Melting of DNA Nonoriented Fibers: A Wide-Angle ... - ACS Publications

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Melting of DNA Nonoriented Fibers: A Wide-Angle X‑ray Diffraction Study Federico Sebastiani,†,‡ Alberto Pietrini,† Marialucia Longo,†,§ Lucia Comez,†,‡ Caterina Petrillo,† Francesco Sacchetti,†,‡ and Alessandro Paciaroni*,† †

Dipartimento di Fisica, Università degli Studi di Perugia, Via Pascoli, I-06123 Perugia, Italy CNR, Istituto Officina dei Materiali, Unità di Perugia, c/o Dipartimento di Fisica, Università degli Studi di Perugia, I-06123 Perugia, Italy § Elettra − Sincrotrone Trieste, I-34149 Basovizza, Trieste, Italy ‡

ABSTRACT: The melting transition of A- and B-DNA has been investigated by wideangle X-ray diffraction. A significant crystalline phase is present in both the systems, even if the fibers have not been artificially aligned. The behavior of the intramolecular Bragg peaks of both A- and B-DNA as a function of the temperature clearly reveals the unfolding structural transition of the double helix. This transition occurs at the same temperature as the melting of the crystalline phase. The trends of the intramolecular correlations and the index of crystallinity are nicely described by the Peyrard−Bishop−Dauxois model for DNA melting. A description of the processes taking place at a microscopic level, i.e., double-helix deformation, crystalline dilation, and collapse, on approaching and during thermal melting is proposed.

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neutron scattering experiments on a B-form DNA fiber sample across the melting process.10 This kind of study in highly condensed nucleic acid samples (films, semicrystalline fibers, gels) is of crucial importance because the state of nucleic acids in vivo can be viewed as a kind of highly concentrated multicomponent solution.11,12 In fact, since the early fifties it has been noted that even the measured X-ray patterns from intact biological materials, such as sperm heads and bacteriophage particles, are quite similar to those from oriented fibers;13,14 i.e., they reflect a 3-dimensional order. It was shown that the ordered DNA packing may be involved in the formation and stabilization of more complex structures, such as nucleosomes and chromatin.15−17 More recently such features have been extensively observed in bacteriophages, chromosomes of dinoflagellates, and bacteria and in eukaryotic spermatozoa.18−20 On this ground, we report here the results of an X-ray diffraction investigation performed on nonoriented A-form and B-form fibers from salmon testes DNA as a function of the temperature. This investigation has been performed with the aim of studying the structural properties of DNA as the system goes through thermal unfolding in the absence of artificial fiber orientation. In both the DNA forms we evaluated the index of crystallinity and its thermal behavior, which is related to the intermolecular order.21 In addition, we selected characteristic

INTRODUCTION A key physical and chemical feature of DNA is its capability to maintain a double-helical structure and for this structure to undergo the well-known heat-induced melting denaturation.1 Thermal denaturation of DNA in solution is a highly cooperative process, meaning that the corresponding structural change is abrupt so that no intermediate state exists between the fully native and fully denatured states. This one-dimensional first-order phase transition2 is accompanied by disruption of Watson−Crick base pairs, unstacking of the bases, and disordering of the peculiar form of the backbone.3 Modeling of DNA melting requires the description of breathing fluctuations of the double helix, i.e., local denaturation and reclosing of the double-stranded structure.4 These breathing fluctuations are limiting steps to the key biological processes of DNA replication5,6 and transcription. During such processes indeed, the DNA molecule is severely deformed: the double helix is locally bended, untwisted, stretched, and compressed, and the base-pair patterns are locally destroyed. Local thermal fluctuations in DNA at melting have been further studied to identify the dynamical processes and the conformational arrangements underlying DNA folding.7 The role of thermal fluctuations was taken into account in the so-called Peyrard−Bishop−Dauxois (PBD) model,8,9 where statistical mechanics calculations and constant-temperature molecular dynamics were applied to the case of a very long homogeneous DNA chain to satisfactorily describe the corresponding experimental melting curve. Quite recently, the PBD model has been successfully used to interpret the trend of © 2014 American Chemical Society

Received: November 11, 2013 Revised: March 11, 2014 Published: March 14, 2014 3785

dx.doi.org/10.1021/jp411096d | J. Phys. Chem. B 2014, 118, 3785−3792

The Journal of Physical Chemistry B

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Figure 1. SEM micrographs of A-DNA (panels a and b) and melted DNA (panels c and d) at different magnifications. Marker sizes are indicated in the pictures.

