Molecular Chain Orientation of DNA Films Induced by Both the

Biomacromolecules 2004, 5, 2297-2307

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Molecular Chain Orientation of DNA Films Induced by Both the Magnetic Field and the Interfacial Effect Nahoko Morii,*,†,‡ Giyuu Kido,†,§ Hiroyuki Suzuki,† Shigeki Nimori,§ and Hisayuki Morii| Nanomaterials Laboratory and Tsukuba Magnet Laboratory, National Institute for Materials Science (NIMS), Tsukuba, Ibaraki 305-0003, Japan, and Protein Dynamics Research Group, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan Received June 17, 2004; Revised Manuscript Received August 9, 2004

DNA films showing highly homogeneous orientation of molecular chains were successfully prepared by drying a semidiluted solution in a horizontal magnetic field. Most of the molecular chain elements in the obtained film were found to be one-dimensionally oriented, as shown by X-ray diffraction, polarization microscopy, and linear dichroism spectroscopy. Because a DNA chain is theoretically expected to orientate only in divergent directions perpendicular to a magnetic field, this result suggests that the DNA chains were aligned not only by a magnetic field but also by the interfacial effect that induced the chains to fit along the air-liquid interface. The descent speed of an air-liquid interface by evaporation was faster than the estimated diffusion rate of DNA, suggesting an emergence of a concentrated layer near the surface. As proved by polarization microscopy, this emergence led to the transitional formation of a nematic-like liquid crystalline phase, which resulted in a DNA film with good chain alignment and unitary orientation. This mechanism underlying chain alignment was supported by molecular weight dependency, in which higher molecular weight DNA is more likely to evince chain alignment that exhibits a higher degree of birefringence. Low molecular weight components have such high thermal motility that it would be difficult to fit them along the air-liquid interface in the early stage of drying. For chain alignment, it was preferable to use an initial concentration of DNA lower than a critical concentration for liquid crystal formation so that the possible diffusion and assembly in a diluted solution would be essential for chain alignment. The DNA film exhibited obvious linear dichroism, indicating the potential for further applications. Introduction Because DNA involves the essential information of animate beings, it is one of the most important polymers from the aspect of biological and medical science. In recent years, the specific structural characteristics of DNA have led to a lot of interest in applying it toward nano-functional materials. The stacked nucleic-acid base pairs inside the double-stranded helical backbone are expected to have some optical and electronic functions. Moreover, the intercalation of a planar organic compound to stacked base pairs can lead to the development of additional functions. Various approaches to develop new functional materials have been reported, such as a two-dimensional array of gold or magneto-nanoparticles constructed with the aid of a nanoaddressed designation and the complimentarity of DNA,1 a photoinduced switching device conjugated with the azobenzene group,2 molecular tools to analyze a single nucleotide polymorphism in a genome,3 and organic electro-luminescent devices.4 DNA is also attracting a lot of attention as an organic conductive material.5-7 The electrical conductivity * To whom correspondence should be addressed. Phone: +81-29-8635359. Fax: +81-29-863-5571. E-mail: [email protected]. † Nanomaterials Laboratory, National Institute for Materials Science. ‡ Project of Support and Cooperation for Prior Researches, committed by Japan Science and Technology Corporation. § Tsukuba Magnet Laboratory, National Institute for Materials Science. | National Institute of Advanced Industrial Science and Technology.

of a single DNA molecule has been measured with a scanning tunneling microscope and a noncontact atomic force microscope.5 In some of these applications, the use of anisotropic material, such as a membrane with molecular orientation, would be promising to enhance functionality. To realize the orientation or chain alignment for DNA, researchers have developed a meniscus-driven method along with the translation of the air-liquid interface,8 the Langmuir-Blodgett method using a monolayer of cationic lipids,9 a method using dielectric polarization in a solution,10 and a mechanical stretching method for DNA-lipid conjugates.11 However, even if the DNA chains are oriented in a bulk solution or a gel, disordering caused by the drying process still makes it difficult to fabricate a solid film having a chain orientation. Additionally, some methods are limited to the formation of monolayer materials. In this work, therefore, we tried to overcome these problems by using a semidiluted solution method in a magnetic field to prepare homogeneous DNA films made up of well-aligned molecular chains. The nanoscopic structures of materials have been extensively developed as well as the methodologies for their observation. The surface induced ordering is one of the effective principles to provide the nanostructured materials. It is generally recognized that the amphiphilicity and the liquid crystallinity of molecules bring about the ordered

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molecular orientation. The controlled structures were reported for the Langmuir monolayer12 on the mercury surface and polypeptide chain13 at the liquid-air interface, for example. In that case, specific chain orientation at the surface seems to be induced by rigidity of the moiety of molecules such as 1,3-bis(ethynylene)benzene.14 The rigid rodlike polymer chain also contributes to parallel alignment of the molecules as reported for poly(benzyl-L-glutamate), which has lateral orientation without external pressure.15 Similarly, it was reported that the microphase formed by block copolymer is affected by the surface to exhibit characteristic patterns in the 10-100-nm regime.16,17 Thus, the surface has an important role in forming nanostructures, which would be true of our DNA films. Moreover, in some cases, these surface-induced structures are regulated by the solvent evaporation. It was revealed that the morphology of polystyrene depends on the organic solvents used for the preparation of thin polymer films.18 The solvent evaporation would cause the solvent gradient normal to the surface, which may further affect the nanostructures. Recently, the magnetic method has drawn special attention in the fields of aligned carbon nanotubes19 and the crystallization of proteins.20 These examples utilize the anisotropic diamagnetism of organic substances. More than a few basic studies have reported on the magnetic orientation of crystalline and/or liquid crystalline polymers, which involve the aromatic moieties fixed in the main chains.21-23 The aromatic group is thought to bear a larger diamagnetism in the direction perpendicular to its plane, which may drive a molecular orientation in a magnetic field. Because a DNA molecule includes conjugated bonds in its nucleic-acid bases, a similar response to a magnetic field can be expected. In fact, the formation of a cholesteric liquid crystal was reported for highly concentrated DNA solution.24 However, creating a one-dimensional orientation of DNA chains over a wide region of the material has been difficult, probably because the magnetic effect causes orientation to only divergent directions perpendicular to a magnetic field. In this paper, we describe the successful preparation of DNA films with highly aligned molecular chains and the confirmation of this development by using X-ray diffraction, polarization microscopy, and linear dichroism spectrometry. It should be noted that our DNA film has homogeneous chain orientation in a whole region, which would be brought about by both a magnetic field and an interfacial effect, as discussed in the text. Experimental Section Preparation of DNA Films. Commercial DNA material derived from salmon sperm was used throughout the study. Fibrous DNA sodium salt, purchased from Yuukigousei Co. (Japan), was dissolved in water and then sonicated at below 30 °C to prepare several classes of samples with different averaged molecular weights. These DNA solutions were lyophilized into puffy white solids. The obtained DNA samples were redissolved in still-standing water for 2 weeks before use. The concentration of DNA solutions was adjusted to 60 mg/mL at pH 6.8 without any additional buffer component.

