Radiationless Transitions of G4 Wires and dGMP - The Journal of

Jul 16, 2008 - Steady-state and time-resolved emission techniques were employed to study the nonradiative process of deoxygunaosine monophosphate (dGM...
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J. Phys. Chem. C 2008, 112, 12249–12258

12249

Radiationless Transitions of G4 Wires and dGMP R. Gepshtein,† D. Huppert,*,† I. Lubitz,‡ N. Amdursky,‡ and A. B. Kotlyar‡ Raymond and BeVerly Sackler Faculty of Exact Sciences, School of Chemistry, and George S. Wise Faculty of Life Sciences, Department of Biochemistry, Tel AViV UniVersity, Tel AViV 69978, Israel ReceiVed: April 16, 2008; ReVised Manuscript ReceiVed: May 26, 2008

Steady-state and time-resolved emission techniques were employed to study the nonradiative process of deoxygunaosine monophosphate (dGMP) and novel uniform continuous G4 wires containing hundreds of stacked tetrads. We found that the time-resolved emissions of both dGMP and G4 wires decay nonexponentialy. At room temperature, the short-time decay of the G4 wires is about 10 ps. At low temperatures in ice, the fluorescence quantum yields of both dGMP and G4 wires increase as the temperature decreases. For the G4 wires, the fluorescence quantum yield increases from about 10-3 at room temperature to about 0.03 at liquidnitrogen temperatures. The asymptotic long-time decay of the lifetime-corrected emission of the G4 wires obeys a power law. At all temperatures, the average fluorescence decay time of G4 wires is longer than that of dGMP. We successfully used an inhomogeneous nonradiative model to fit the experimental results. Introduction Ultraviolet irradiation causes mutations and death for many microorganisms. The wavelength dependence of the damaging biological effects has been shown to correspond to the absorption spectrum of DNA. Therefore, it was concluded that photodamage centers produced in DNA might have lethal or mutagenic consequences. In the late 1960a, Eisinger and Shulman1 examined the photophysical properties of DNA. They made a connection between the low fluorescence quantum yield of DNA and the short lifetime of the lowest excited state of a single base. They found that the fluorescence quantum yield of DNA nucleobases at room temperature is on the order of 10-4, whereas at liquid-nitrogen temperature, the quantum yield increases to about 0.01. For a pure radiative lifetime of about 10 ns, their results indicated a nonradiative rate constant of 1012 s-1, i.e., 〈τnr〉 ) 1 ps. Recently, femtosecond techniques were used to monitor the ultrafast nonradiative decay2–11 of nucleobases and DNA structures. A review by Kohler and co-workers summarizes the recent achievements in the field.7 Kohler and co-workers6,7 reported transient absorption measurements performed on four DNA nucleosides in aqueous solution. After excitation at 266 nm, transient absorption spectra were recorded in the whole UV-vis region. They observed different characteristic decay times for the transient absorption observed in the UV (close to the excitation wavelength) and visible regions and explained the difference in the results as arising from a two-step relaxation pathway leading to hot vibrational levels in the ground state. All excited-state decay signals showed a major component shorter than 1 ps. More recently, Kohler and co-workers8,11 reported the lifetimes of the S1 electronic states of cytosine and some of its derivatives. They found the same lifetimes, 1.0 and 0.2 ps, respectively, for the base and nucleoside of each of these molecules. Direct measurements of the excited-state lifetimes of four nucleosides and four nucleotides in aqueous solution were measured by Peon and Zewail9 using fluorescence upconversion. The lifetimes given * Corresponding author. Fax/phone: 972-3-6407012. E-mail: huppert@ tulip.tau.ac.il. † School of Chemistry. ‡ Department of Biochemistry.

for the nucleosides ranged between 0.5 and 1.0 ps, close to the values given in ref 10. Sharonov et al.2 reported excited-state lifetimes of several DNA constituents in aqueous solution obtained by the fluorescence upconversion technique, including the thymine homologous series, the adenine homologous series, and a comparative study of eight DNA nucleosides and nucleotides. The results led the authors to the conclusion that the fluorescence decays cannot be characterized by a monoexponential decay, implying a more complex excited-state relaxation process. The quantum yield of DNA is generally higher than that of the nucleobases, but the intrinsic fluorescence from DNA is still very weak. Crespo-Hernandez and Kohler11 showed that the fluorescence quantum yield of poly(dA) is 2.5 times higher than previously reported for the poly(A) ribonucleotide polymer. Ge and Georghiou12,13 demonstrated large differences in the fluorescence emission of alternating poly(dA-dT)-poly(dT-dA) and nonalternating poly(dA)-poly(dT) polynucleotides that were composed of equal amounts of the T and A bases and differed only in the spatial arrangement of the bases. The authors suggested that the fluorescence emission from DNA structures is sequence-dependent and is determined by the ground-state base-stacking morphology. Electronic energy coupling that occurs between the bases in the base-stacked regions of DNA thus seems to support a long-lived electronic excitation, and as a result, there is an increase in the fluorescence lifetime and intensity. Double-stranded B-DNA is the most common form of DNA found in living organisms. However, many other forms of DNA, with molecular structures different from the classical double helix, are known. One of those structures is G4 wires, also known as G-quadruplexes, which comprises a series of stacked guanine tetrads, held together by Hoogsteen and Watson-Crick hydrogen bonds.14,15 These structures (see Scheme 1) are present in biologically significant regions of the genome and play an important role in the processes of aging and cancer.16–18 The larger surface area and seemingly enhanced interplanar packing between the tetrads19 might lead to improved π-stacking in G4 wires with respect to the stacking between bases in native DNA. Onidas et al.2 studied the photophysical properties of triguanosine oligonucleotide (G3). They showed

