydrogen bonding. The NMR solution structure, as determined from the experimental nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC) data, has essentially the B-DNA form (see Materials and Methods and SI). Conformational flexibility is introduced by the abasic site opposite to the HNF residue.45-50 Although we see only one NOE data set for the duplex as a whole, the subset relating to the HNF chromophore cannot be described by a single orientation. Instead, two structures of the same duplex are needed with different orientations of the chromophore, one where the fluorene methylene group points toward the major groove (face-up) and one where it points to the minor groove (face-down). Interchange between them involves a 180° flip around the long HNF axis. This can take place only during transient opening of the formal HNF-abasic site base pair, which, for natural base pairs, is observed on a

millisecond time scale.51 We conclude that the HNF chromophore undergoes orientational change on a millisecond time scale, and this leads to an averaged NOE data set in agreement with our observations. A 1:1 population ratio between the two orientations is estimated from the integrals of the HNF H1-H1′′ and H3-H1′′ NOE peaks (see Scheme 1 and SI). One could argue that the additional NOE peaks are caused by spin diffusion; however, this can be ruled out because the ratio of the two integrals does not depend on mixing time. Interestingly, the RDC restraints allow both orientations equally well. This can be explained by the orientational degeneracy of RDC data when measured in just one orientational medium.52 Simulated annealing calculations for the two orientations produced two families of structures. The best (minimum-energy, violationfree) 10 of each were used for generating the averaged structures, which are shown in Figure 3. Coordinate fluctuations are 0.66 and 0.47 Å (rms) for the face-up and face-down structures, respectively. The HNF probe fits into the helical fold and stacks with residues 6, 8, 21, and partly 19. This is illustrated by the NMR spectra in Figure 4, where the imino protons of the HNFsubstituted duplex (a) are compared to those of the same duplex containing a central AT (b) or GC (c) base pair. While T21 is fully stacked with the HNF moiety, the T19 residue is turned outward somewhat, away from HNF (see SI). Thus stacking interaction does not stabilize the T19-A8 base pair as well as the other base pairs, and the T19 imino proton is shifted upfield and broadened, similar to the semiterminal imino protons. The T21 imino proton on the other hand, though also shifted upward, exhibits a very sharp resonance due to the ring current of HNF and the stabilizing effect of strong stacking interactions with it.

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Figure 4. Imino proton spectra of the middle nucleobases reflect the stacking of the HNF moiety. The artificial duplex (a) is compared to one with a central AT (b) or GC (c) base pair. The broad T19 and sharp T21 resonances in (a) are shifted upfield from (b and c) considerably.

In the past, base pair mimics devoid of hydrogen bonding have been demonstrated to be incorporated by DNA polymerases with comparable efficiency and even higher selectivity than natural bases due to steric complementarity.53 For example, the pyrene nucleotide can sterically mimic a Watson-Crick base pair and is incorporated into DNA duplexes without distortion of structure or decrease in duplex stability.54,55 However, pyrene has a pronounced effect on the local dynamics of adjacent base pairs, indicated by the presence of two interconverting resonances for the thymine imino proton to the 5′ side and broadening of the imino proton of the adjacent GC base pair.54 The fact that for the HNF-containing DNA duplex we do not observe interconverting signals but substantial broadening of the T19 imino proton indicates that the perturbation of local dynamics is weaker than that for a pyrene residue although local flexibility is induced. Further validation of the calculated structures is achieved by computing the theoretical NOESY spectrum from them and comparing it to the experimental one. Only when the back-calculated NOESY spectra of the duplex with both orientations of the HNF residue are overlaid, the experimental spectrum is reproduced (SI). This supports the idea that both orientations are sampled, leading to an averaged structure. 2.2. Femtosecond Transient Absorption. Transient absorption spectra ∆OD(λ,t) are presented in Figure 5 for the time window until ∼10 ps (all spectra shown refer to H2O buffer solution). For the earliest spectrum in panel (a), the probe interacts with the sample 48 fs after the pump pulse, at which time the induced absorption is almost free from electronic coherence effects.56,57 Negative induced optical density ∆OD in the fluorescence region indicates stimulated emission S1 f S0 (SE). The prominent peak at 430 nm is caused by excitedstate absorption Sn r S1 (ESA), and ground-state bleaching (BL) is expected to make a negative contribution58 at 380 nm. A red shift of the emission band is indicated by horizontal arrows in panel (b). In panels (a) and (c), the shift is overlaid by some rise and decay, respectively, symbolized by the slopes of the arrows. Transient absorption after ∼24 ps is shown in Figure 6. By this late time, we find uniform decay of the characteristic singlet ESA and SE bands, while the known triplet absorption spectrum39 rises to a small amplitude. All spectra shown here were recorded with a magic angle between the pump and probe polarization vectors. The anisotropy (∆OD| - ∆OD⊥)/(∆OD| + 2∆OD⊥) has a well-defined maximum, of 0.34, around 620 nm in the emission band. This measure stays constant until ∼40 ps, when it comes under the influence of triplet absorption.