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Figure 1. Elution profiles by size exclusion chromatography for the DNA samples before and after sonication. The profiles for SD-29 and SD-6 are shown with solid and dotted curves, respectively. The bottom abscissa represents the ratio of elution volume to the total column volume. The top axis denotes the estimated molecular weight represented by the unit of base pairs. The elution around 0.35 of Ve/ Vt corresponds to the exclusion limit of column packings (20 kbp for DNA).

The molecular weight distributions of the DNA samples were analyzed by size-exclusion chromatography with a column of Sephacryl S-1000-SF (Amersham Pharmacia Biotech). The weight-averaged molecular weights were calculated according to the elution curves. The molecular weight of nonsonicated DNA was estimated to be 29 kilobase pairs (kbp), which was about 5 times larger than the averaged logarithmic molecular weight corresponding to the chromatogram peak (5.3 kbp). The DNA samples were named SD-x, where x indicates the weight-averaged molecular weight in kbp. They are SD-29 (29 kbp as the weightaveraged value, 5.3 kbp as the peak), SD-23 (23 kbp, 4.2 kbp, shown in a similar manner as before), SD-22 (22 kbp, 4.1 kbp), SD-9 (8.8 kbp, 1.8 kbp), SD-6 (5.6 kbp, 1.2 kbp), and SD-1 (1.4 kbp, 0.26 kbp).25 The representative chromatograms are shown in Figure 1. The DNA solution was put onto a 25 × 25 × 0.10 mm silica plate, which was cleaned in advance.25 Just after the solution was deployed, the sample was introduced into the central spot of a magnet and was kept there until a dry film had formed. Two forms of starting solution for the films were examined: a 0.05-mm-thin layer solution widened to a 20 × 20 mm square region and a 1.3-mm-thick layer covering an 8-mm diameter circular region. The former brought about homogeneous chain orientation, but the latter resulted in the orientation with a specific pattern (see Results and Discussion). The latter system was used to investigate the magnetic intensity dependence, because a certain amount of time is necessary to ensure the influence of a magnetic field for DNA chains. The film was prepared under the usual conditions, at 25 °C and 40-50% humidity. Magnetic Field. The magnetic field was generated with a cryogen-free superconducting magnet, JASTEC-10T 100 mm (Japan Super Conductor Technology, Inc.). An almost homogeneous magnetic field with the accuracy within 1.5% was achieved in a spheric region with a diameter of 40 mm. The magnetic intensity was regulated to be 2, 4, 6, 8, and 10 T for this work. The magnet has a cylindrical shape bearing a central bore with a 100-mm diameter as a sample port. The samples were introduced into the central spot along

Chain Orientation by Magnetic Field in DNA Films

the magnetic field line. The horizontal or vertical magnetic field was realized by rotating the body of the magnet by 90 or 0° from the gravitational direction, respectively. Using a balanced holder, the plate with a cast DNA solution was always kept in a horizontal plane regardless of the horizontal or vertical directions of a magnetic field. X-ray Diffraction Analysis. The X-ray diffraction data were collected by a transmission Laue camera method with a MAC Science M18XC. For DNA films detached from the plates, the selective Cu KR ray at 40 kV and 80 mA was irradiated through a 0.3-mm-diameter collimator during 120 min. The obtained data at a diffraction angle of about 27° corresponding to 0.34 nm were accumulated with R-AXIS software (Rigaku) and were converted to the β-I plot of the X-ray diffraction intensity, which represents the dependence of the intensity on the azimuthal angle. Polarization Microscopy. The dried DNA films were characterized by a polarization microscope (Olympus 60BX) equipped with a crossed-nicols. To observe the mode of chain orientation, a coloring method with a 530-nm sensitive tint plate was adopted. The birefringence (∆n) was evaluated according to both the R value determined by a Berek compensator and the film thickness as measured with a Sony U30A digital indicator. The orientation patterns of the films prepared in the presence of a magnetic field were analyzed by recording color pictures with a sensitive tint plate at every 10° rotation of the sample plate. Then, the most probable directions of chain orientation were manually determined according to the changes in color tones. Linear Dichroism. The ultraviolet (UV) light absorption spectra were measured with a Shimadzu UV-1200 spectrophotometer. The incident UV radiation was polarized through a Glan-Taylor prism. The DNA film for UV measurement was prepared from a 0.09-mm-thin layer solution at a 12 mg/mL concentration. Preparation of Single-Stranded DNA Samples. To clarify the importance of double-stranded structure, the DNA samples with single-stranded chains were prepared by the thermal denaturation and/or alkali treatment for SD-29. The former protocol for the 30 mg/mL DNA solution employed the heat treatment at 95 °C for 30 min to dissociate the double strands followed by quick chilling. The temperature is higher than the reported melting temperature, 87.5 °C, of salmon DNA.26 The latter protocol was carried out by mixing 60 mg/mL DNA solution and the equal volume of aqueous 1 M NaOH solution for 15 min. The alkali treatment was quenched by the addition of a 100-fold volume of ethanol to precipitate DNA, which was collected by centrifugation. The solid DNA was washed five times with ethanol to remove excess sodium ions and then dried in vacuo. The formation of single-stranded DNA was qualitatively confirmed by UV measurement on the basis of the hyperchromic effect. Calculation of Diffusion. To assess the dynamic behavior of DNA molecules, the diffusion constants were calculated with theoretical equations at 25 °C (T ) 298 K). The