10.1021/jp803301r CCC: $40.75  2008 American Chemical Society Published on Web 07/16/2008

12250 J. Phys. Chem. C, Vol. 112, No. 32, 2008 SCHEME 1: Chemical Structure of the Guanine Tetrad, in Which Four Guanines Are Arranged in a Plane through Hoogsteen Hydrogen Bondsa

Gepshtein et al. display a solute-solvent configuration such as a hydrogen-bond length or an angle or a generalized coordinate that expresses the hydrogen-bond energy. The potentials of the ground and excited electronic states (V0 and V1, respectively) in this coordinate are assumed to be biharmonic, that is

V0(x) ) R0(x-x0)2 V1(x) ) R1x2 - R0x02

a

R groups are for the ribose of the molecule’s backbone.

that, in the presence of a high concentration of Na+, the fluorescence quantum yield of the G3 solution is about 3 times higher than that of the monomer, deoxyguanosine monophosphate (dGMP). It is known that short G-rich oligonucleotides form polydisperse aggregates arising from the self-association of various numbers of molecules. This association of G-rich oligonucleotides yields various nonuniform higher-ordered structures bonded by guanine-guanine base pairs, base quartets,14,15 pentads,20 and hexads21 that can contribute differently to the overall fluorescence of the sample. We recently reported the synthesis of novel uniform continuous monomolecular G4 wires composed of single self-folded poly(dG) strands containing hundreds of stacked tetrads.19 The G4 wires have a uniform structure and size and are composed of hundreds of tetrads.19 They originate from intramolecular folding of a long (thousands of bases) poly(dG) strand four times on itself in an antiparallel fashion. The G bases in the wire are highly ordered, which leads to an improved π-π stacking, and as a result, the electrical properties of the wires improve in comparison to those of classical double-stranded DNA.22 In this study, we report that, at room temperature, the fluorescence intensity of G4 wires is about 10 times stronger than those of dGMP and poly(dG)-poly(dC). In addition, the time-resolved fluorescence of G4 wires is much longer than that of dGMP. We also conducted a temperature dependence study of the fluorescence decay over a wide range of temperatures from 80 to 280 K. We found that, at all temperatures, the fluorescence decay is nonexponential. We also measured the photophysics of the monomeric unit dGMP for comparison with the results obtained for the G4 wires. As in the case of the G4 wires, we found that, in this temperature range, the fluorescence decay for dGMP is nonexponential. At a particular temperature in the frozen sample, the average decay time of dGMP is shorter than that of the G4 wires. We successfully used the same model of nonradiative processes that we used recently for a model compound of the green fluorescence protein (GFP) chromophore p-hydroxybenzylidene dimethylimidazolone (p-HBDI)23 to fit the fluorescence decay of both G4 wires and dGMP. Model for the Nonradiative Process In this section, we describe a simple theoretical model for nonradiative rates leading to nonexponential fluorescence decay. This model was already used in our previous study on p-HBDI,23 the GFP synthetic chromophore, for the dynamics of internal conversion. This model assumes a single floppy coordinate, x, that controls the nonradiative process of the electronically excited system. In this study, we propose that the x coordinate

(1)

The ground-state potential intersects the excited-state potential at its minimum, for simplicity defined as x ) 0. Because the time-resolved emission contains no distance scale, for simplicity, it is chosen as the distance unit for which R0 ≡ 1. In the case of nonrediative decay of nucleobases such as guanine, the nonradiative process can involve an intermediate excited 1nπ* state.24 The 1ππ* state is strongly coupled to the 1nπ* state, and the 1ππ* population crosses first to the 1nπ* state, which does not carry a large oscillator strength with the ground electronic state and hence is a “dark” state. The formalism of our model is also valid for this case. The inhomogeneity arises from the equilibrium distribution on the ground-electronic-state surface V0(x) prior to excitation. It is given by the Boltzmann distribution and, for a harmonic potential, exhibits a Gaussian shape

p(x) )

1

√2πσ

[

exp -

(x - x0)2 2σ2

]

(2)

where σ2 is the distribution width. In the static limit, no dynamics takes place along the x coordinate, so that p(x) remains time-invariant. The nonradiative process is inhomogeneous, and therefore, instead of having a single rate constant, knr, each x configuration decays to a hot vibrational state in the ground electronic state at a rate constant knr(x). In the model, it is assumed that knr(x) obeys an “energy-gap law”27

k(x) ) A exp[-γ∆V(x)]

(3)

where A is a preexponential with units of 1/time and γ is a characteristic parameter. According to the gap law, there are two limits for the dependence of k(x) on x: (i) k(x) is an exponential function of x and (ii) k(x) depends quadratically on x. We treated the experimental data of the DNA nucleobase as belonging to the second case, which leads to a Gaussian dependence of x given by

k(x) ) Aexp(-bx2)

(4)

where A is the nonradiative rate constant at x ) 0, which is the equilibrium position of the ground state. b is quadratic prefactor and is independent of temperature. In our previous study, 23 we argued that the only temperaturedependent term in the model is the exponential term b(T). We found that b increases linearly as a function of 1/T.23 In such a case, the activation energy of the nonradiative process depends on the value of x. In the fitting of the radiationless process of HBDI, the activation energies for x ) 0 and x ) 2 are 0 and 18 kJ/mol, respectively. The experimental data of the current nucleobase study indicate that the temperature dependence of the nonradiative process is quite complex. In nonviscous liquids, the nonradiative rate is ultrafast even at rather low temperatures. The nonradiative rate in ice matrix is much lower than that in the liquid state at the same temperature. Therefore, in this study, we argue instead that the temperature dependence of the distribution as a whole has a single value that is independent