Figure 5. Early transient absorption spectra of HNF linked into the 13mer DNA duplex, in H2O buffer solution. Optical excitation at 403 nm was followed by white-light probe pulses with 89 fs time resolution. Induced optical density OD < 0 in the fluorescence region outlines a band for stimulated emission S1 f S0 (SE). Excited-state absorption Sn r S1 (ESA) is dominated by a narrow band at 430 nm. It overlays the bleached ground-state absorption (BL), which is responsible for the local minimum at around 380 nm. The shift of the emission band with time (arrows) is the basic observable to be extracted from the measurements. Shown are the raw spectra (after time correction).

Figure 6. Transient absorption after ∼24 ps is characterized by uniform decay (arrows) of the singlet ESA and SE bands while the known triplet absorption spectrum rises to a small amplitude (conditions as those in Figure 5).

The ability of the probe to report on instantaneous polarity appears to be impaired by excited-state decay and photochemical change. On the other hand, substituted fluorene chromophores excel in some relevant properties (see Discussion). Also, despite the aforementioned shortcomings, their solvatochromic behavior

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Figure 7. Species-associated spectra and kinetics (inset) from spectral evolution after 24 ps (Figure 6). The excited S1 state decays with a 35 ps time constant to the hot ground state S*0 and simultaneously to the triplet state T0, which is formed with 12% quantum yield. Dashed lines show the corresponding spectra for the 2′-deoxyriboside of HNF in methanol, in which case the T1 state is populated with 100% yield.41

before 50 ps can be isolated quite accurately; this will be done next. The analysis begins with the decay that is seen in Figure 6. A constant offset and a single exponential component with decay time k1-1 ) 34.8 ps mostly describe the data, but a small contribution by a second exponential component with k2-1 ) 239 ps is also needed. Therefore, at least three species are involved. The last recorded spectrum, at 800 ps, can confidently be assigned to the HNF triplet state T1, which must be included in any kinetic scheme. We find that ISC

S1

98

T1

IC

98

S*0

(1)

cool

98

S0

is the most likely scenario. Figure 7 shows the spectra and timedependent amplitudes (inset) for the species involved. The cooling process corresponds to recovery of the baseline, so that the spectrum associated with S0 is zero by design. The S1 and T1 spectra for the 2′-deoxyriboside dRi-HNF in methanol39 are shown as dashed lines for reference. In that case, intersystem crossing has a quantum yield of ΦISC ≈ 100%, and this observation can be used to calibrate ΦISC. Returning to HNF-DNA in H2O buffer, we find that ΦISC ) 12% generates a T1 spectrum which closely resembles the reference spectrum. From this one obtains kISC ) ΦISCk2 ) 1/290 ps and kIC ) (1 ΦISC)k1 ) 1/39.5 ps, while kCool ) k2 ) 1/239 ps.59 To see the spectral evolution which is complementary, the data of Figure 5 are subjected to two corrections. First is the removal of the rising T1 and S0* absorption, which is known by virtue of their spectra and kinetics (from the decay analysis) by backextrapolation.60 Second is the simulation of the constant S1 population by setting the ESA maximum ) 1. The result is shown in Figure 8, which exhibits the red shift of the SE band and the smaller, simultaneous, blue shift of the ESA band clearly. In the last paragraph of this section, we look for a spectral oscillation which may be attributed to the supramo-