Bragg peaks in the high-wave-transfer Q range that give information on the behavior of intramolecular structural correlations when the system crosses the melting point. We find that the inter- and intramolecular order is lost simultaneously when the melting occurs, as measured by differential scanning calorimetry (DSC).22 Quite strikingly the melting trend for inter- and intramolecular correlations in both B- and A-DNA is excellently described by the PBD approach, even though such a model is supposed to describe only the stability of the B-DNA double-helix structure.

the X-ray temperature-dependent measurements performed under vacuum. We checked that the number of water molecules remained constant by weighing the sample before and after the measurements. Finally, a sample of dry DNA was prepared, ensuring to have exposed to the beam about the same amount of biological matter. The mass of each sample was about 0.055 g. The measurements were carried out in a temperature range between 300 and 375 K. Monochromatic Co Kα radiation (λ = 1.79 Å) was used. The scattering angle was varied in steps of Δ2θ = 0.2° over an angular range 2θ = 3°−50° corresponding to an exchanged wave-vector Q interval going from 0.18 to 3 Å−1. An angular resolution of Δ2θ = 0.77° was achieved, corresponding to about ΔQ = 0.047 Å−1 in all the investigated angular ranges. Experimental data have been corrected for cell and sample environment contributions and sample transmission. Two different kinds of diffraction patterns were measured. At room temperature and after melting temperature high statistics diffraction patterns were collected, resulting from an acquisition time of 6 h. On the other hand, in the temperature ranges from 300 to 360 K and from 300 to 370 K for, respectively, A- and BDNA, low-statistics diffraction patterns were measured during a heating ramp with a rate of about 0.2 K/min to minimize kinetic effects during melting. The microscopic morphology of DNA was inspected also by means of high-resolution scanning electron microscopy (SEM). SEM images were acquired by a FEG LEO 1525, at the

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MATERIALS AND METHODS DNA sodium salt from salmon testes was purchased from Sigma-Aldrich (St. Louis, MO) and used with no further purification. To remove completely the residual water the samples were dehydrated under vacuum in the presence of P2O5 for three days. The remaining water, as estimated by thermogravimetric analysis, is h = 0.07 (h = grams of water/grams of dry DNA). To obtain DNA in the A- and B-forms, two samples were humidified under 75% and 95% relative humidity (r.h.), respectively, using oversaturated salt solutions of distilled water. Indeed, it has been established that in Na-DNA fiber samples the A-form is induced between 40% and 85% r.h., while the B-form is attained at higher humidity (from 92% r.h. upwardly).22 After the preparation, the samples were sealed in a 2 mm thick flat aluminum cell with Be windows, which was used for 3786



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acceleration voltage of 0.5−2 kV by an in-lens detector and with magnifications up to 85 000. The samples were sputtercoated with chrome at 120 mA for 20 s. SEM micrographs of A-DNA and melted DNA samples are shown in Figure 1. A rather uniform and ordered fibrous phase is revealed in the former case (panels a and b), while the latter sample is devoid of any noticeable structural feature (panels c and d). In addition, elemental analysis was achieved using energy-dispersive X-ray measurements (EDX) with a Bruker Quantax EDS and demonstrated that about 9 sodium atoms are present every 11 phosphate groups.