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of the value of x. The temperature dependence of the nonradiative rate enters through the term A(T) in eq 4, whereas we held the b term fixed for all of the experimental data for both dGMP and the G4 wires. In the static limit, the probability P(t) that the excited state has not decayed by time t after excitation is given by

P(t) ) exp(-t ⁄ τf)

∫0∞ p(x) exp[-k(x)t] dx

(5)

The integral represents the inhomogeneous nonradiative decay kinetics. The decay of P(t) is nonexponential and depends on σ2, the width of the population p(x), the value of A(T), the quadratic prefactor b, and the pure radiative rate constant. The observed normalized transient fluorescence signal, I(t), is a convolution of the instrument response function (IRF) with the theoretical decay function given in eq 5. Experimental Section Time-resolved fluorescence was acquired using the timecorrelated single-photon-counting (TCSPC) technique, the method of choice when sensitivity, large dynamic range, and lowintensity illumination are the important criteria in fluorescence decay measurements. The experimental details of the optical setup are given elswere.24 The sample was irradiated by the third-harmonicgeneration (THG) frequency, in the spectral range of 260-290 nm with a relatively low repetition rate of 500 kHz and a pulse width of about 250 fs. The temperature of the irradiated sample was controlled by placing the sample in a liquid-nitrogen cryostat with a thermal stability of approximately (1 K. Results The steady-state emission spectra of aqueous solutions of the G4 wires, poly(dG)-poly(dC), and dGMP are shown in Figure 1a. The 2800-base-pair poly(dG)-poly(dC) and the 5000-base G4 wires were synthesized using enzymatic procedures described in our recent publications 19,25 The emission spectra of all of the above samples were recorded from solutions with absorptions of approximately 0.2. As seen in Figure 1a, the fluorescence intensity of the G4 wires is much higher than those of dGMP and poly(dG)-poly(dC). The relatively high fluorescence intensity of the G4 wires, compared to those of doublestranded poly(dG)-poly(dC) and dGMP, might be due to cooperative π-π stacking interactions between hundreds of tetrads in the wire and should thus depend on the ground-state base stacking morphology. The π-π interactions between the stacked tetrads in the wire might cause delocalization of the excitation, thus leading to a considerable decrease of the nonradiative decay rate and a considerable increase of the fluorescence emission compared to that of dGMP.26–28 As seen in the figure, adding K+ to the G4 wires results in a strong increase of the fluorescence intensity. We have shown that the structure and optical properties of the G4 wires are strongly affected by K+.19 Figure 1b shows the normalized steady-state emission spectra of dGMP, the G4 wires, and the G4 wires in the presence of 10 mM K+ ions. The individual spectra differ from each other. The peak of the emission spectrum of G4 shifts to the red, and the red side of the spectrum shows a large increase of its width. As seen in Figure 2, adding K+ also leads to a dramatic change in the circular dichroism (CD) spectrum of the G4 wires: the negative band at 280 nm completely disappeared from the CD spectrum of the molecules when K+ was added (compare solid and dashed lines in Figure 2). These changes reflect

Figure 1. (a) Steady-state fluorescence emission spectra of poly(dG)poly(dC), dGMP, G4 wires, and G4 wires in the presence of 10 mM KCl. (b) Normalized steady-state fluorescence emission spectra of dGMP, G4 wires, and G4 wires in the presence of 10 mM KCl.

Figure 2. Effect of inserting K+ ions into G4 wires molecules on the CD spectrum.

differences in the secondary structure of the G4 wires induced by the cation. Thus, the results presented in Figures 1 and 2 show that the fluorescence is sensitive to the ground-state morphology of the G4 wires and suggest that the G4 wires have different decay pathways for a singlet excitation energy, unlike a single G base whose decay pathway is determined by the ground-state base stacking of the DNA. Time-resolved fluorescence measurements at room temperature of aqueous solutions of the G4 wires, poly(dG)-poly(dC), and dGMP in which the time-correlated single-photon-counting (TCSPC) technique was employed are presented in Figure 3.

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Figure 3. Time-resolved fluorescence measurements at room temperature of aqueous solutions of G4 wires, poly(dG)-poly(dC), and dGMP.