Figure 8. Early transient absorption spectra (from Figure 5) after subtraction of the small and contribution for every time t. Spectra were then normalized at the ESA peak for this figure. In such ways, the SE band becomes accessible (through spectral decomposition, Figure 10).

lecular structure around (and including) the probe. However, before, we proceed to quantify the spectral shifts for solution in H2O and D2O buffer. The temporal shift of the emission band is a key observable through which the relaxation of the environment is accessed.22-27 To obtain this function, each transient spectrum of Figure 8 is decomposed into its BL, SE, and ESA terms. Examples are given in Figure 9 where an early (a) and late (b) transient absorption spectrum is shown (black lines), and the procedure is fully described in the SI. Thus, the SE line shape is obtained in log-normal form,61 with peak position ν˜ P depending on delay time t. In Figure 10, the peak positions in H2O and D2O are plotted on linear and logarithmic time scales.62 As major aim of this work, we look for oscillatory behavior of the emission frequency at early time. To conserve information, it is best to analyze the transient spectra of Figure 8 directly, rather than employing a spectral decomposition whose uncertainties63 can no longer be tolerated at this stage. For example, the absorption trace at 19900 cm-1 (Figure 11, black solid line) reflects the initial sweep, to lower energy, of the blue SE flank. We focus on the evolution for 0.100 e t e 2.5 ps and 450 e λ e 680 nm, where the emission band is prominent. When n ) 4 exponential decay functions are used in a global fit, one obtains a simulated trace at 19900 cm-1 (among all others) which is shown as a red line in the panels of Figure 11. However, systematic residuals remain, which are magnified in the upper part of each panel (gray); they do not disappear when n ) 5 or 6. On the other hand, a complete description is achieved (green lines) by including a time function cos{2πcν˜ osct} exp{-t/τosc}, with optimum frequency ν˜ osc ) 26.4 cm-1 and damping time τosc ≈ 0.56 ps. The amplitude spectrum of the cosine contribution in H2O is shown in Figure 12 (green line), where the stimulated emission band at early time is also provided (red). The oscillation amplitude is seen to be dispersive, like the

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ν˜ (t) ) ∆ν˜ · S(t) + ν˜ (∞)

Figure 9. Decomposition of transient absorption spectra (black lines) into constant bleach and time-dependent contributions from stimulated emission and excited-state absorption. (The T1 and S*0 contributions had been removed as before, and the resulting spectra were normalized to a constant S1 population, as detailed in the SI).

(2)

contains an empirical shift function S(t). The latter may be identified with the autocorrelation function C(t) of the S1-S0 energy gap, as calculated by MD with the probe in the electronic ground state. So far, only overdamped spectral relaxation has been observed in TRSS experiments. Using Coumarin 102 in place of a nucleobase, Berg and co-workers26,27 found that empirical S(t) is not multiexponential with distinct time scales, as believed earlier,24,25 but instead follows a power law to 40 ns and beyond. In a careful analysis of ν˜ (t) in terms of underlying C(t) and its contributions,34 the authors showed that the observed power law behavior can be attributed to solvation by water, whereas the associated contribution from DNA is small (4%) and decays like a 30 ps stretched exponential. The important water is located within 15 Å of the probe;34 its anomalous dynamics (compared to the bulk) suggests confinement in the grooves and/or electrostatic perturbation by the phosphates. Simulations by Pal et al.31 confirmed the existence of multiple time scales for the solvation energy of a probe, taken to be one of the nucleobases in the electronic ground state. Fluctuations of the DNA structure contribute only neglibly. Water reorientation, translational diffusion, and hydrogen bond lifetimes were further evaluated by Bagchi and co-workers,32 who provided the corresponding time correlation functions in triexponential form. Hydrogen bond lifetimes are longer in the minor groove than those in the major groove by almost a factor of 2. Hydrogen bonds with phosphate oxygen have substantially shorter lifetimes than those with the groove atoms. A probe which binds in the minor groove, H33258, was used instead in experiments by Zewail and co-workers.24 In discussing their simulation of this system, Corcelli and co-workers35 came to the opposite conclusion of Pal et al.;31 water plays a negligible role in the slow response. Note that H33258 is a flexible molecule, and conformational relaxation must be included for an accurate description of solvation.35 The simulated DNA