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translational diffusion constant (Dtr) was evaluated with the Stokes-Einstein equation for a sphere, as follows, Dtr ) kT/(6πηr)

(1)

where k, T, η, and r represent the Boltzmann constant, temperature, viscosity coefficient, and radius of an object, respectively. Using the diffusion constant, the mean diffusion distance (x) within a certain time (t) is estimated with the following: x2 ) 2Dtr t

(2)

On the other hand, the rotational diffusion constants (Drot) were calculated with the Stokes equation for a sphere. Drot ) kT/(8πηr3)

(3)

The rotational correlation time (tc) can be related with the diffusion constant as expressed with eq 4. tc ) 1/(6Drot)

(4)

Then, the rotational Brownian motion can be described with the mean change in a spatial direction angle (δ) within a short time (t), as follows, (3 mean[cos2 δ] - 1)/2 ) exp(-t/tc)

(5)

where the function “mean” represents the statistical average. These calculations were carried out for a random-coiled DNA chain consisting of more than 1000 base pairs. According to the theory of polymer physics, the overall radius (r) of the spreading random-coiled chain can be estimated with the following equation, r ) (Lu)1/2/2

(6)

where L and u represent the full length of a polymer and the size of a statistical chain element, respectively. The value of u is roughly estimated to be 100 nm for double-stranded DNA. Results and Discussion Analysis of X-ray Diffraction. For the DNA films prepared in the magnetic field, a couple of intensive halos were observed at a region corresponding to 0.34 nm (Figure 2). A similar X-ray diffraction pattern was reported for uniaxially extended DNA fiber by Wilkins et al. about half a century ago.27 As revealed by Dickerson et al. for the DNA oligomer crystal with a length of 12 base pairs,28 the major conformation of DNA in a well-hydrated condition is the B form, which is constructed with a double-stranded helical backbone and base pairs stacked at intervals of 0.34 nm. Our DNA films also exhibited an intensive diffraction at 0.34 nm so that the DNA chains are likely to take the B form even in dried solid films. The β-I plot of the X-ray diffraction intensity for 0.34 nm is shown in Figure 2A. Strong and sharp diffractions were observed at +90 and -90° for the DNA film prepared in a horizontal magnetic field in the direction of 0° in relation to the film. This result means

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Figure 2. Dependence of X-ray diffraction intensity on the meridian angle for DNA films prepared in a 10-T magnetic field. The plot A represents the diffraction obtained by the irradiation along the normal of SD-9 DNA film. The plots B and C represent the data obtained with the irradiation for the tilted films by 30° around the axis of the magnetic field and by 60° around the axis of the oriented chains, respectively. The data were accumulated along diffraction angles of about 26.5°, corresponding to the base-pair stacking. The Gaussian curve (solid curve) was fitted onto the observed plots (closed circles). The top illustration for DNA film prepared in a magnetic field (m.f.) explains the directions of X-ray irradiation. The direction of the magnetic field accords with the zero point of the abscissa in parts A-C.

that the stacking of base pairs is in the direction perpendicular to the magnetic field. To evaluate the extent of molecular orientation, Gaussian curves were fitted to the observed β-I plots. The halfbandwidths (w1/2) for DNA samples with different molecular weights, SD-1 and SD-9, were 33.8 and 28.5°, respectively. The DNA with higher molecular weight seemed to bring about a more ordered orientation, resulting in a narrower half-bandwidth. Using the equation for the extent of orientation order, that is, f ) (180 - w1/2)/180, the extents of the order are estimated to be 81 and 84%, respectively, for the SD-1 and SD-9 samples. A later section discusses the detailed

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dependence of chain orientation on molecular weight from the aspect of birefringence. Interestingly, the DNA film obtained by the trailing-dry method, which involves the mechanical drawing of viscous DNA solution, resulted in insufficient orientation showing 41.6° of half-bandwidth. Thus, the preparation of DNA films in the magnetic field is superior to the mechanical one. To survey the inner distribution of the chain orientations, the X-ray diffraction data were collected for the DNA film tilted to two different directions. One of them is the direction B in Figure 2, in which the normal of the film was tilted by 30° as for the incident X-ray by rotating around the axis of magnetic field vector. Another is the direction C tilted by 60° by rotating around the expected predominant axis of DNA chains. The former gave 58° of the half-bandwidth w1/2 (Figure 2B), which reflects the apparent compression of the distribution angles in chain orientation. Most importantly, the two peaks of the β-I plot in Figure 2B revealed a significant difference in peak heights. This means that the chain orientations in the cross-sectional plane of the film are well coincided with each other. The X-ray diffraction in Figure 2C showed 32° of the half-bandwidth, which is almost similar with the result obtained by the irradiation from a normal. These results suggest the highly ordered chain orientation in the direction not only perpendicular to the magnetic field but also parallel to the film plane. Therefore, it is concluded that the DNA chains have unique orientation by a horizontal magnetic field. On the other hand, the DNA film prepared without a magnetic field gave only a flat pattern in the β-I plot, showing no molecular chain orientation in the planar directions. The remarkable Debye ring observed for the DNA quasicrystal is attributed to the base-pair stacking. This X-ray diffraction band is essentially broad, because various diffractions of the atoms within nucleic acid bases are overlapped in addition to the variety of DNA sequences. The shape and broadness of the Debye ring for our DNA films agree well with those reported previously for DNA quasicrystals. Therefore, it can be concluded that the DNA chains in the solid films prepared under the magnetic field are as highly ordered as the quasicrystals. As for molecular orientation by the magnetic effect, the crystallization of naphthalene in the solution provides a typical example of the molecular behavior in a magnetic field.29 The aromatic rings of naphthalene in the obtained crystal were found to be quite parallel to the magnetic field. Considering that the plane of the DNA base pair is parallel to the magnetic field as described above, the base pair moiety would play a major role in magnetic orientation and in a similar manner as naphthalene does. Observation with the Polarization Microscope. DNA films prepared from 60 mg/mL of solution in a 10-T horizontal magnetic field revealed a uniform texture and optical anisotropy, which were observed by polarization microscopy under a crossed-nicols condition. Figure 3 shows a microscopic photograph with a sensitive tint plate and tilted images. Because the color changed to yellow and blue when the film was tilted by +45 and -45°, the birefringent feature of the oriented DNA chain perpendicular to the magnetic