The samples were excited by a short pulse of about 300 fs and 270 nm at 500 kHz, and the fluorescence was monitored at 350 nm, where the center wavelength of the broad emission bands of both the G4 wires and dGMP is approximately located. As seen in Figure 1, the emission decay of the G4 wires (trace 1) clearly shows a much longer decay time than do those of dGMP and poly(dG)-poly(dC) (traces 2 and 3, respectively). The decay is nonexponential and could be fitted by three exponentials. The lifetime of the main and shortest component was approximately 10 ps. The emission decays of the latter two compounds follow the instrument response function (IRF) limit, trace 4, pointing out that the emission relaxation rates are shorter than 5 ps, the resolution of the TCSPC technique. Figure 4a shows the time-resolved emission of the G4 wires and dGMP in aqueous solution at several temperatures. The G4 wire sample was buffered with 2 mM Tris buffer (pH 7.4 at room temperature), whereas the dGMP samples were in neutralpH, HPLC-grade water. Each part of the figure contains two decay curves: that of the G4 wires and that of dGMP. As seen in the figure, at a particular temperature, the average decay time of the G4 wires is much longer than that of dGMP. For both compounds, the fluorescence decay curves are nonexponential. The nonexponential decay is also observed at liquid-nitrogen temperatures (∼80 K). Figure 4b shows a double logarithmic plot of the lifetime-corrected decay curves for the G4 wires and dGMP presented in Figure 4a. As seen in Figure 4b, the decay curves of the G4 wires at long times obey a power law (straight line in the figure), whereas the dGMP decays as a stretched exponent. The slope of the log-log plot is 1.2 ( 0.2, and its temperature dependence is weak in the frozen sample. The lower the temperature, the larger the slope. At T ) 237 K the slope is 0.98. In the inhomogeneous model used in this study, the slope of the power-law decay depends on the population width σ2. The larger the width, the smaller the slope. Fit of the Experimental Results with the Inhomogeneous Radiationless Transition Model. We used the inhomogeneous model (see eq 5) to calculate the time-resolved fluorescence of the DNA and dGMP. Figure 5 shows the fitting curves (solid lines) of the experimental time-resolved emission data (dots) of the G4 wires at several temperatures in the range of 80-280 K. As seen in the figure, the model fits the experimental data nicely at all temperatures. The fitting parameters used were A(T), the preexponential factor of the rate constant k(x), and σ2, the width of the distribution p(x) (eq 2). x0, the position of the center of both the excited-state population Gaussian distribution and

Gepshtein et al. the excited-state potential minimum with respect to the origin of the x coordinate, was placed at x0 ) 1 Å. The exponential distance dependence of the quadratic term b of the rate constant k(x) (see eq 4) for both systems was kept fixed at all temperatures, with b ) 1.08 Å. We used the value τf ≈ 34 ns of the pure radiative excited-state lifetime for both the G4 wire and dGMP samples. The value is about twice as long as the jet-cooled measured lifetime.29–31 For the numerical calculations of the computed fits, we used the user-friendly graphic program SSDP (version 2.63) of Krissinel and Agmon.32 An additional parameter in the fit, which only slightly affects the long times of the signal, is the diffusion constant along the x coordinate of the population distribution, D. The distribution of p(x) on the excited-state potential surface immediately after the short pulse excitation is determined by the ground-state conformation, as well as by processes in the excited state prior to or competing with the radiationless process, such as excess energy intermolecular dissipation and intramolecular redistribution, as well as intermolecular solute-solvent coordinate time dependence such as hydrogen bonding and solvent fluctuations. We assumed that some rearrangement processes that affect the distribution take place on a long time scale. At low temperatures the processes that affect p(x) are probably on a time scale of tens of picoseconds. From the best fit to the experimental results, we find that, for dGMP, the value of the diffusion constant in the high-temperature region is about 2 × 10-11 cm2/s. In the low-temperature region of T < 175 K, the D value is ∼1 × 10-11 cm2/s. The use of a diffusion constant allows the population along the x axis in our model to change slightly within the experimental time window of 10 ps < t < 10 ns. In our model calculations, we imposed a weak harmonic (parabolic) potential around the equilibrium position x0 of the form U(x) ) c(x - x0)2. We set a value of 1.4 kJ/(mol · Å2) for c. The diffusion constant that we used for the fit of the experimental data obtained with the G4 wires was smaller, i.e., D ≈ 5 × 10-12 cm2/s. The small D value used for the fit of the G4 wires extends the time-resolved emission signal to longer times and tends to keep the long-time decay of the signal as a power law, t-d, where the d values are around 1.2 ( 0.2 over the temperature range 80 K < T < 180 K. A(T) is the temperature-dependent nonradiative rate constant at the curve crossing points of S1 and S0, i.e., x ) 0. The width, σ2, of the population distribution p(x) also depends on the temperature. For a system in thermal equilibrium in a harmonic potential, the mean square of the distribution σ2 scales with the force constant, k, of the potential energy, and the width is given by σ2 ) kBT/k. In the ice samples for both dGMP and the G4 wires, we found that the width of the population decreased with the temperature. The fitting parameters of the experimental data are given in Tables 1–3. We also include in the tables the average fluorescence lifetimes. To diffentiate between a pure reactive temperature effect (over the barrier crossing) and solute-solvent frictional effects, we used mixtures of water and monols to decrease the freezing point of the samples. Figure 6a,b shows the time-resolved fluorescence of dGMP in methanol-rich (66% by volume) methanol-water mixtures and ethanol-rich (66% by volume) ethanol-water mixtures, respectively. These samples freeze at -75 and -40 °C, respectively. In the liquid state, the effective lifetime in both samples is shorter than the instrument response function of the TCSPC technique. In the solid state, the fluorescence lifetime is much longer and depends on the temperature. The freezing point of the methanol-rich methanol-water mixture is about -75 °C. In polycrystalline ice samples of pure water, the

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Figure 4. (a) Semilogarithmic plot of the time-resolved emission of G4 wires and dGMP in frozen aqueous solution at several temperatures. (b) log-log plot of the lifetime-corrected decay curves of G4 wires and dGMP shown in part a.