frequency derivative of the SE band. A similar result is obtained with the HNF-DNA duplex in D2O buffer solution. To check whether the oscillation is absent when the DNA environment is removed, one would repeat the experiment with the bare HNF chromophore in water. However, even with dRi-HNF, the solubility in water is too low for quality transient absorption measurements. Therefore the spectral evolution of dRi-HNF in methanol was re-examined39,64 with the same method instead, in which case no comparable frequency modulation was found (Figure 11c). It follows that the spectral oscillation of the emission band is caused by the supramolecular environment. 3. Discussion The time-resolved Stokes shift from a fluorescent probe in place of a nucleobase has been used intensely to study the dynamics of ds oligonucleotides. In general, the following conditions should be met to enable MD simulations of the aqueous DNA probe system and thus a microscopic interpretation: (1) the macromolecular structure is known sufficiently, (2) the probe is relatively rigid, and (3) the interaction change, upon optical S1 r S0 excitation, of the probe with its environment is dominated by electrostatics. To distinguish experimentally between different interpretations, one also needs (4) a large total shift ∆ν˜ ) ν˜ (0) - ν˜ (∞) and (5) fluorescence at late time (symbolized by t ) ∞) when spectral relaxation has ceased. The instantaneous emission frequency

Figure 10. Dynamic red shift of stimulated emission (SE) from HNF-DNA in H2O and D2O by following the peak position ν˜ P of the line shape. Multiexponential fits are shown as underlying smooth lines. For comparison, the relaxation from 100 fs to the quasistationary state was calculated by simple continuum theory (see text).

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Figure 12. The amplitude spectrum of the low-frequency HNF-DNA oscillation, for example in H2O (Figure 11a, green line), changes sign at the center of the stimulated emission band (red). Therefore a frequency modulation of the SE band is observed.

Figure 11. The HNF-DNA absorption trace at 19900 cm-1 (black lines in a,b) reflects the initial sweep of the blue SE flank. Even with a four-exponential global fit over the limited time and wavelength window of 450 e λ e 680 nm (resulting in the red lines), systematic differences remain (gray, shown magnified). An extended fit suggests a damped 26 cm-1 spectral oscillation of the emission band (green lines in a,b). 2′-Deoxyriboside dRi-HNF in methanol shows no oscillation for the equivalent trace at 20820 cm-1 (c).

contribution becomes dominant at long times in this case. The discrepancy could be caused by the different location and direction of the probe relative to the helical axis.34 Intriguingly, a weak oscillatory recurrence at 1 ps is predicted,35 to which we will return later. Changing the nucleobase sequence allows one to modify the coupling of water with phosphate oxygen, for example, and thus the solvation structure and dynamics.27 Using this approach with DNA films, Elsaesser and co-workers65-67 burned spectral holes in the IR region 3000-3600 cm-1 of the N-H/O-H stretching modes and examined the subsequent spectral diffusion. IR spectral hole burning obviously removes the need for substitution of a nucleobase by a polarity probe. The lack of “chemical” localization, however, must be compensated by systematic variation of sequence, hole-burning frequency, and hydration level. For the remainder, let us return to observations with a fluorescent polarity probe. Ernsting and co-workers39 considered the 13mer duplex of the present study, in which 2-amino-7nitrofluorene replaces a central base pair. Water was not explicitly included in Monte Carlo simulations which allowed changes in the ribose-phosphodiester backbone and base-base coordinates only. The emphasis was on low-frequency oscilla-