Figure 3. Polarization microscopic observation of SD-9 DNA samples situated at three different angles. The 45° tilted images are shown at either side of the normal one. The situation of the film is marked by a rectangle in each photograph and is accompanied by the representative orientation of the DNA chain if any. The pictures, observed with a sensitive tint plate, show DNA films prepared in a horizontal magnetic field (A), in a vertical magnetic field (B), and by the trailingdry method (C). The red and blue arrows denote the direction of the magnetic field and the trailing operation, respectively. The polarizing filters and the sensitive tint plate were used in manner similar to that shown in Figure 4.

Figure 4. Photograph of the SD-29 DNA film prepared from a concentrated solution in a 10-T magnetic field. The picture was observed with a sensitive tint plate (STP), a polarizer filter (Po), and an analyzer filter (An) situated in the directions indicated in the side square. The small rectangle represents the situation of the film as described above.

field was judged to be the negative characteristic. This is consistent with the result of the DNA film prepared by the trailing-dry method, in which the chain orientation was parallel to the direction of the chain drawing (Figure 3C). In contrast, the film prepared under the vertical magnetic field showed no clear molecular orientation (Figure 3B). The polarizing microscopic view of this sample was almost entirely similar to that of the DNA film prepared without a magnetic field. This result also suggests that DNA molecules cannot be oriented homogeneously by a magnetic field alone. To survey the effect of initial DNA concentration, DNA films prepared from 6, 20, 60, 120, and 200 mg/mL solutions were compared. Among these, the DNA film obtained with the liquid crystal formed by a 200 mg/mL solution was found to have a pattern of stripes at intervals of 2 µm under crossednicols, as shown in Figure 4. It resembled the characteristic liquid crystal DNA with almost uniform length (100 base pairs) prepared in a magnetic field.24 Brandes and Kearns mentioned that DNA solution enclosed in a slide-glass cell took on a granular structure at a concentration of 248 mg/ mL under a 5.9-T magnetic field. Also, they found a


conversion to a multiple-banded structure after letting the DNA sample stand in a magnetic field for several days. Twofold of the observed stripe interval (2.5 µm) was attributed to the pitch of the cholesteric liquid crystal by means of optical rotation and NMR. Therefore, it is considered that our DNA solid film obtained from the concentrated solution is the fixed material of the cholesteric liquid crystal, though the DNA chain is extremely long in our experiments, unlike the case in theirs. Starting from the low DNA concentrations other than 200 mg/mL, the obtained DNA films exhibit homogeneous texture. Moreover, these films had almost similar birefringence about 0.02-0.025, indicating a common mechanism of the chain alignment. A phase diagram for temperature and concentration was determined by Strzelecka and Rill.30 Those authors showed that DNA with about 146 base pairs represents three distinguishable phases and intervening biphasic states. The initial concentration of 60 mg/mL used for our representative film preparation seems to correspond to the isotropic phase, because in their system the cholesteric phase appears from the isotropic solution above a concentration of 125 mg/mL. The different results indicated that the hexagonal packing of the DNA chain occurred locally while the concentrated DNA solution was cooling.31 In our system, the homogeneous film formation from the diluted solution may have undergone this hexagonal packing in a similar manner, even though the chain length differed. Thus, the polarizing microscopic images of DNA films fabricated in a magnetic field apparently depend on the initial concentration of DNA. These results are closely related to a critical concentration for the formation of liquid crystal so that the chain orientation of DNA in different mechanisms is suggested to depend on the initial phases of DNA solution. Probably, the mobility of a DNA chain at the diluted concentration would be responsible for the well-ordered chain packing. Linear Dichroism in UV Region. The degree of orientation in the DNA films prepared in the magnetic field was evaluated using an UV absorption spectrometer equipped with a polarizer to detect linear dichroism. DNA film prepared in a 10-T magnetic field exhibited pronounced absorption of polarized light perpendicular to the direction of molecular chain orientation, as shown in Figure 5. Linear dichroism was found not only at a 260-nm band arising from the π-π* transition but also at the region of less than 230 nm, which corresponds to the absorption band of carbonyl groups involving the plane of a base pair. Generally, π-π* electron transition moments of conjugated systems lie alongside the plane of conjugated double bonds so that the polarized light parallel to this plane is well absorbed. Because most conjugated bonds of DNA exist in the base pairs, this model can easily explain the observed linear dichroism. The experimental result also shows a small but not negligible absorption of polarized light parallel to the molecular chain. This suggests a local disorder, such as in the loop region, in addition to the slight leaning of base pairs with respect to the chain axis of B-form DNA. Some loop regions would remain locally as turning strands even after global chain orientation.

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Figure 5. Linear dichroism spectra of the SD-23 DNA film. The film was positioned in parallel (bold curve) or perpendicular (thin curve) to the polarizer for the direction of the 10-T magnetic field given for the DNA solution. The wavelength was limited to 220 nm as a result of significant absorption by the polarizing prism.