nonradiative rates are much slower than corresponding rates measured in the liquid phase of the methanol-rich (66%) methanol-water mixture in the temperature range from 240 to 200 K. The solid-state (T < 190 K) methanol-water mixture structure is crystalline, for which we find (see in Figure 6a) that the nonradiative rate is much slower than that in pure ice dGMP samples. For high-methanol-concentration water-methanol

mixtures in the solid state, the nonradiative rate temperature dependence is relatively small over the temperature range of 86 K < T < 177 K. The water-ethanol mixture sample freezes already at about -40 °C. As seen in Figure 6b, in the liquid sample, the fluorescence decay of dGMP is ultrafast, whereas in the solidstate phase, the decay is rather long, longer than that in pure-

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Gepshtein et al. is a significant decrease in the nonradiative rate when dGMP is dissolved in 1% methanol-doped ice. Discussion

Figure 5. Model fitting (solid lines) of the experimental time-resolved emission data (dots) of G4 wires at several temperatures (top to bottom): 121, 126, 148, 197, 237, and 260 K.

water dGMP ice samples. Water-ethanol mixtures at low temperatures are known to form glasses, and hence, the decay time (below -40 °C) depends much more on the temperature than in the polycrystalline water-methanol mixture. These findings clearly show that solute-solvent friction has a profound importance in controlling the nonradiative rates of both dGMP and G4 wires. Figure 7 shows the time-resolved emission of dGMP samples at several temperatures in 1% methanol-doped ice and in methanol-rich (66% by volume) methanol-water mixtures. At 227 K, the methanol-rich methanol-water mixture is still a liquid, whereas the 1% methanol-doped ice sample is a polycrystalline ice. As seen in the figure, the decay of the liquid sample is much faster than that of the solid. At 129 K, both samples are in the frozen phase. The decay of dGMP at T < 129 K in the methanol-rich methanol-water mixture is much longer than that of the 1% methanol-doped ice sample. These results indicate that the nonradiative processes might be coupled to solute-solvent rotational transitional motions. Effect of Methanol Concentration in Methanol-Doped Ice on the Nonradiative Rate. Figure 8 shows the time-resolved emission of dGMP in two samples of methanol-doped ice containing 0.1% and 1% methanol by mole ratio. Each panel shows two signals: that of dGMP in 0.1% methanol-doped ice and that of 1% methanol-doped ice samples measured at a particular temperature. As seen in the figure, the decay curves are nonexponential. The average decay rate strongly depends on the methanol concentration in the ice samples; the higher the methanol concentration, the longer the decay time. The decay rate of the time-resolved emission of dGMP in pure ice (no methanol) is somewhat higher than that in a 0.1% methanoldoped ice sample. The figure also shows a computer fit using the inhomogeneous nonradiative model of the time-resolved emission of dGMP. At a particular temperature, the preexponential constant A(T) is the only parameter that was varied in the fit of the experimental data obtained for the two samples. At a particular temperature T, A(T) is much larger for the 0.1% methanol-doped ice sample than for the 1% methanol sample. In a recent study,24 we found that both the rate of proton transfer from a photoacid in ice and the proton mobility in ice are strongly dependent on the methanol concentration of methanoldoped ice. Proton mobility is 3 times smaller in 1% methanoldoped ice than in 0.1% methanol-doped ice. We therefore propose that methanol plays an important role because of its specific binding to the nucleobase. The overall effect produced

Bagchi, Fleming, and Oxtoby (BFO)33 suggested a nonradiative decay model via a nonadiabatic curve crossing. In this model, a rapid internal conversion is assumed to occur only at the intersection of the potential curves (Figure 1 of ref 23). It is therefore described by a delta-function “sink term”. The distribution, after being excited to the upper state, first has to diffuse to the intersection point in order to decay nonradiatively back to the ground state. This suggests that, at medium viscosities, delay in the nonradiative process is expected. In highly viscous solvents, no internal conversion should occur, because the distribution cannot diffuse to the intersection point. Thus, at sufficiently low temperatures, the excited state decays radiatively at an exponential rate. The BFO model’s prediction contradicts experimental observations of the present study as well as the data obtained in our previous study on p-HBDI23 of a fast (picosecond) nonexponential internal conversion also in viscous solvent glasses at low temperatures and in solid polycrystalline ice. Therefore, the curve-crossing frame requires the sink term to be nonlocal, thus making internal conversion even at very short times after excitation possible. It is coupled to an inhomogeneous distribution in the same coordinate, which, by a photoexcitation process, is transferred from the ground state to the excited state. Clear evidence for inhomogeneous broadening comes from the shape of the very broad and structureless steady-state fluorescence spectra of both G4 wires and dGMP. In a recent work,23 we used a time-resolved emission technique to monitor the nonradiative transition of the anion of p-HBDI, a model chromophore of the green fluorescence protein, in viscous glycerol-water mixtures over a large range of temperatures. We found that, at all temperatures, the decay of the fluorescence is nonexponential and the average decay rate strongly depends on both the temperature and the solution viscosity. We used a one-dimensional model to explain and fit the efficient radiationless transition of the p-HBDI molecule in frozen water-glycerol mixtures. Similar inhomogeneous kinetics is included in ligand binding to myoglobin.34 In this study, the time profiles of the time-resolved emissions of both the G4 wires and dGMP exhibit nonexponential decays. The decay rates strongly depend on the phase of the medium of the sample. In polycrystalline and frozen glass samples, the decay rates are significantly lower than those in fluid liquid samples. Role of the Host in the Nonradiative Process. Domcke et al.35 and Sobolewski et al.36 carried out ab initio electronicstructure calculations on nucleobases and related heterocyclic compounds containing NH2 and OH. From these calculations, they drew a general mechanistic picture of the nonradiative decay of nucleobases and other similar structured molecules. An important part of their model is an intermidiate excited singlet state with πσ* character that has repulsive potentialenergy functions with respect to the stretching of OH or NH bonds. The 1πσ* potential-energy functions intersect not only the bound potential-energy functions of the 1ππ* excited states, but also the potential-energy functions of the electronic ground state. An ultrafast internal-conversion process takes place via predissociation of the 1ππ* state to the 1πσ* state, and subsequently, a conical intersection with the ground state ends the photocycle. In protic solvents, the 1πσ* states spur a hydrogen-transfer process from the chromophore to the solvent. Calculations for chromophore-water clusters have shown that