tions on the TRSS which can be expected from the DNA scaffold. The local infrared absorption spectrum in the range of 0-10 THz is related through time derivations. It has prominent vibrational structure and large changes when neighboring base pairs are altered, suggesting that a sequence can be optimized for time domain spectroscopy with a molecular probe. The time-resolved Stokes shift is now examined. The linked 2-hydroxy-7-nitrofluorene moiety fulfills conditions (1)-(4) for a polarity probe in the 13mer DNA duplex,39 but it fails dramatically on (5) since the S1 lifetime is only 35 ps. On the other hand, the magnitude of the shift is large (condition 4); the peak position ν˜ P of the emission line shape shifts by 2660 cm-1 (C102: 960 cm-1) between 0.1 and 50 ps, the time range shown in the upper panel of Figure 10. By the end of that range, HNF registers ∼80% of the shift which is expected until 40 ns, the present limit of measurements with C102 in dsDNA. To characterize the subpicosecond and picosecond dynamics, the curves in Figure 10 are therefore fitted with triexponential time functions, imposing identical time constants for the slowest term. The results (underlying smooth lines in the figure) are

ν˜ P(H2O) /cm-1 ) 789 exp{-t/0.221 ps} + 1681 exp{t/2.35 ps} + 663 exp{-t/18.7 ps} + 15640 ( 23 ν˜ P(D2O) /cm-1 ) 638 exp{-t/0.324 ps} + 1508 exp{t/2.95 ps} + 828 exp{-t/18.7 ps} + 15640 ( 19 The optimal parameters are correlated so that characteristic times for solutions in H2O and D2O should not be compared by themselves. However, one can see the deuterium effect directly; half of the observed shift is reached in H2O solution at 1.79 ps and in D2O solution at 2.48 ps. The retardation by heavy water is much larger than expected for solvation in free water, as will be shown next. The dielectric dispersion of bulk water is represented in Figure 13 (green line). Bulk H2O has a structured THz-FIR spectrum68-71 with Debye processes at τ1 ) 8.3, τ2 ) 0.25 ps, an intermolecular O · O stretching mode at ν˜ S ) 176 cm-1 (5.3 THz), and an intermolecular librational mode at ν˜ L ) 490 cm-1 (14.7 THz). The dispersion for bulk D2O is also shown (red line).68-70,72 Actually, for the figure we calculated the susceptibility (having

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Figure 13. Solvation response spectra. The dynamic Stokes shift of HNF-DNA (Figure 10) is represented by black lines. The weak oscillatory band at 26 cm-1 (which was inferred from Figures 11 and 12) is also shown separately. Free water responds to dipolar change in a cylindrical cavity at higher frequencies; green and red lines (for H2O and D2O) were calculated from the dielectric dispersion involving Debye terms τ, the intermolecular O · · · O stretching band νS, and the intermolecular librational band νL. Limited time resolution is represented by a spectral filter (blue line).