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RESULTS AND DISCUSSION Figure 2 illustrates the X-ray diffraction pattern for A- and BDNA nonoriented fibers. These patterns show several rather sharp peaks, whose presence suggests the existence of an extended intermolecular order. The diffraction curve is made by the superposition of many semicrystalline contributions, thus making it impossible to distinguish each single reflection. Nevertheless, the classical pictures shown in Figure 3 of X-ray diffraction patterns for crystalline-oriented fibers of A- and BDNA from Landgridge et al.23 and Fuller et al.,24 respectively, are of invaluable help in interpreting our data. For B-DNA, the main three peaks in Figure 2 at 0.28, 0.53, and 1.88 Å−1 correspond just to (1,1,1), (2,1,2), and (0,0,10) reflections as calculated for the hexagonal lattice formed by Na salt at 92% r.h.23 In particular, the third peak is related to the characteristic B-DNA base-pair stacking of 3.34 Å. On the other hand, the melting transition, whose temperature dependence will be discussed in detail below, leads to a practically featureless diffraction pattern, also shown in Figure 2. As for A-DNA the Bragg peaks appear to be less defined and intense than in the B-form diffraction pattern, thus suggesting that the crystalline phase is partially lost in this case, hence assigning the correct Miller indices to the peaks observed in Figure 2 is rather difficult. In fact, some of them seem to originate from the superposition of different reflections: for instance the Bragg peak at 0.42 Å−1 can be related to the (1,1,1) reflection, while the other found at 0.57 Å−1 is probably due to both the (1,1,2) and (1,3,0) reflections, following the estimates coming from the monoclinic structure found for DNA-oriented fibers at low hydration in the presence of Na salts.24 At higher Q-values a triplet of peaks appears between 1.5 and 2.0 Å−1, resulting from the tilt of the base pairs relative to the molecular axis and already found by Franklin and Gosling.25 In addition, it is worth noting that, conversely to what is observed in ref 22, no B-form contamination is observed in the diffraction pattern of the present nonoriented fibers of A-DNA, as there is no trace of the very intense peak found at 0.28 Å−1 for B-DNA. In both the diffraction patterns of B- and A-DNA, a large low-intensity bump, typical of amorphous systems, is superimposed to the Bragg peaks all over the spectra. The abovementioned findings can be interpreted as originating from nonoriented fibers of DNA composed of crystalline-powderlike regions (discrete lines) embedded in an amorphous, mainly single-strand, matrix (broad band). If we suppose that the amorphous phase of the sample is reasonably represented by the diffraction pattern from melted DNA, we can estimate the fraction of the crystalline DNA component by calculating the crystallinity index21 Xc = Icr/(Icr + Iam), where Iam and Icr are the integrated intensities of the completely amorphous sample and of the native sample after the subtraction of the amorphous

Figure 2. Diffraction patterns of the A-form (a) and the B-form (b) DNA at room temperature (open circles) and after melting (full line). Results for A-DNA are shown in blue and for B-DNA are plotted in red. The subtraction of the measured spectra before and after thermal denaturation is also reported (full triangles). Insets: a comparison between the spectra of dry (dashed line) and hydrated (full line) samples is shown. Arrows indicate the intramolecular peaks chosen for subsequent analysis (see text).

contribution, respectively. We find that these fractions at room temperature correspond to Xc = 0.24 and Xc = 0.50 for A-DNA and B-DNA, respectively. Such values are consistent with what is found in other fibrous biological systems like cellulose.26 This result suggests that the crystalline conformation of the two natural forms of the nucleic acid at low hydration is strongly dependent upon the amount of accessible solvent, the 3787



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that zipper-like strand-groove alignment is an innate feature of interactions between rod-like macromolecules with helical patterns of surface charges, such as phosphate groups in DNA. In particular, in dense DNA aggregates this alignment should be caused by an attraction of the phosphates of strands of a double helix with cations adsorbed in the grooves of another juxtaposed DNA.29,30 An estimate of the DNA crystallite size can be done if we describe aggregates as paracrystals.31,32 In this case the limiting size of the diffracting DNA clusters is given by the simple relationship √Nσ = α*d, where N is the number of net planes in the paracrystal, σ the distance fluctuation between two neighboring net planes, α* an adimensional constant, and d the average plane separation.33 In B-DNA, σ has been calculated to be 0.55 Å from conformational analysis, on assuming it to be equal to the structural disorder perpendicular to the double-helix axis, once thermal fluctuations are neglected.34 Since d ∼ 25 Å in dense DNA aggregates35 and conventionally α* = 0.14, we obtain N ∼ 40; i.e., in B-DNA the diffracting domains at the equilibrium are made by aggregates of densely packed double helices with a linear cluster size of about N*d = 100 nm. Analogously in ADNA, where σ = 0.7 Å,34 the packing number of double helices results to be N ∼ 25, corresponding to clusters of a total linear size of 60 nm. Our findings about the higher crystallinity of BDNA with respect to A-DNA support this result about the estimate of the crystalline domain size. To investigate the thermal behavior of intramolecular structural correlations we focused on two Bragg reflections, namely, the peaks at 1.5 Å−1 for A-DNA and at 1.85 Å−1 for B-DNA (as indicated by arrows in the insets of Figure 2), corresponding to the 8th and 10th helical layer order, respectively. Actually, the contribution to these peaks from the intermolecular term can be neglected as it decreases exponentially with the layer order, even in the case of strong azimuthal correlations.36 This is why these features,

