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Orientation of DNA Double Strands in a Langmuir-Blodgett Film Yoshio Okahata,* Takuya Kobayashi, and Kentaro Tanaka Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama, 226 Japan Received June 30, 1995. In Final Form: November 6, 1995X A cationic amphiphile monolayer was spread on a subphase containing DNA double strands and intercalating dyes, and the polyion complex DNA/dye/cationic monolayer was transferred to a substrate by the vertical dipping method. It was confirmed by X-ray diffraction patterns and polarized absorption spectra that the dye-intercalated DNA strands were aligned along the dipping direction in the LB film. DNA strands are not aligned in the LB film without intercalating dye molecules. When the DNA-lipid monolayers were deposited by a horizontal lifting method instead of a vertical dipping method, DNA strands were randomly oriented in the film. The orientation of DNAs was decreased when the monolayer packing was decreased by using a low surface pressure or an expanded monolayer. Thus, the rigid, dyeintercalated DNA strands could be oriented during the vertical dipping process.
Introduction DNA is well-known as a source of biological information depending on its base sequences. DNA is also interesting as a material that exists as double helical rodlike molecules consisting of base-pair (bp) stacking. Rodlike polymers such as polyglutamate,1-3 polysiloxane,4 alkylated cellulose,5 and discotic crystals6 have been reported to form Langmuir-Blodgett (LB) films in which rodlike molecules aligned in one direction during the compression process on the subphase7,8 or the deposition process of monolayers.9 DNA is a good candidate to form an oriented LB film in the same manner. However, DNA is soluble only in a water phase due to the anionic phosphate backbone. In this paper, we describe the orientation of DNA strands along the dipping direction of LB films (see Figure 1). The polyion-complex-type monolayers were prepared between cationic monolayers and anionic DNA strands intercalated with dye molecules in the subphase. When the DNA/ dye/monolayer was transferred by a vertical dipping method, the rigid, dye-intercalated DNA strands were aligned parallel to the dipping direction. Ordering of DNA strands in LB films was confirmed by X-ray diffraction patterns and polarized absorption spectra. Formation of polyion complexes between charged lipid monolayers and water-soluble polyions has been studied to increase the stability of monolayers on the subphase and transferred LB films.10-14 Abstract published in Advance ACS Abstracts, February 1, 1996. X
(1) Duda, G.; Wegner, G. Macromol. Chem. Rapid Commun. 1988, 9, 495. (2) Hickel, W.; Duda, G.; Jurich, M.; Kro¨hl, T.; Rockford, K.; Stegeman, G. I.; Swallen, J. D.; Wegner, G.; Knoll, W. Langmuir 1990, 6, 1403. (3) Adue, N.; Ringenbach, A.; Stevenson, I.; Jugnet, Y.; Duc, T. M. Langmuir 1993, 9, 3567. (4) Erbach, R.; Hoffmann, B.; Schaub, M.; Wegner, G. Sens. Actuators 1992, B6, 211. (5) Gaines, G., Jr. Langmuir 1991, 7, 834. (6) Karthaus, O.; Ringsdorf, H.; Tsukruk, V. V.; Wendorff, J. H. Langmuir 1992, 8, 2279. (7) Malcom, B. R. Adv. Chem. 1975, 145, 338. (8) Jones, R.; Tredgold, R. H. J. Phys. D., Appl. Phys. 1988, 21, 449. (9) Schwiegk, S.; Vahlenkamp, T.; Xu, Y.; Wegner, G. Macromolecules 1992, 25, 2513. (10) Shimomura, M.; Kunitake, T. Thin Solid Films 1985, 132, 243. (11) Higashi, N.; Kunitake, T. Chem. Lett. 1986, 105. (12) Nishiyama, K.; Kunihara, M.; Fujihira, M. Thin Solid Films 1989, 179, 477. (13) Kajiyama, T.; Zhang, L.; Uchida, M.; Oishi, Y.; Takahara, A. Langmuir 1993, 9, 760. (14) Seki, T.; Tohnai, A.; Tamaki, T.; Ueno, K. J. Chem. Soc., Chem. Commun. 1993, 1876.