To evaluate the linear dichroism quantitatively, parameters r12 and Dp (direct ratio and degree of polarization) were calculated according to the absorbances A1 and A2, which are observed with polarized light perpendicular or parallel to the oriented chain axis, respectively. r12 ) A1/A2

(7)

Dp ) (A1 - A2)/(A1 + A2)

(8)

For an oriented DNA film prepared under a magnetic field, r12 and Dp were 3.13 and 0.52, respectively. On the other hand, the parameters for the oriented film by the trailingdry method were 1.74 and 0.27, respectively. Though the values for the latter film are not always constant because of the difficulty in controlling the mechanical process, it is obvious that the chain orientation by the magnetical method is more efficient than that by the trailing-dry method. Tanigaki et al. originally developed the friction-transfer method to prepare thin films of poly(dimethylsilylene) and poly(p-phenylenesulfide), which have the remarkable feature of linear dichroism owing to an electronic transition moment along aligned polymer chains.32 Our DNA films obtained under a magnetic field also exhibited enhanced linear dichroism. It is noteworthy that our films are fabricated simply by drying them from aqueous solution. Moreover, the dichroic absorption of our film is characteristic because the wavelengths of absorption bands are shorter than 300 nm. The typical functional group with an absorption spectrum of less than 300 nm is that of short aromatics such as naphthalene. Though a polymer involving bicyclic aromatics in its main chain can be aligned, the longitudinal axis of this aromatic group would not be oriented completely along the axis of the polymer chain because of the constraint of chemical topology. On the other hand, the DNA base pairs are oriented almost precisely perpendicular to the axis of the molecular chain. Thus, this framework might be an advantage in the formation of a higher linear dichroism. As expected, the films made with a vertical magnetic field produced no dichroic material. Anisotropy of Magnetic Susceptibility. Both the direction of chain orientation by the magnetic field and the degree of magnetically induced torque originate in the magnetic

anisotropy of the molecules. Because the diamagnetic effect of the organic molecules is generally weak, the anisotropy of magnetic susceptibility is also small. However, arranging the anisotropic moieties in the same direction would make it possible to orient the molecules by magnetic effect. To clarify the mechanism by which the magnetic field orients the DNA, the magnetic susceptibility of the base pairs was first predicted. Maret et al. measured the magnetic anisotropy of a DNA molecule in a diluted solution by using the Cotton-Mouton method.33 By measuring its dependence on the temperature, they found a steep decrease in the CottonMouton constant at 87 °C, which was attributed to the dissociation of DNA double strands. Thus, the doublestranded structure of DNA is essential for achieving magnetic orientation. This also means that the alignment of base pairs in a chain synergistically enhances the anisotropy of magnetic susceptibility. Because DNA is made up of successively stacked base pairs, a DNA chain is relatively rigid up to about 100 nm.34 On the other hand, at sizes of more than 1 µm the DNA seems to behave as a flexible chain, which may enable magnetic orientation in diluted solution. Veillard et al. reported the magnetic anisotropy of a DNA nucleotide with the measure of ∆χ/∆χbenzene, which is the anisotropy relative to that of benzene. As for the base pairs A-T and C-G, the relative magnetic anisotropies are evaluated to be 1.098 and 0.827, respectively.35 The component of magnetic susceptibility for benzene is estimated as -95 × 10-6 emu/mol in the direction of the Z coordinate, which is defined as the normal to the aromatic ring. On the other hand, the magnetic susceptibilities for X and Y coordinates parallel to the plane are almost equal to each other, at -35 × 10-6 emu/mol. Therefore, on the basis of these values the magnetic anisotropy ∆χ for benzene is -60 × 10-6 emu/mol.36 Thus, the direction of magnetic anisotropy for aromatic groups is along the normal. This can be explained by classical physics in that the degree of the Lorenz force of π electrons becomes highest for the magnetic field in the direction of the normal. Similarly, the magnetic anisotropies of the DNA base pairs coincide with the normal for the plane of the nucleic base, and the ∆χ values are calculated to be -50 × 10-6 and -66 × 10-6 emu/mol for A-T and C-G, respectively. Because the salmon DNA used in this work was composed of 59% A-T and 41% C-G,26 the averaged magnetic anisotropy of our sample was estimated to be -56 × 10-6 emu/mol per base pair, roughly comparable to the value for benzene. According to the additivity rule of Pascal concerning the elemental parameters of magnetization, the magnetic susceptibility for the nucleic base is estimated to be -220 × 10-6 emu/mol per base pair unit. Also, that for the backbone consisting of ribose and phosphate groups is -175 × 10-6 emu/mol. Though it is difficult to evaluate the anisotropy of the backbone because of the complicated distribution of the charges involving the countercations, most of the anisotropies for the covalent bonds seem to be compensated because of the dispersion of these spatial directions. Therefore, as is presumed from the experimental results, the nucleic bases would be the main contributors to the intensive anisotropy of DNA along the chain axis. From this, we estimated that


the magnetic susceptibility of a base pair is about -111 × 10-6 emu/mol and -55 × 10-6 emu/mol for the directions perpendicular and parallel to the plane of the base pair, respectively. Thus, the magnetic anisotropy is twice as large in the direction along the chain axis than it is in equatorial direction. Moreover, the base-pair stacking would extremely enhance the overall magnetic anisotropy, playing a significant role in magnetically induced chain orientation. Mechanism of Chain Orientation. As described above, a solid film with well-aligned chains was obtained by drying the isotropic solution of DNA in the magnetic field parallel to the substrate. In principle, however, the magnetic effect is arranging the DNA chain only to divergent directions perpendicular to the magnetic field. In other words, the DNA chains are equally stable no matter the direction of their orientation within the perpendicular plane. Therefore, it is not likely that the effect of magnetic anisotropy alone well aligns DNA chains in film. There should be another effect that helps orient the chains in a coinciding direction in the course of drying. We recently found that DNA solution is concentrated near the air-liquid interface by the evaporation of solvent.25 By observing a droplet as it dries, we observed that the descent rate of the air-liquid interface is faster than the average translational diffusion of a DNA molecule with 22 000 base pairs, whose speed was calculated to be 0.008 mm/min. This means that large DNA molecules are likely to remain near the air-liquid interface of the solution and to be concentrated. DNA molecules with 146 base pairs are known to form a liquid crystal fully above the concentration of 150 mg/mL. Therefore, the emergence of the concentrated DNA phase close to the air-liquid interface suggests the local formation of the liquid crystal phase. In fact, this could be proved with the sample which was taken out from a magnetic field halfway through the drying process. It was found that the DNA molecular chains in the thick-layer solution were orientated even in its undried region. The direction of chain orientation was perpendicular to the given magnetic field as determined by polarization microscopy. This orientation was conserved during several minutes after removing the magnetic field. These results suggest that the transition to the liquid crystalline phase occurs after the chain orientation in a solution by the effect of the magnetic field. From its homogeneous appearance, this liquid crystal seems to be on the nematic phase. Livolant reported the formation of hexagonal packing of DNA.31 Probably, the DNA chains would be forced to orientate along the air-liquid interface. This may be called an interfacial effect. The calculation for the rotational diffusion could provide an additional insight for the behavior of DNA chains. The mean diffusion time of the 22 kbp DNA chain is estimated to be only 0.03 s for the orientational change by 30°. Except for the liquid crystalline region, where the viscosity seems extremely high, the chain fragment seems to move rapidly so as to lie along the interface or liquid crystalline layer. So, it is supposed that the molecular chain alignment can be easily performed within a short time. Thus, the interfacial effect would be supported not only by the slow translational diffusion but also by the rapid rotational diffusion of DNA