Figure 3. Fiber diffraction patterns of A-DNA (a) and B-DNA (b).23,24 Selected peaks with corresponding Miller indices are marked.

crystallinity fraction being larger for higher water contents. On the other hand, hydration is also crucial to induce intramolecular double-helix arrangement and intermolecular longrange order, which actually are not revealed in the dry sample, as shown in the insets of Figure 2. In the latter case no distinguishable peaks can be seen, and only a broad bump centered at about 1.5 Å is predominant, corresponding to the average distance between bases in the single-strand nucleic acids. In fact, this conformation, sometimes called P-form DNA, is devoid of base stacking and has no secondary structure, while a condensed tertiary structure is present.27,28 It is also worth mentioning that the existence of a significant fraction of the crystalline phase even in nonoriented fibers is quite reasonable, if we consider that the packing symmetry and molecular alignment in dense DNA aggregates are mainly induced by electrostatic interaction. Indeed, theory suggests

Figure 4. Examples of scans and corresponding fits of intramolecular peaks in A-DNA (a) and B-DNA (b) at 300 K. Full circles: experimental data. Black solid lines: global fitting functions, accounting for a Gaussian component (color solid line) and a smooth background (black dashed line) due to amorphous DNA and superposition with the possible wings of adjacent Bragg peaks. 3788



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which result from double-helix periodicity in the axial direction, give direct information on the molecular melting. The experimental data have been fitted to a Gaussian function, also accounting for a smooth background due to the amorphous DNA component and superposition with the possible wings of adjacent Bragg peaks. The nice agreement between experimental data and fitting function for A- and BDNA is shown in Figure 4(a) and (b). Figure 5(a) and (b) shows the temperature dependence of both the intramolecular Bragg peak intensity and the crystallinity index for A- and B-DNA, which give a measure of the fraction of folded double-strand DNA and crystalline component in the sample, respectively. The melting temperature, defined as the temperature where the intramolecular peak intensity drops by 50%, turns out to be about Tc = 352 K for ADNA and Tc = 366 K for B-DNA, in good agreement with DSC measurements.22 A theoretical description of the trend of the intensity values as a function of the temperature can be given in terms of the Peyrard−Bishop−Dauxois (PBD) model.8,9 By this approach one can estimate the temperature dependence of open basepair fraction, thus describing the thermodynamic behavior of the system as it approaches the melting temperature Tc, when half of the base pairs are open. Indeed, such a model has been recently extended to describe diffraction data from salmon testes B-DNA fibers,10 i.e., the same average sequence as the sample we measured. This makes it reasonable using exactly the same theoretical curves reported by Wildes et al.,10 where the parameters in the PBD model were obtained from a 2.8 × 105 bp long sequence which is part of the full genome of Pyrococcus abyssi.10 The denaturation temperature calculated from the PBD model using these parameters is about 362 K, quite close to the melting temperatures we found for the A- and B-DNA samples, as reported above. The theoretical parameters were then used to calculate the scattering profile for a 103 bp long DNA,10 i.e., the same order of magnitude as the size ∼3 × 103 bp of salmon testes DNA. First of all, it is interesting to focus on the intensities of Bragg peaks, which scale with the number of closed base pairs. The evolution versus temperature of the integrated intensity of the selected peaks is rather similar in both A- and B- DNA samples, as shown in Figure 5. The diffracted intensity slightly changes on passing from room temperature to 330 K, thus suggesting that DNA undergoes minor conformational changes also in this temperature range before melting. This small transition, not considered in the PBD model, is also confirmed by DSC measurements on A-DNA at low hydration.22 On the contrary, the same transition is not present in B-DNA, probably because the minor structural rearrangements mentioned above have already occurred at room temperature due to the additional degrees of freedom available at higher water content. An analogous phenomenon has been also seen in natural DNA,37,38 where it was related to a premelting transition observed in Arich oligonucleotides and accompanied by straightening of the duplex.39 Here a precise assignment of this transition is actually quite difficult, as the presence of adjacent peaks can possibly produce some small uncertainties in this delicate analysis, especially in the case of A-DNA. At temperatures higher than 330 K the integrated intensities are constant until the melting process occurs, as revealed by a fast decrease to zero. In the case of B-DNA this decreasing trend, that starts slightly above 360 K, is quite sharp and in agreement with the theoretical PBD model. Indeed, both the