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TABLE 1: Temperature Dependence of the Kinetic Parameters of the Nonexponential Dynamics Model for G4 Wires in H2O 1

T (K) 108 114 121 126 131 136 141 160 a

/2σp2a (Å2)

Ab (109 s-1)

〈t〉 ( ns)

T (K)

3.65 3.55 3.55 3.45 3.45 3.30 3.15 3.15

4.5 4.9 5.5 5.8 6.3 6.7 8.2 9.0

2.20 1.125 1.02 0.985 0.950 0.920 0.820 0.676

173 185 197 210 222 235 247 260

1

/2σp2a (Å2)

Ab (109 s-1)

〈t〉 ( ns)

2.95 2.85 2.65 2.35 2.35 2.10 2.10 2.28

11.5 13.0 15.5 27.5 27.5 35.0 50.0 90.0

0.572 0.551 0.42 0.37 0.34 0.30

See eq 2. b See eq 4.

TABLE 2: Temperature Dependence of the Kinetic Parameters of the Nonexponential Dynamics Model for dGMP Methanol-Doped Ice, 1% Mole Fraction 1

T (K) 82 99 105 129 141 153 a

/2σp2a (Å2)

Ab (109 s-1)

〈t〉 ( ns)

T (K)

2.65 2.65 2.65 2.65 2.65 2.65

5.0 5.6 6.2 7.4 8.0 8.75

1.460 1.338 1.210 1.130 0.108 0.910

165 177 190 202 215 240

1

/2σp2a (Å2)

Ab (109 s-1)

〈t〉 ( ns)

2.65 2.65 2.50 2.12 2.12 2.42

9.40 10.5 13.5 22.5 26.5 80

0.860 0.815 0.685 0.519 0.434 0.174

See eq 2. b See eq 4.

TABLE 3: Temperature Dependence of the Kinetic Parameters of the Nonexponential Dynamics Model for dGMP Methanol-Doped Ice, 0.1% Mole Fraction 1

T (K) 82 93 105 117 129 a

/2σp2a (Å2)

Ab (109 s-1)

〈t〉 ( ns)

T (K)

3.6 3.0 2.6 2.48 2.42

13 18 27 32 37

0.300 0.292 0.277 0.273 0.256

141 153 165 177 190

1

/2σp2a (Å2)

Ab (109 s-1)

〈t〉 ( ns)

2.35 2.28 2.28 2.2 2.0

46 66 80 150 220

0.217 0.164 0.135 0.119

b

See eq 2. See eq 4.

a charge-separation process takes place spontaneously in the solvent shell, producing a microsolvated hydronium cation and a microsolvated electron. The experimental results of the current study show that the nonradiative rates of both G4 wires and dGMP strongly depend on the phase of the matrix in which the nucleobases are positioned.37 At room temperature, in the liquid phase, the nonradiative rate is ultrafast for dGMP (∼1 ps) and is somewhat slower for the G4 wires (about 10 ps). Most of the temperature dependence measurments of this work were performed in polycrystalline ice. Ice is generally a bad solvent, and thus, guest molecules tend to be expelled from the crystalline bulk and to form aggregates at the grain boundaries. When a pure aqueous solution of hydroxyl-containing aryls (photoacids24) and similar large fluorescent dye molecules freeze, the strong fluorescence observed in the liquid state disappears. The fluorescence quenching is explained by the exclusion of the dye molecules from the bulk and their aggregation at the grain boundaries.24 In a previous study,23 we found that a small amount of methanol in water (about 1% by volume) prevents the exclusion of many molecules from the ice bulk. In the case of dGMP and the G4 wires in ice samples prepared by freezing a pure water solution, we were unable to detect the exclusion of the chromophores from the bulk ice. When the samples freeze, the fluorescence intensity increases dramatically, and the excited-state effective lifetime increases as well. We explain the strong effect of the phase change on the luminescence properties by the fact that a large portion of the nonradiative process depends strongly on the solute-solvent interactions. The larger the stiffness of the host matrix in which the nucleobase is dissolved, the slower the nonradiative rate. Probably hindered translational and orien-

tational motions linked to hydrogen bonds between the host and the guest control the nonradiative rates. These low-frequency intermolecular modes might be responsible for the strong coupling between the 1ππ* state and 1nπ* states,38 or according to Sobolewski et al. and Domke et al.’s theory, the population is first transferred from the 1ππ* states to the 1πσ* state and then by conical intersection back to the ground state, S0. The low-frequency modes are heavily overdamped, and thus, the crossing rate is not a single rate constant because a nonlocal sink term should replace the traditional local sink term resulting in a nonexponential decay rate. Similar nonexponential behavior is also observed in solvation dynamics of large dye molecules in hydrogen-bonding liquid solvents such as monols, diols, and glycerol and also in ice.39 The G4 structure in the presence of K+ ions is robust and contains within the wire a limited number of H2O molecules. The structure is held together by interactions between the monomeric units. The main interactions are Hoogsteen and Watson-Crick hydrogen bonds and π-stacking. We propose that the lower nonradiative rate of G4 wires compared to dGMP at room temperature arises from the slower dynamics of both water molecules surrounding each individual guanine residue and hydrogen bonding in the G4 structure. The overall effect of the structured G4 wire is a reduction of the nonradiative rate. Temperature Dependence of the Nonradiative Rate. Figure 9 shows an Arrhenius plot of ln[A(T)] versus 1/T for the G4 wire samples in ice. A(T) is the nonradiative rate constant at x ) 0. It is the largest nonradiative rate constant for a Gaussian distribution of the population p(x) placed at x ) 1 Å from the origin (the crossing point of the ground and excited states; see eq 2). As seen in the figure, the slope of the Arrhenius plot is