Figure 6. Schematic representation of a probable mechanism for chain alignment. The direction of a magnetic field is perpendicular to the plane of a front view. Each cylinder denotes a straight region as a structural chain block shorter than the statistical chain element. The time course of the changes in chain orientation due to the evaporation from DNA solution is indicated in the downward order (from A to B and then C). The magnetic field is already given for DNA solution at the stage A. After the progress of evaporation (B), the condensation near the surface (air-liquid interface) occurs. At the subsequent stage C, the further formation of the liquid crystalline phase is shown. The looping regions were not indicated because of complication. The liquid portion is displayed with a light blue background.

molecules. The nematic phase formation would result from both the magnetic effect to orient the chains perpendicular to the horizontal magnetic field and the interfacial effect to align them parallel to the air-liquid interface. Consequently, most DNA chains become oriented in the concordant direction. Then, as the solvent evaporates, the well-packed liquid crystal phase would grow to form DNA film with almost coordinated alignment of the chains. The probable mechanism of chain alignment is illustrated in Figure 6, which shows chain elements of about 30 nm of DNA. The statistical chain element, which is defined as the minimum size conserving the correlation between the chain directions at both ends, is considered to be about 100 nm for DNA.34 This means that, for example, a 30-nm chain of DNA corresponding to about 100 base pairs is relatively rigid in a solution. As discussed above, both the magnetic effect and the interfacial effect are indispensable to the good alignment of these statistical chain elements. Moreover, it should be noted that the diluted concentration of the initial solution allows both of these effects in chain alignment, thus,

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Figure 7. Polarization microscopic images of SD-29 DNA films prepared from the thick-layer solution at various intensities of magnetic field. The photographs show the center regions of circular DNA films. The films were situated normally (X-1 and X-2, where X denotes the code of the row) and were tilted by 45° (X-3 and X-4) for the polarization system indicated schematically at the bottom. Images observed with a sensitive tint plate (STP) are shown in columns X-2 and X-4, which correspond to pictures X-1 and X-3 obtained without the STP, respectively.

alleviating the problem of chain entanglement. Exceptionally, a small amount of a disordered region, such as a loop, may occur between the aligned chain elements, because the whole chain length of actual DNA is much longer than the statistical chain element. Orientation Pattern of Films Prepared from a ThickLayer Solution. DNA films with good chain alignment were obtained by drying the 0.05-mm layer solution under the magnetic field as described above. On the other hand, the films obtained from the 1.3-mm layer DNA solution, which had been placed as a droplet, exhibited special patterns of chain orientation. This phenomenon was found even if the film had been prepared without the magnetic field (Figure 7A). Typical polarization microphotographs are shown in Figure 7 for DNA films prepared with magnetic intensities of up to 10 T in 2-T increments. The obtained films exhibited isotropic chain orientation in none of these cases, instead showing cross patterns. The microphotographs with a sensitive tint plate revealed the symmetrical patterns of chain orientation in these films. The 45° tilted images of the films prepared above 4 T show characteristic zonal regions at the film centers, which have relatively intensive chain orientation (columns of X-3 and X-4 in Figure 7). The directions of chain orientation in a film depend significantly on both the distance and the azimuthal angle from the center of the circular droplet, as well as on the magnetic intensity. In the condition without a magnetic field, the cross pattern found by the polarized imaging method would be attributable

Morii et al.

Figure 8. Orientation patterns of DNA chains in the circular films prepared from the thick-layer solution. The curves were drawn along the representative chain orientation at respective positions (illustrations in the right column). The photographs in the left column indicate the corresponding polarization microscopic views, which are the same as in Figure 7. The specific points discussed in the text are represented with italic characters (o, d, p, t, and e). In the grayish region, the chain orientation was ambiguous or disordered. The arrows in the left column denote the direction of a magnetic field.

to a phenomenon consisting of a series of unit processes, as follows.25 (i) DNA molecules are concentrated near the airliquid interface as a result of evaporation. (ii) The DNA chains are transported to the peripheral edge of the droplet. (iii) The concentrated DNA chains form a liquid crystal phase along the edge curve. (iv) As drying progresses, the region with annular chain alignment becomes a film (points o, d, and p in Figure 8A). (v) The translational rate of the solidliquid interface speeds up to form a less-ordered region as a transient change (point t in Figure 8A). (vi) High-speed translation brings about a radial chain orientation by the trailing effect (point e in Figure 8A). The DNA films prepared with 2-T and 10-T magnetic intensities gave highly deformed orientation patterns as illustrated in parts B and C of Figure 8, respectively. Each film appears to consist of a number of domains, each with a characteristic orientation. The pattern of the film prepared in the 2-T magnetic field somewhat resembled that prepared in the 0-T field. However, at point o by 2 T (Figure 8B), the extent of the chain alignment was extremely lower than at the other points and different from that at point o in the 0-T film (Figure 8A). Both the magnetic field given along a