Figure 5. Integrated intensities of selected Bragg reflections in diffraction patterns of DNA natural configurations as a function of temperature. The points show the thermal behavior of the intramolecular peaks (open squares) at 1.85 Å−1 for B-DNA (b) and at 1.5 Å−1 for A-DNA (a), respectively. Results for A-DNA are shown in blue and for B-DNA are plotted in red. The index of crystallinity Xc is also reported for both samples (full triangles). The full line plots the Peyrard−Bishop−Dauxois model calculations,10 while differential scanning calorimetry results (dashed line) are also shown on an arbitrary scale.22 Insets: ratio of the peak positions with respect to that measured at 300 K (Qp/Qp(300 K)) in A-DNA and B-DNA. The points show the thermal behavior of the intermolecular (full triangles) and intramolecular peaks (full squares) at 0.28 Å−1 and at 1.85 Å−1 for B-DNA (b) and at 0.57 Å−1 and at 1.5 Å−1 for A-DNA (a), respectively. Solid lines are guides to the eye. 3789



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of the intramolecular (0,0,10) peak in the absence of any azimuthal order.36 Conversely, the quite distinctive trend of the (1,1,1) peak comes from the intermolecular contribution to nonzero layer lines due to strong azimuthal correlations. In addition, undulations have been shown to play a crucial role in the stabilization of DNA aggregates as contributing to the delicate balance between structural and thermal fluctuations and electrostatic interactions.42 This equilibrium is indeed greatly perturbed when the system approaches and goes through the melting transition, as witnessed by the dilation of the helical pitch and the expansion of the crystalline component discussed above. Consequently, in this regime a strong amplification of DNA undulations is likely taking place. Moreover the analysis of the experimental width of the Bragg peaks provides information about the thermal behavior of the size of the scattering units that standard observations of DNA denaturation, such as DSC, cannot. Figure 6 illustrates the

experimental data and the theoretical curve show a very low fraction of open base pairs until the vicinity of the transition is reached (T/Tc ≈ 0.98). Especially in the early stage of the transition the theoretical curve follows quite well the experimental decay of the peak intensity on approaching Tc (T/Tc ≈ 1), while above the melting the agreement is less noticeable. As for A-DNA the intramolecular peak intensity starts decreasing at about 340 K, less sharply than B-DNA. It is worth recalling that the theoretical curves in Figure 5(a) and (b) both refer to the PBD trend for B-DNA; that is, they have been only rescaled by the respective melting temperature Tc to the reduced temperature variable T/Tc. Quite interestingly, this theoretical trend is able to fit quite nicely B-DNA and fairly well also the A-DNA thermal behavior, thus suggesting that the features of this one-dimensional first-order phase transition are rather independent of its native structural conformation. It is worth mentioning that the DNA melting transition as detected by DSC and X-ray occurs at a similar critical temperature dependence, likely due to the fact that X-ray diffraction and calorimetric measurements have been done on a comparable time rate, i.e., ∼100 s/K. From Figure 5 it is also clear that the thermal behavior of the crystallinity index Xc in both the DNA configurations parallels that of the corresponding intramolecular peaks. This similarity that suggests that crystalline and helical melting take place at the same time at least within the experimental errors is not trivial, and as in other biological systems at low hydration, such as lysozyme crystals, inter- and intramolecular melting seem to occur at different temperatures.40 In this regard Cherstvy and Kornyshev predicted that intermolecular electrostatic interactions would possibly prevent DNA melting in dense aggregates.41 In fact, in excellent agreement with their calculations for an interhelical distance of 25 Å we observe an upward shift of about 5.5 K (1.5%) in the melting temperature of B-DNA with respect to that measured in dilute solutions of DNA sodium salt from salmon testes (about 360.5 K), as reported by Sigma-Aldrich (St. Louis, MO). Of course there is some degree of approximation in comparing directly DNA solutions with DNA-hydrated fibers because some experimental conditions such as the ionic strength may significantly affect the results. In the insets of Figure 5(a) and (b) we report also the temperature dependence of the peak positions for the intramolecular and intermolecular Bragg reflections of ADNA and B-DNA, respectively. In the first case, the intramolecular peak center slightly decreases over the whole range, without presenting an anomalous behavior in proximity of melting. This trend suggests that the fraction of persisting folded A-DNA only shows a small (less than 1%) dilation of the helical pitch. On the other hand, the intermolecular peak center coming mainly from the (1,3,0) line displays a much more marked decrease after about 340 K, i.e., in correspondence of the drop of the intensities of intra- and intermolecular peaks. This behavior indicates that when the DNA double helices progressively melt the residual crystalline component is characterized by an increasing expansion (about 2.5% at 350 K), probably due to the more compact volume occupied by molten DNA domains. A similar trend is observed for B-DNA intra- and intermolecular peak centers, thus suggesting that melting occurs in a similar way as A-DNA. In this case, the intermolecular peak position is also informative of possible azimuthal correlations between DNA molecules.36 Indeed, as we mentioned before, such a peak represents the nonzero layer line (1,1,1), whose temperature dependence should parallel that