12256 J. Phys. Chem. C, Vol. 112, No. 32, 2008

Gepshtein et al.

knr1(T) ) k01 exp(-Ea1/RT) knr2(T) ) k02 exp(-Ea2/RT)

(6)

where k01 . k02 and Ea1 . Ea2. At a certain temperature, the total nonradiative rate constant is given by

ktot)k1+k2

Figure 6. Time-resolved fluorescence of dGMP at several temperatures in (a) methanol-rich (66% by volume) methanol-water mixtures in the liquid and polycrystalline solid and (b) ethanol-rich (66% by volume) ethanol-water mixtures in the liquid and the glass state.

Figure 7. Time-resolved emission of dGMP in 1% (by volume) methanol-doped ice and in methanol-rich (66% by volume) water mixtures.

not constant. At low temperatures, the slope is much lower than at high temperatures. A plausible nonradiative mechanism that might explain the non-Arrhenius temperature dependence of the nonradiative rate includes two coordinates, rather than one, that control the overall nonradiative rate. In this case, we can assign a rate constant for each coordinate (k1 and k2). The temperature dependence of each rate constant follows an Arrhenius law. Let us assume that k1 and k2 are substantially different from each other in both their activation energy and their preexponential factor

(7)

In such a case (by choosing the precise parameters), the main nonradiative channel at high temperatures is that of the rate constant k1, whereas at low enough temperatures, a switchover occurs where k2 g k1. Figure 9 shows the dependence of ln[A(T)], the inhomogeneous nonradiative model parameter, on 1/T for G4 wires, along with the calculated curves of ktot, k1, and k2 (solid curves). The fit is rather satisfactory at both high and low temperatures. We used k01 ) 2 × 1013 s-1, k02 ) 2.5 × 1010 s-1, Ea1 ) 13 kJ/mol, and Ea2 ) 1.5 kJ/mol. Figure 10 shows a plot similar to Figure 9 but for dGMP in 0.1% and 1% methanol-doped ice. The fitting parameters using the inhomogenous nonradiative model are given in Tables 2 and 3. We also include in the tables the average fluorescence decay time, 〈τ〉, of the dGMP samples. As in the case of the G4 wires, A(T) depends strongly on temperature in the hightemperature region (T > 180 K), whereas at low temperatures, T < 180 K, the preexponential rate A(T) is far less dependent on the temperature. The parameters for fitting the 0.1% methanol-doped ice sample are k01 ) 6 × 1014 s-1, k02 ) 1.4 × 1011 s-1, Ea1 ) 13 kJ/mol, and Ea2 ) 1.5 kJ/mol. The parameters for the 1% methanol-doped sample are k01 ) 2.5 × 1013s-1, k02 ) 1.24 × 1010 s-1, Ea1 ) 13 kJ/mol, and Ea2 ) 0.6 kJ/mol. The preexponential parameter, k01, is more than an order of magnitude larger for 0.1% methanol-doped ice than for the 1% methanoldoped ice. In a recent study,39 we examined the solvation statics and dynamics of Coumarin 343 and the strong photoacid (pK* ≈ 0.7) 2-naphthol-6,8-disulfonate (2N68DS) in methanol-doped ice (1% molar concentration of methanol) in the temperature range of 160-270 K. Both probe molecules showed relatively fast solvation dynamics in ice, ranging from a few tens of picoseconds at 240 K to several nanoseconds at 160 K, whereas in doped ice below T < 175 K, we observed a sharp decrease of the dynamic Stokes shifts of both Coumarin 343 and 2N68DS. The value was approximately equal to only 200 cm-1 at ∼160 K, far less than the value of about 1100 cm-1 at T g 200 K (at times longer than t > 10 ps). At a particular temperature, the rate of the solvation dynamics in 1% methanoldoped ice is comparable to the nonradiative rate of dGMP in 1% methanol-doped ice. We therefore propose that the nonradiative rate in nucleobases is determined by the solvent translational and rotational motions. In the liquid state, these solvent motions are rather fast. The solvation dynamics in water is bimodal. The long component is about 1 ps,40 the same time scale as the nonradiative rate of nucleobases in water at room temperature.2,6,7 The similarity of the time scales of the solvation dynamics in ice and the nonradiative rate of the nucleobases fits nicely to Sobolewski et al.’s36 and Domcke et al.’s35 modelbased calculations on the origin of the large nonradiative rate of nucleobases and similar compounds. Their findings show that the 1πσ* state promotes a hydrogen-transfer process from the chromophore to the solvent. G4 Wire Fluorescence Measurements As a Tool for Biological Applications. Guanine-rich segments are found in biologically significant regions of the genome, such as telomeres,41 immunoglobulin switch regions,42 gene promoter

Radiationless Transitions of G4 Wires and dGMP

J. Phys. Chem. C, Vol. 112, No. 32, 2008 12257

Figure 8. Time-resolved emission at several temperatures of dGMP in two samples of methanol-doped ice containing 0.1% and 1% methanol by mole ratio (green and red lines, respectively) and model fit (black lines).