horizontal line and the annular edge effect as described above are thought to play major roles in aligning the DNA chains. The former effect would orient the chain vertically for the baseline of this figure, whereas the latter effect would align the chains at point o horizontally. Because these effects are orthogonal at point o, they are supposed to conflict and to form a less-ordered region around this point. Point e also apparently suffers from the magnetic effect, which seems to disturb only horizontal trailing effects, as recognized from the change for the shape like a gourd. Under the 10-T magnetic field, the obtained DNA film showed a very biased pattern of chain orientation (Figure 8C). At the vertical region, including the center point e of the film, the magnetic effect would overcome the annular edge effect, resulting in a highly ordered region with vertical alignment. The regions around point p for Figure 8A-C commonly exhibited almost completely vertical chain alignment, because the orientation by the magnetic effect is in accord with that by the annular edge effect. The cooperation of these concordant effects much enhances the extent of chain alignment. Though the magnetic effect by the 10-T field was significant for chain orientation in this case, it is interesting that the annular edge effect remained at region d. As shown in Figure 8C, pseudo-boundaries between regions bearing characteristic chain orientations were found, probably due to the balance between these representative effects. Both the magnetic effect with concentrating at the surface and the regulation by the solid-liquid interface are responsible for the characteristic orientation pattern of the DNA film obtained from a thick-layer solution. On the other hand, the dry process of the thin-layer solution proceeds within a relatively short time so that the shift of solid-liquid interface is too rapid to form chain orientation due to the regulation at the interface. Consequently, the orientation pattern from the thin-layer solution would be defined only by the magnetic effect, which resulted in the formation of the solid film with homogeneous chain orientation in a wide region. Dependence of Chain Alignmetn on Magnetic Intensity. To elucidate the dependence of the degree of chain alignment on the magnetic intensity, the birefringence of the obtained films was analyzed. For this purpose, a droplet of DNA solution with about 1.3-mm thickness was used. This method was modified slightly from that described in the early sections, in which the solution was in the form of a 0.05mm layer. The thick-layer solution is suitable for evaluating the magnetic effect, because it can be sufficiently processed in a magnetic field before the material is completely dry. On the contrary, the overall magnetic condition for thinlayer solution is difficult to be regulated because of the rapid progress of drying. The birefringence was measured at positions 2 mm from the center. Specifically, three representative positions were selected for the observation. They are located on the central lines perpendicular, parallel, and tilted by 45° to the magnetic field and on the same circle as shown in the inlet of Figure 9, which we call points o′, p′, and d′,respectively. The observed birefringences of the films were plotted against the provided magnetic intensities (Figure 9). In the 0-T field, the DNA chains were aligned only by the annular


Figure 9. Dependence of birefringence in the SD-22 DNA film on the magnetic intensity. Birefringence was measured at three positions defined in the circular film prepared from the thick-layer solution as shown in the inset, where the arrow represents the direction of the magnetic field. These positions (closed circle, triangle, and open circle) are on the same circle with a 2-mm radius.

edge effect so that the birefringence at each of the three observation points was 0.006. Interestingly, the birefringence values by the 2-T field were reduced to almost nothing at points o′ and d′. This suggests that the magnetic effect by 2 T and the annular edge effect are almost balanced at points o′ and d′, where the two effects act on the DNA from different directions. In contrast, the value at point p′ remained about 0.006. In the magnetic field above 3 T, the birefringence increased sharply and became saturated at the 8-T field. Such a change, involving a steep increase and saturation, was demonstrated for a general case theoretically.37 This can be interpreted as a phenomenon that depends on the balance between the order and the disorder caused by thermal motion. Therefore, with regard to the diluted DNA solution, it is suggested that a magnetic field of about 4 T can compensate for the thermal randomizing. Among the observation positions, the birefringence at point p′ was highest, with a value of 0.027. On the other hand, the value at point o′ resulted in a birefringence of only 0.012 even in a 10-T field. Thus, as with the thin-layer system, both the magnetic effect and another one, such as the annular edge effect, would cooperatively enhance the chain alignment for the thick-layer droplet system. For a portion of the structural change reaction described with a two-state model isolated by a relatively high transition state, the magnetic intensity and its action time may affect the extent of molecular alignment synergistically. In contrast, the chain alignment from the solution system as investigated in this work is not a simple but rather a processive reaction. Thus, the magnetic field must pass a certain threshold intensity to overcome the vigorous thermal motion disrupting the orientation. This would be the most essential condition for the magnetic effect on chain alignment. Influence of Molecular Weight. To clarify the contribution of molecular weight on chain alignment, DNA solid films were prepared under a 10-T magnetic field and analyzed in a similar manner as above. DNA films were obtained from the samples whose molecular weights ranged from 1.4 to 29.1 kbp, as designated in the experimental section. The birefringence at point p′ of these films is plotted in Figure 10. The higher the molecular weight of a sample,

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Figure 10. Molecular weight dependence of birefringence. The abscissa axis represents the weight-averaged molecular weight of DNA, and the ordinate shows the birefringence at point p′ of the circular DNA film prepared from the thick-layer solution in the 10-T magnetic field.

the more significant its birefringence. There seems to be an almost linear relationship between birefringence and the logarithmic molecular weight. This result is in line with that of the DNA films prepared without a magnetic field, whose annular orientation was enhanced by the higher molecular weight.25 As described above, we verified experimentally that the liquid crystal phase forms near the surface while the solution is drying. This was also supported by the calculation of the average diffusion rate. The diffusion of average DNA chains in samples SD-1 and SD-29 are estimated to be 0.016 and 0.008 mm/min, respectively. The former diffusion rate is almost comparable with the declining speed of the air-liquid interface (ca. 0.02 mm/min) so that some portion of such short chains could possibly get away from the interfacial layer. Moreover, the shorter DNA chain is estimated to have the larger rotational diffusion constant as expressed by eq 3 so that its rotational motion may overcome the chain alignment by the interfacial orientation. In a liquid crystalline phase, the thermal disorientation energy of DNA chains is competing with the stabilization energy by the formation of liquid crystalline. Though the latter energy per base pair is almost independent of the molecular weight, the former energy is not. Because the lower molecular weight chains have larger disorientation energy per base pair, this would reduce the degree of chain orientation. Thus, the samples, including low molecular weight DNA chains, are at a disadvantage in reaching a well-ordered chain alignment. The dependence of birefringence on the molecular weight was consistent with the result of X-ray diffraction as discussed in the previous section, but here the proof was more explicit. Influence of Single-Stranded DNA Chains. The solid films prepared from thermally unwound DNA and those from the usual one were compared by means of polarization microscopy (Figure 11). Though the usual DNA film showed an obvious chain orientation pattern induced by a magnetic field and along the periphery curve as described above, the thermally denatured DNA resulted in only weak orientation. The distinct difference observed by polarization microscopy was also found for the films prepared from these two DNA samples without a magnetic field. Because the content of single-stranded DNA is reported to be about half in the thermally denatured sample,26,38 our denatured samples may