imaginary part χdip ′′ ) for solvation of a dipole using simple continuum theory.22 For a spherical cavity, the water solvation spectrum has been observed in the time domain where a weak oscillation corresponds to ν˜ S.40 However, in the present case the geometry is different. In the most simple view, the double helix is modeled as an infinite cylinder having refractive index nDNA inside, surrounded by bulk liquid with dielectric dispersion 2 ) εDNA ≈ 3 is ε(ν˜ ). An effective dielectric constant nDNA 29 employed for the base stack zone. The HNF solute is represented by a dipole on the axis, pointing at a right angle to it. Upon optical excitation at t ) 0, the dipole is suddenly increased, and the time-dependent reaction field is calculated.73 The resulting relaxation curves, scaled to match the experimental data at 0.1 and 50 ps, are also shown in Figure 10.74 The shoulder at 200 fs corresponds to the intermolecular stretching mode at ν˜ S, as with the spherical geometry. Such a shoulder is not observed with HNF-DNA, even though the photometric resolution appears to be sufficient. The large difference between solvation curves for HNF-DNA in H2O and D2O is seen to be characteristic of the biopolymer (compared to aqueous solvation of HNF alone). Its presence until 12 ps, covering most of the resolved Stokes shift, implies that only bound water is observed on this time scale. Another contribution to the observed deuterium effect may come from H/D exchange of nucleobase protons which are involved in base pairing. Corresponding N-H modes of nucleobases are expected to be coupled to intermolecular stretching modes,65 with the consequence that H/D exchange would also affect the collective low-frequency response of the duplex structure. The oscillation of the emission at early time (Figure 11) represents a new observation with a biopolymer. It is faintly seen even after the first recurrence at 1.26 ps and does not change measurably upon H2O/D2O exchange. The corresponding amplitude spectrum (Figure 12) matches the ν˜ derivative of the stimulated emission band at early time; it was already concluded that a modulation of the emission frequency is being observed. From the modulation phase and amplitude, one finds that at t ) 0.1 ps, the band is located ∼6 cm-1 above ν˜ P(0.1 ps) of the

Dallmann et al. diffusive dynamics.75 Therefore, the oscillation reflects an additional relaxation process about which we can only speculate. For H33258 bound in the minor groove of an A-tract dodecamer d(CGCAAATTTGCG)2, Corcelli and co-workers35 simulated a partial solvation relaxation function C0DNA(t) which has contributions from the AT region to which H33258 is attached. They find a weak 1 ps recurrence which involves the backbone and bases equally. However, the structure of our system is substantially different because the HNF chromophore replaces a central base pair; hence, we probably observe other modes. For such a geometry, the DNA contribution to the TRSS was estimated to be 4%,34 which, in the present case, corresponds to ∼140 cm-1. The observed oscillation amplitude fits in here, and assignment to DNA motion would be consistent. The hypothesis can be examined with the help of simulation results for our system. We showed previously39 that the energy gap of the chromophore fluctuates with supramolecular coordinates in a susceptibility band which peaks at 18 cm-1.76 That position agrees reasonably with experiment, but the broad width of the band does not; the relaxation is predicted to be overdamped. As a final possibility, water could be observed which is doubly bound to phosphate O or to atoms of nearby nucleobases. It has higher energy and lower entropy compared to singly bound or free water.77 Intermolecular O · · · O stretching motion terminating on doubly bound water would be down-shifted significantly, while reorientational Debye processes are expected to be suppressed. Altogether, a narrow resonance of bound water, strongly coupled to a low-frequency mode of the backbone, is conceivable. A conclusive assignment requires accurate MD simulations of the entire system including the probe and water. 4. Conclusion The molecular probe 2-hydroxy-7-nitrofluorene (HNF) was incorporated into dsDNA opposite an abasic site as a base pair surrogate. The probe occupies a well-defined position inside of the double helix, stacking with adjacent base pairs. The incorporation does not distort the helical B-form, but flexibility in this part of the sequence is enhanced. Femtosecond optical pump-probe experiments are used to measure the time-resolved Stokes shift of emission from the probe. A weak 26 cm-1 spectral oscillation of the emission band is observed which is not present when the probe is free in solution and is therefore caused by the supramolecular structure (DNA and hydration water). We thus demonstrated that THz vibrational activity of dsDNA can be measured locally, on a 15 Å length scale as inferred from simulations,34 with an embedded molecular probe. 5. Materials and Methods 2-Hydroxy-7-nitrofluorene was synthesized in four steps78-81 (SI). The 2′-deoxy riboside dRi-HNF was prepared by reaction with 1′R-chloro-3′,5′-di-O-toluoyl-2′-deoxy-D-ribose in the presence of activated molecular sieve. The R-glycoside was formed predominantly, and it was purified by column chromatography. The phosphoramidide was reached by standard methods.82 Fixed-phase synthesis of the labeled DNA strand (BIOTEZ) required a four-fold increase over the normal reaction time for coupling dRi-HNF. Strands were purified by reversephase HPLC. After hybridization, size exclusion chromatography and ammonia treatment removed low-molecular-weight impurities (mainly Et3N buffer from HPLC). Samples were prepared at pH 7 in aqueous solution containing 10 mM Na2HPO4/NaH2PO4 and 150 mM NaCl. NMR spectra were acquired on a Bruker Avance 600. At a duplex concentration of 3 mM, NOESY, COSY, and HMQC