Figure 1. Schematic illustrations of the formation of a DNAoriented LB film by using a polyion complex of DNA/intercalater and cationic lipid monolayers.
Experimental Section Materials. DNA-Na+ from Salmon testes (Sigma Co., ca. 2000 bp’s) was cut to a short strand by irradiation using ultrasonic power (40 W) of an aqueous solution of commercial DNA (100 mg/50 mL with 0.1 M NaCl) for 10 min under N2 bubbling and cooling. The shortened DNA was purified by reprecipitation in ethanol, and the average length was confirmed by agarose gel electrophoresis to be 300 ( 50 bp. DNA was dried as a fibrous powder by freeze-drying. Preparation of dialkyl amphiphiles, dioctadecyl N-(((trimethylammonio)methyl)carbonyl)glutamate chloride (2C18-glu-N+ Cl-) and bis(11-oxaheptadecyl)-N-(((trimethylammonio)methyl)carbonyl)glutamate chloride (2C10-O-6-glu-N+ Cl-) were reported elsewhere.15 Organic solvents for a stock solution of LB film-forming amphiphiles were purchased as spectra analytical grade and (15) Ariga, K.; Okahata, Y. J. Am. Chem. Soc. 1989, 111, 5618.
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used without further purification. Water for the subphase was purified with a Milli-QII system (Millipore, Co.) whose specific resistance was ca. 18 MΩ cm. Langmuir-Blodgett (LB) Films. Measurements of pressure-area (π-A) isotherms and the transfer of monolayers on a substrate to prepare LB films were carried out by using the computer-controlled film balance system FSD-20 (USI system, Co., Fukuoka).15,16 The trough surface and the moving barrier were coated with Teflon, and the subphase was temperature controlled with a thermostat (20 ( 0.5 °C). A 10 µM bp-1 amount of DNA and then the respective concentration of intercalating dye molecules were solvated in the subphase of Mili-QII water. A chloroform solution of cationic dialkyl amphiphiles was spread on the subphase. After 1 h, a monolayer was compressed and π-A isotherms were measured. Monolayers were transferred to the hydrophobic quartz plate surface treated with 1,1,1,3,3,3hexamethyldisilazane by using a conventional vertical dipping and a horizontal lifting method. The transfer process of the DNA-lipid complex was followed by both the conventional barrier movement on the trough and the quartz crystal microbalance (QCM) method. QCMs are known to be very sensitive mass measuring devices because their vibration frequency decreases upon the deposition of mass on the Au electrode to the nanogram level.17-20 We already reported that the transferred mass of LB films can be directly followed from the frequency decrease (mass increase) on the QCM substrate.16 Calibration of the QCM used in this experiment gave the following equation according to Sauerbrey’s equation.15-18
∆m ) -(1.15 ( 0.01) × 10-9∆F
Figure 2. π-A isotherms of 2C18-glu-N+ monolayers on (a) pure water, (b) [DNA] ) 10 µM bp-1 in the solution, and (c) [DNA] ) 10 µM bp-1 and [proflavine] ) 10 µM in the solution at 20 °C.
(1)
The Au electrodes of the QCM were hydrophobized by treatment with a self-assembled monolayer of dodecanethiol, to transfer LB films. X-ray Diffraction and Polarized Absorption Spectra. The orientation of DNA strands in the LB film was measured by use of an X-ray diffraction instrument that recorded on an imaging plate (Rigaku-Denki Co., Tokyo, model R-AXIS). The ca. 100 layers of the LB film were deposited on an amorphous polystyrene sheet. Polarized absorption spectra of LB films (ca. 80 layers) of DNAlipid complex deposited on a quartz plate were measured by a diode array photospectrometer (Hewlett Packard, Co., Tokyo) by using polarized I2 light at 0° and 90°.