Morii et al.

Figure 11. Polarization microscopic images of the films prepared from native (A), thermally denatured (B), and alkali-treated (C) DNA samples. The solid films were dried from the thick-layer solutions with and without 10-T magnetic field, which were represented at the top and the bottom of this figure, respectively. The films with 6-mm diameter were prepared from 15 µL of DNA solution at the concentration of 30 mg/mL. The photographs show the periphery regions of the circular films. The films were tilted by 45° for the polarization system indicated at the side.

contain both single- and double-stranded DNA to some extent. Therefore, single-stranded DNA would be not only difficult to form chain alignment but also disruptive to the chain orientation of the coexisting double-stranded component. On the other hand, the alkali-treated DNA gave no chain orientation as shown in Figure 11C. This is a little different from the result of thermally denatured DNA. This may be attributed to that the alkali treatment caused the chain scission of phosphate bonds as well as the dissociation of doublestranded coils. On the contrary, thermally denatured DNA would involve only the dissociation. The system containing the single-stranded DNA might be complicated because of the rearrangement of base pairs which brings about crosslinking sites or heterogeneous domains. In summary, the single-stranded DNA is unfavorable to the alignment and the orientation of the chains. In contrast, it is suggested that the rigidity of double-stranded DNA chains and the wellordered base-pair stacking are responsible for chain alignment. Conclusion DNA films having highly aligned molecular chains can be fabricated by drying the solution in a horizontal magnetic field. To realize a well-ordered chain alignment, it is essentially important that the initial concentration of DNA solution is lower than that bringing about the liquid crystal formation before evaporation. The largest factors in orienting the chains along a unitary direction are thought to be both the use of a magnetic field and an interfacial effect that induced the chains to fit along the air-liquid interface. The anisotropic DNA film developed in this work may have various other applications. Moreover, the method involving the mechanism for chain alignment would be applicable to enhance the functionality of other polymers. Acknowledgment. We thank Dr. Hiroyuki Morioka (NIMS) for the kind support in X-ray measurement and Dr. Miyuki Ishimura (AIST) for helpful discussions on DNA


structures. This work was supported in part by a research grant from the Foundation for the Promotion of Material Science and Technology of Japan (MST), which is greatly appreciated. The authors are also grateful for a grant-in-aid provided by the Ministry of Economy, Trade and Industry, Japan. References and Notes (1) Gu, J.; Cai, L.; Tanaka, S.; Otuka, Y.; Tabata, H.; Kawai, T. J. Appl. Phys. 2002, 92, 2816-2820. (2) Liang, X.; Asanuma, H.; Komiyama, M. J. Am. Chem. Soc. 2002, 124, 1877-1883. (3) Takenaka, S.; Yamashita, K.; Takagi, M.; Uto, Y.; Kondo, H. Anal. Chem. 2000, 72, 1334-1341. (4) Uemura, S.; Shimakawa, T.; Kusabuka, K.; Nakahira, T.; Kobayashi, N. J. Mater. Chem. 2001, 11, 267-268. (5) Maeda, Y.; Kawai, T. Jpn. J. Appl. Phys. 1998, 38, L12111212. (6) Magan, J. D.; Blau, W.; Croke, D. T.; McConnell, D. J.; Kelly, J. M. Chem. Phys. Lett. 1987, 141, 489-491. (7) Yang, C. Y.; Yang, W. J.; Moses, D.; Morse, D.; Heeger, A. J. Synth. Met. 2003, 137, 1459-1460. (8) Bensimon, A.; Simon, A.; Chiffaudel, V.; Heslot, F.; Bensimon, D. Science 1994, 265, 2096-2098. (9) Ijiro, K.; Shimomura, M.; Tanaka, M.; Nakamura, H.; Hasebe, K. Thin Solid Films 1996, 284, 780-783. (10) Kabata, H.; Kurosawa, O.; Arai, I.; Washizu, M.; Margarson, S. A.; Glass, R. E.; Shimamoto, N. Science 1993, 262, 1561-1563. (11) Okahata, Y.; Kobayashi, T.; Tanaka, K.; Shimomura, M. J. Am. Chem. Soc. 1998, 120, 6165-6166. (12) Kraack, H.; Ocko, B. M.; Pershan, P. S.; Sloutskin, E.; Tamam, L.; Deutsch, M. Langmuir 2004, 20, 5375-5385. (13) Higuchi, M.; Takizawa, A.; Kinoshita, T.; Tsujita, Y.; Okochi, K. Macromolecules 1990, 23, 363-365. (14) Lederer, K.; Godt, A.; Howes, P. B.; Kjaer, K.; Als-Nielsen, J.; Lahav, M.; Wegner, G.; Leiserowitz, L.; Weissbuch, I. Chem.sEur. J. 2000, 6, 2173-2183. (15) Fukuto, M.; Heilmann, R. K.; Pershan, P. S.; Yu, S. M.; Griffiths, J. A.; Tirrell, D. A. J. Chem. Phys. 1999, 111, 9761-9777. (16) Krausch, G.; Magerle, R. AdV. Mater. 2002, 14, 1579-1583.

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Molecular Chain Orientation of DNA Films Induced by Both the

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