Local THz Spectroscopy of DNA spectra were measured in D2O (99.98%), a NOESY spectrum in H2O (H2O/D2O 90:10), and a HMQC spectrum in D2O with 20 mg/mL Pf1 phage added in order to induce weak alignment. NOESY spectra in H2O and D2O were each acquired with 16 transients, 4096 (F1) × 2048 (F2) data points, 150 ms mixing time, and COSY spectra with 4096 × 2048 data points and 16 transients. The decoupled HMQC spectrum was measured with 4096 × 512 data points and 128 transients, and the d2 delay was optimal for 1J(CH) ) 145 Hz. The coupled HMQC spectra with and without addition of Pf1 phage were sampled with 128 transients, 8192 × 512 data points, and d2 delay corresponding to aromatic coupling constants 1J(CH) ) 200 Hz. All spectra were processed with the Bruker TOPSPIN program and standard methods. The NMR solution structure was determined from NOE and RDC data by simulated annealing calculations with XPLORNIH v2.21. The experimental data were supplemented with standard hydrogen bond distance restraints from crystal structures and with loose ((50°) dihedral restraints to confine the conformational space to B-DNA. With dihedral restraints for A-DNA, the measured data could not be calculated, and the H1′-H2′/H2′′ coupling constants also suggest pure B-DNA conformation. The HNF residue and the abasic site were subjected to experimental restraints alone, that is, all supplementary constraints were omitted for these residues. Parameters for the HNF residue were taken from DFT calculations performed with the program GAUSSIAN03, with TZVP as the basis set and the CHelpG algorithm to derive atomic charges via the molecular electrostatic potential.83 Stationary optical absorption spectra were recorded on a Cary 300 spectrophotometer. Fluorescence spectra were obtained with a Spex 212 fluorometer by comparison with the emission from a secondary standard lamp (Gigahertz Optic). Transient absorption spectra were induced by 50 fs, 0.6 µJ excitation pulses at 403 nm and measured with time-delayed supercontinuum probe pulses.84 The spot diameter for the pump and probe was 150 and 50 µm, respectively. Delays were stepped 6 fs up to 2.5 ps, 200 fs to 100 ps, and 2 ps to 800 ps. The solution (0.7 mM in the buffer described above with pure H2O) had absorbance of 0.63 at the pump wavelength in a flow cell of 1 mm internal path length. The laser repetition frequency was f ) 920 Hz, and the pump beam was chopped at f/2 to record pump-induced spectral changes. 50 individual records were averaged for a given probe delay of a time scan, and results from 2 to 6 scans were averaged. The resulting spectra were then time-corrected for the chirp of the supercontinuum.82 Baseline noise was better than 5 × 10-4 rms. Measurements were performed at parallel, perpendicular, and magic angles between the pump and probe polarizations, both with the sample solution and with the pure solvent. The latter give the pump-probe intensity cross correlation, which can be described by a 89 fs (fwhm) Gaussian shape over the entire probe region. A spectral resolution of 440 cm-1 (fwhm) is inferred from the width of the water Raman band. Scatter of ∼1-3 mOD at the pump wavelength was constant; it was recorded at negative delays and subtracted from the data. Species-associated spectra were determined as described in ref 85. Acknowledgment. We thank the Deutsche Forschungsgemeinschaft for a grant (ER 154/9-1) to N.P.E., Evonik Industries for a scholarship to M.P., and Dr. G. Herrmann (BIOTEZ) for optimization of the oligonucleotide coupling protocol. Supporting Information Available: The structures of Figure 3 have been deposited in the Protein Data Bank under PDB ID

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