Results and Discussion Figure 2 shows π-A isotherms of cationic 2C18-gluN+ amphiphiles spread on pure water, on a DNA solution (10 µM bp-1), and on a DNA solution (10 µM bp-1) with an intercalating dye (proflavine, 10 µM). These π-A isotherms of 2C18-glu-N+ monolayers were hardly affected by the presence of DNA and dye molecules in the subphase, and the minimum area per molecule was obtained to be ca. 0.4 nm2 consisting of the molecular area of the dialkyl chains. The collapse pressure of the monolayer increased in the presence of DNA and/or dyes. These results indicate that cationic 2C18-glu-N+ amphiphiles can form stable monolayers even in the presence of DNA with or without intercalated dye molecules in the subphase and that polyion complex formation with DNAs does not expand monolayers and seems to stabilize the monolayer. The 2C18-glu-N+ monolayer spread on the solution of 10 µM bp-1 DNA and 10 µM proflavine was transferred to the hydrophobized quartz plate or QCM plate at a (16) Ariga, K.; Okahata, Y. Langmuir 1994, 10, 3255. Okahata, Y.; Ariga, K. J. Chem. Soc., Chem. Commun. 1987, 1535. (17) Sauerbrey, G. Z. Phys. 1959, 155, 206. (18) Okahata, Y.; Ebato, H.; Taguchi, K. J. Chem. Soc., Chem. Commun. 1987, 1363. (19) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986, 58, 1206. (20) Evansole, R. C.; Miller, J. A.; Moran, J. R.; Ward, M. D. J. Am. Chem. Soc. 1990, 112, 3239. (21) Okahata, Y.; Ariga, K.; Tanaka, K. Thin Solid Films 1992, 210/ 211, 702.
Figure 3. Vertical dipping processes of DNA-/proflavine/2C18glu-N+ LB films. (A) Barrier movement on the trough, (B) frequency decrease (mass increase) of a QCM substrate during the vertical dipping process, and (C) linear correlation between transferred weight of DNA-/proflavine/2C18-glu-N+ LB films obtained from the QCM measurements and deposition cycles. [DNA] ) 10 µM bp-1, [proflavine] ) 15 µM, at 20 °C, at the surface pressure of 35 mN m-1, and at the dipping speed of 10 mm min-1.
surface pressure of 35 mN m-1 at 20 °C. Figure 3 shows barrier movements of the monolayer area and the frequency decrease (mass increase) on the QCM substrate depending on dipping cycles. From the moving area of the barrier, two layers of the monolayer was confirmed to be transferred in each cycle (Y-type deposition). From the QCM measurement, 283 ( 10 ng of substance was calculated to be transferred to the substrate at each cycle from eq 1. A good linear correlation was obtained between the transferred weight of DNA/dye/lipid LB films and dipping cycles (see Figure 3c). The mass of two layers of
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Figure 4. X-ray diffraction patterns of DNA-/proflavine/2C18glu-N+ LB films (ca. 100 layers). Open two-headed arrows show the vertical dipping direction, and closed arrows show the incident beam of the X-ray.
the 2C18-glu-N+ monolayer is calculated to be 203 ( 10 ng from the area of the QCM plate and the area per lipid molecule. Therefore, 80 ( 10 ng of DNA with intercalated dyes was calculated to be deposited with two monolayers. Since the amount of intercalated dyes can be obtained from absorption at 450 nm (described later: 6 ( 2 ng), the amount of transferred DNA was estimated to be 74 ( 5 ng, which means ca. 95% of the monolayer area was covered by DNA molecules. The LB films of the DNA-/proflavine/2C18-glu-N+ complex were transferred to an amorphous polystyrene sheet with ca. 100 layers (ca. 50 cycles), and X-ray diffraction patterns were measured. As shown in Figure 4, two spots with a distance of 41 Å (the diameter of a DNA-lipid strand) were clearly observed perpendicular to the dipping direction when the beam was exposed both parallel to the side edge or perpendicular to the LB film plane. When the beam was irradiated parallel to the top edge of the LB film, the circular reflection with 41 Å diameter was observed. These findings clearly show that DNA-lipid strands were aligned along the dipping direction in the LB film as shown in the illustration. The LB film of the DNA-/proflavine/2C18-glu-N+ complex was transferred with 44 layers (22 cycles) on one side of the hydrophobized surface of a quartz plate (a total of 88 layers on both sides), and the polarized absorption spectra were measured in aqueous solution (see Figure 5a). The LB film did not swell or remove from the substrate in the aqueous solution. The absorption of the intercalated proflavine at 450 nm for the light polarized perpendicular to the dipping direction was 2.6 times larger than that for the parallel polarized light (A⊥/A| ) 2.6). This indicates that the transition moment of the intercalated dyes between base-pairs is aligned perpendicular to the dipping
Figure 5. Polarized absorption spectra of DNA-/proflavine/ 2C18-glu-N+ LB films (88 layers) deposited by (a) the vertical dipping method and (b) the horizontal lifting method.