Figure 9. Arrhenius plot of ln[A(T)] versus 1/T for G4 wire sample.

regions,43,44 and sequences associated with human disease.45 Many of these sequences have been shown to form quadruplex structures in vitro. However, the existence of these structures in vivo still remains to be verified. Many cellular events including replication, recombination, and transcription are known to occur during the cell cycle and to involve local separation of the strands of the double-stranded DNA, providing an opportunity for the G-rich strand to fold into quadruplex structures. Steady-state and time-resolved fluorescence studies on cells or/and nuclei could provide important information supporting the formation of quadruple DNA structures in vivo at different stages of the cell cycle. Human cells at birth contain telomeres of 15-kb length, consisting of repeating 5′-TTAGGG units. They are shortened, by approximately 50-100 bases, upon cell division, until they become so short that they are no longer capable of protecting chromosome ends, leading to cell death. In cancer cells, in contrast to normally proliferating cells, a constant telomere length is maintained during cell divisions, the property that makes cancer cells immortal. Fluorescence

Figure 10. Plot of ln[A(T)] as a function of 1/T for dGMP in 0.1% and 1% methanol-doped ice.

techniques might provide a useful analytical tool for differentiating between normally proliferating cells and cancer cells, as well as between young and mature cells. Several proteins are known to specifically bind to G-quadruplexes.46–52 Interaction with the G4 wire structures, as well as other physicochemical characteristics, affects their stability and will most likely influence the fluorescence emission of the DNA. Change in the fluorescence intensity and in the fluorescence decay rate could thus be used to measure the interaction between proteins and G4 wires. Summary Steady-state and time-resolved emission techniques were used to study the nonradiative process of dGMP and novel, uniform, continuous poly(dG) strands containing hundreds of stacked tetrads. We found that the time-resolved emissions of both dGMP and the G4 wires decay nonexponentialy. At room temperature, the short-time decay of the G4 wires is about 10

12258 J. Phys. Chem. C, Vol. 112, No. 32, 2008 ps, 10 times longer than that of dGMP. At low temperatures in ice, the fluorescence quantum yields of both dGMP and the G4 wires increase as the temperature decreases. At all temperatures, the average fluorescence decay time of the G4 wires is longer than that of dGMP. The fluorescence quantum yield of the G4 wires increases from about 10-3 at room temperature to about 0.03 at liquid-nitrogen temperatures. The asymptotic long-time decay of the lifetime-corrected G4 wires emission obeys a power law. We successfully used an inhomogeneous, nonradiative model to fit the experimental results. The model assumes a single floppy coordinate, x, governing the nonradiative process of the system. The results clearly show that the nonradiative rate is strongly dependent on solute-solvent interactions. The nonradiative rates in the solid polycrystalline state and in the glass state are much lower than that in the liquid state. We suggest that a large portion of the nonradiative process strongly depends on the solute-solvent friction; specifically, at large friction values, the nonradiative rate is reduced. The hindered translational and orientational motions between the host and guest most likely control the nonradiative rates. These low-frequency intermolecular modes might be responsible for the strong coupling between the 1ππ* and 1nπ* states,38 or between the 1ππ* and 1πσ* states.36 The nonradiative rate of dGMP in 1% methanol-doped ice corresponds to the solvation dynamics in ice that we observed recently in the same solvent mixtures of probe molecules such as photoacids and coumarin dye.39 Acknowledgment. A.B.K. acknowledges financial support from the European Union through IST Future and Emerging Technologies projects DNA NANOWIRES and DNA NANODEVICES. D.H. is grateful to Dr. Boiko Cohen and professor Haim Diamant for their helpful and fruitful suggestions and discussions. This work was supported by the Binational U.S.-Israel Science Foundation and the James-Franck German-Israel Program in Laser Matter. References and Notes (1) Eisinger, J.; Shulman, R. G. Science 1968, 161, 1311. (2) Gustavsson, T.; Sharonov, A.; Onidas, D.; Markovitsi, D. Chem. Phys. Lett. 2002, 356, 49. (3) Onidas, D.; Markovitsi, D.; Marguet, S.; Sharonov, A.; Gustavsson, T. J. Phys. Chem. 2002, B106, 11367. (4) Markovitsi, D.; Onidas, D.; Gustavsson, T.; Talbot, F.; Lazarotto, E. J. Am. Chem. Soc. 2005, 127, 17130. (5) Markovitsi, D.; Gustavsson, T.; Talbot, F. Photochem. Photobiol. Sci. 2007, 7, 717–724. (6) Pecourt, J. M. L.; Peon, J.; Kohler, B. J. Am. Chem. Soc. 2000, 122, 9348. (7) Crespo-Herna˘ndez, C. E.; Cohen, B.; Hare, M. P.; Kohler, B. Chem. ReV. 2004, 104, 1977. (8) Malone, R. J.; Miller, A. M.; Kohler, B. Photochem. Photobiol. 2003, 77, 158. (9) Peon, J.; Zewail, A. H. Chem. Phys. Lett. 2001, 348, 255. (10) Pecourt, J. M. L.; Peon, J.; Kohler, B. J. Am. Chem. Soc. 2001, 123, 10370.

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