Figure 6. Widths of selected Bragg reflections in diffraction patterns of DNA natural configurations as a function of temperature. The points show the experimental results for the intramolecular peaks at 1.85 Å−1 for B-DNA (open red circles) and at 1.5 Å−1 for A-DNA (open blue squares), respectively, after deconvolution with the experimental resolution. The full and the dashed lines are guides to the eye. Inset: widths of selected intermolecular Bragg peaks at 0.28 Å−1 for B-DNA (open red circles) and at 0.57 Å−1 for A-DNA (open blue squares), respectively. No deconvolution with the instrumental resolution has been performed in this case (see text).

measured widths of the intramolecular reflections, at 1.85 Å−1 and 1.5 Å−1 in B- and A-DNA, respectively, deconvoluted from instrumental resolution. It is worthy of notice that a large contribution to the measured Bragg peak widths comes from the isotropic disorder of the system, which has to be considered as a polycrystalline powder-like sample. This is why in our diffraction patterns the (0,0,10) B-DNA reflection fwhm is definitely larger than both theoretical and experimental widths found along the helix axis direction for oriented DNA fibers.10 The linewidths are essentially constant from 300 K until the melting temperature where the integrated intensities abruptly drop off. At the melting transition they suddenly increase just in 3790



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and provides further validation of this theoretical calculation, even if such a model is supposed to describe only the base-pair opening process in B-DNA double helices. Moreover, it might be speculated that the simultaneous melting of both the inter- and intramolecular structural conformations is induced by interhelical interactions in dense DNA aggregates at low hydration, which are already known to play a key role in some biological processes, such as the formation and stabilization of nucleosomes. The present results give also new insights into the local denaturation and reclosing of the double-helix structure, when intermolecular interactions and aggregation effects are taken into account, i.e., possibly closer to physiological conditions.

correspondence to the disappearance of the peaks themselves. An analogous trend is also observed for the width of the intermolecular reflections, at 0.28 and 0.57 Å−1 for B-DNA and A-DNA, respectively, that are shown in the inset of Figure 6. This similarity suggests that the base-pair openings, which become very significant at the melting point, do not cause a sharp decrease in the size of diffracting clusters until complete denaturation has occurred. We want to remark that for the intermolecular peaks the deconvolution from the instrumental fwhm is a more delicate process with respect to the one employed for the intramolecular reflections because in this case the widths assume values comparable with the resolution, as can be seen in the inset. If in any case we estimate the average value of the deconvoluted widths Γ for B-DNA, we can tentatively evaluate the diffracting cluster linear size by the relationship (2π/Γ = 40 nm), which is of the same order of magnitude as the above estimated crystallite size. On the contrary, this approach cannot be applied to A-DNA, as the intermolecular peak comes from the superposition of two distinct Bragg peaks, i.e., the (1,1,2) and (1,3,0) reflections, thus providing unreliable deconvolution results. Overall, the present results give new insights into the effect of thermal fluctuations on the interactions between DNA molecules, which play a key role in some biological processes, such as DNA−DNA assembly in nucleosomes.17,43 During thermal unfolding a partially melted long DNA is a disordered sequence of alternating melted and intact helical domains.10 As the single strands are very flexible, the remaining double-helix segments are embedded in a liquid-like medium of entangled single strands which quickly becomes the dominant phase in the sample as temperature increases. When the lengths of the melted fragments become substantial (close to and above the melting transition), DNA molecules undulate up to a critical value, at which a structural collapse occurs, in a sort of Lindemann criterion based description.44,45 In fact, being confined by neighboring DNA molecules, this overundulation can cause the well-known entropic repulsion, leading to a simultaneous disappearance of inter- and intramolecular order.41