direction. Thus, DNA strands in the LB film are aligned parallel to the dipping direction. The absorption spectra hardly changed after soaking in the aqueous solution at least for 2 days: reorientation of DNA strands and/or removal of intercalated dyes hardly occurred. The two-dimensional orientation parameter of the dye molecules intercalated perpendicular to DNA strands was calculated to be ca. 0.5 from dichroic ratio of A⊥/A| ) 2.6. This value is close to the orientation of synthetic rodlike polymers such as poly(arylenes) prepared by a uniaxial stretching method.22,23 The monolayer of the DNA-/proflavine/2C18-glu-N+ complex could be transferred by a horizontal lifting method to the hydrophobized quartz plate. Two lipid monolayers (Y-type) with 95% coverage of DNA were confirmed to be transferred in one cycle of the horizontal lifting process by using the QCM method.21 As shown in Figure 5b, the absorption spectra were independent of the polarization direction of the light. This indicates that DNA strands are not aligned in the LB film deposited with the horizontal lifting method. The advantages of the horizontal lifting (22) Skotheim, T. A., Ed. Handbook of Conducting Polymers; Marcel Dekker: New York, 1986. (23) Yamamoto, T.; Maruyama, T.; Zhou, Z.; Ito, T.; Fukuda, T.; Yoneda, Y.; Begum, F.; Ikeda, T.; Sasaki, S.; Takezoe, H.; Fukuda, A.; Kubota, K. J. Am. Chem. Soc. 1994, 116, 4832.
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Table 1. Effect of Transfer Conditions on the Orientation of DNA Strands in the Transferred DNA/Proflavine/Cationic Lipid LB Filmsa transfer conditions
run
lipid molecules
[proflavine] in the subphase/µM
1 2 3 4 5 6
2C18-glu-N+ 2C18-glu-N+ 2C18-glu-N+ 2C18-glu-N+ 2C18-glu-N+ 2C10-O-6-glu-N+
0 5 10 10 10 10
a
[DNA] ) 10 mM
bp-1
LB films
surface pressure/mN m-1
dipping speed/mm min-1
[dye] per 100 base-pair
surface coverage of DNA/%
dichroic ratio A⊥/A|
35 35 35 5 35 35
10 10 10 10 100 10
0 3.6 20 28
55 47 95 21
57
74
1.0 1.3 2.6 1.3 2.1 2.1
in the subphase; 88 layers of LB films were transferred.