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AUTHOR INFORMATION

Corresponding Author

*E-mail: alessandro.paciaroni@fisica.unipg.it. Phone: +39 075 585 2785. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Alessandro Di Michele is kindly acknowledged for his help with the SEM experiment. This work was supported in part by a grant from the Consiglio Nazionale delle Ricerche − Istituto Officina dei Materiali.

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REFERENCES

(1) Zimm, B. H.; Bragg, J. K. Theory of the Phase Transition between Helix and Random Coil in Polypeptide Chains. J. Chem. Phys. 1959, 31, 526−535. (2) Dauxois, T.; Theodorakopoulos, N.; Peyrard, M. Thermodynamic Instabilities in One Dimension: Correlations, Scaling and Solitons. J. Stat. Phys. 2002, 107, 869−891. (3) Duguid, J. G.; Bloomfield, V. A.; Benevides, J. M.; Thomas, G. J., Jr. DNA Melting Investigated by Differential Scanning Calorimetry and Raman Spectroscopy. Biophys. J. 1996, 71, 3350−3360. (4) Peyrard, M. Using DNA to Probe Nonlinear Localized Excitations? Europhys. Lett. 1998, 44, 271−277. (5) Kornberg, A.; Baker, T. A. DNA Replication, 2nd ed.; W.H. Freeman and Company: New York, 1992. (6) Gai, D.; Chang, Y. P.; Chen, X. S. Origin DNA melting and unwinding in DNA replication. Curr. Opin. Struct. Biol. 2010, 20, 756− 762. (7) Ma, H.; Wan, C.; Wu, A.; Zewail, A. H. DNA Folding and Melting Observed in Real Time Redefine the Energy Landscape. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 712−716. (8) Peyrard, M.; Bishop, A. R. Statistical Mechanics of a Nonlinear Model for DNA Denaturation. Phys. Rev. Lett. 1989, 62, 2755−2758. (9) Dauxois, T.; Peyrard, M.; Bishop, A. R. Dynamics and Thermodynamics of a Nonlinear Model for DNA Denaturation. Phys. Rev. E 1993, 47, 684−695. (10) Wildes, A.; Theodorakopoulos, N.; Valle-Orero, J.; CuestaLópez, S.; Garden, J.-L.; Peyrard, M. Thermal Denaturation of DNA Studied with Neutron Scattering. Phys. Rev. Lett. 2011, 106, 048101. (11) Podgornik, R.; Strey, H. H.; Gawrisch, K.; Rau, D. C.; Rupprecht, A.; Parsegian, V. A. Bond Orientational Order, Molecular Motion, and Free Energy of High-Density DNA Mesophases. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4261−4266. (12) Pelta, J., Jr.; Durand, D.; Doucet, J.; Livolant, F. DNA Mesophases Induced by Spermidine: Structural Properties and Biological Implications. Biophys. J. 1996, 71, 48−63. (13) Wilkins, M. H. F.; Randall, J. T. Crystallinity in Sperm Heads: Molecular Structure of Nucleoprotein in Vivo. Biochim. Biophys. Acta 1953, 10, 192−193.

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CONCLUSIONS We have shown that X-ray diffraction can be used to monitor the melting of both the A- and B-form of nonaligned DNA fibers, providing the spatial information that other methods cannot measure. A significant fraction of crystalline phase, whose features are strongly solvent-dependent, has been found, similarly to intact biological materials. By studying selected Bragg peaks and the crystallinity index, which are associated to the intra- and intermolecular order, respectively, we highlight the simultaneous loss of short- and long-range structural correlations by thermal denaturation in both the A- and B-form of DNA. Melting of double helices is accompanied by a slight dilation of the lattice parameters of the crystalline residual component, while the most relevant changes in the size of diffracting domains, i.e., their collapse, are observed only after complete denaturation has occurred. The trend of the intermolecular peak in B-DNA is consistent with the existence of strong azimuthal correlations between DNA molecules. The comparison of experimental results with the PBD approach excellently works in the case of both A- and B-DNA 3791



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