method is known to be deposition of the real monolayer structure on the subphase, in contrast to the vertical dipping method. Wegner and co-workers reported that rodlike polymers are oriented during compression process by the flow on the subphase.9 It is suggested from these data that DNA strands are not aligned on the subphase and are oriented during the vertical dipping process by the help of the flow of compression. From the dichroic ratio (A⊥/A|) of 2.6 for the DNA-/ proflavine/2C18-glu-N+ LB films, the average tilt angle (θ) of dyes from the dipping direction can be calculated to be 64°,24 and the molar absorption coefficient (film) of dyes in the film was calculated to be 26 000 from the value in the solution (soln ) 29 000). The intercalated amount of proflavine per layer in the LB film was calculated to be 2.9 × 10-9 mol cm-2 from the film value (6 ng/2 layers). From the QCM measurement, the total amount of the transferred LB films (DNA + dye + lipid) could be obtained. Since the amount of transferred lipids and the intercalated dyes can be calculated from the area of the barrier movement and absorption spectra, respectively, each component can be separated. Thus, in the LB film of DNA-/proflavine/2C18-glu-N+ complex, it was found that 20 proflavine molecules were intercalated per 100 base-pairs and that the dye-intercalated DNA was transferred with 95% area coverage of the monolayer (see run 3 in Table 1). Table 1 shows effects of transfer conditions on the transfer and orientation of DNAs in the LB films. In the absence of proflavine in the subphase, the transferred DNA/lipid LB films do not show any dichroic anisotropy, and the transferred amount of DNA was small (run 1). When the concentration of proflavine dye in the subphase is increased, the transferred amount of the intercalated dye and DNA and the orientation of DNAs were increased (runs 1-3). Thus, when dye molecules intercalated into DNAs, DNA strands are getting a rigid rod and easily oriented in the dipping direction. The DNA without dyes seems to be a flexible strand and is difficult to orient during the dipping process. Similar DNA-oriented LB films were obtained when other intercalaters such as acridine orange, ethidium bromide, and safranine T, were employed as shown in Scheme 1 with their dichroic ratio. When the dyes having an acridine ring such as proflavine and acridine orange were employed, a large dichroic ratio was obtained. The effect of the surface pressure of the 2C18-glu-N+ monolayer on the orientation of DNAs in LB films was also studied (runs 3 and 4 in Table 1). The transferred (24) The average tilt angle θ of intercalated dyes in DNAs was calculated according to the following equation, which was introduced for the tilt angle of rodlike polyarenes in the oriented cast film obtained by polarized absorption IR spectra (see ref 23).
(A⊥/A|) - 1 (A⊥/A|) + 2
3 3 ) (cos2 θ) 2 2
Figure 6. π-A isotherms of 2C10-O-6-glu-N+ monolayers on (a) pure water, (b) [DNA] ) 10 µM bp-1 in the solution, and (c) [DNA] ) 10 µM bp-1 and [proflavine] ) 10 µM in the solution at 20 °C. Scheme 1. Alternative Intercalaters
amount of DNA and the dichroic ratio were decreased when the surface pressure was decreased from 35 to 5 mN m-1. Thus, at the high surface pressure, the dyeintercalated DNA can be transferred largely with cationic monolayers and the tightly packed DNA strands can be oriented during the dipping process. These depositions were carried out at a dipping speed of 10 mm min-1. When the dipping speed was increased to 100 mm min-1, the orientation of DNA strands was slightly decreased (runs 3 and 5). Figure 6 shows the π-A isotherms of cationic 2C10-O-6glu-N+ monolayers having an ether linkage in the two alkyl chains, which was spread on pure water, on a DNA solution, and on a DNA with proflavine solution. The 2C10-O-6-glu-N+ amphiphiles show an expanded, liquidlike monolayer due to the ether linkages in the two alkyl chains, and π-A isotherms were hardly affected by DNAs and intercalating dyes in the subphase. When the DNA-/ proflavine/2C10-O-6-glu-N+ LB films were transferred, the amount of intercalated dye, the transferred amount of DNA, and the orientation of the DNA were smaller than those of the DNA-/proflavine/2C18-glu-N+ LB films (see runs 3 and 6 in Table 1). This tendency is similar to the effect of the surface pressure: when the monolayer is expanded, DNAs are not packed tightly and are difficult to orient during the dipping process.
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The effect of the DNA length on the orientation was also studied. Two different lengths of DNA (300 bp, ca. 100 mm length and 2000 bp, 680 nm) were employed. The dichroic ratios (A⊥/A|) were obtained to be 2.1 and 1.9 for the LB films of 300 bp and 2000 bp DNA, respectively. Thus, a difference in DNA length of about 7 times does not affect the orientation of DNA strands during the transfer process. Conclusion We have succeeded in producing DNA-oriented LB films in the presence of dye intercalaters by using a complex
Okahata et al.
formation with cationic monolayers at the air-water interface. When dye molecules intercalated between basepairs, DNA strands seem to be rigid and the transfer ratio with monolayer and the orientation during the vertical dipping process is increased. The DNA-oriented LB film is expected to be a new material for one-dimensional electron transfer and conduction along stacked base-pairs and/or intercalated redox active dyes in double strands. These experiments are underway in our laboratory. LA950529N
