© Copyright 1996 American Chemical Society
AUGUST 21, 1996 VOLUME 12, NUMBER 17
Letters Direct Demonstration of Attraction for a Complementary Pair of Apposed Nucleic Acid Base Monolayers Kazue Kurihara,*,†,‡ Takashi Abe,† and Naotoshi Nakashima§ Department of Applied Physics, School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan; PRESTO, JRDC, Japan; and Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo, Nagasaki 852, Japan Received October 13, 1995. In Final Form: March 15, 1996X Direct measurement of surface forces has been employed for the systematic determination of interaction forces between nucleic acid base monolayers at various pH values from 5.6 to 9.3. Measured forces have been classified as (1) forces at a distance D > 20 nm, which are attractive or repulsive depending on the hydrophobicity and the charge of the surfaces and which can be accounted for in terms of the sum of “double-layer forces” and so-called “very long ranged hydrophobic attraction”; (2) forces at D < 20 nm, which are always attractive; and (3) pull-off (adhesive) forces from contact, which reflect characteristics of molecular contact between monolayers of nucleic acid bases. The forces between complementary pairs (adenine-thymine) are found, for the first time, to be always attractive and stable independently of separation distances as well as pH’s studied. On the other hand, interactions between noncomplementary pairs (thymine-thymine, adenine-adenine) are found to be regulated by the surface charge; thus, notably the long-ranged forces change from attractive to repulsive at pH’s where the nucleobases start to dissociate.
Elucidation of the mechanism which governs complementary nucleic acid base pairing is of fundamental importance in biology and is crucial to our understanding of molecular recognition.1-3 Surface force measurements have been shown to provide a wealth of information on the interaction forces between apposed molecular layers.4 Indeed, specific molecular interactions between molecules of streptavidin-biotin (receptor-ligand)5 and those of * To whom correspondence should be addressed. E-mail address:
[email protected]. † Nagoya University. ‡ PRESTO. § Nagasaki University. X Abstract published in Advance ACS Abstracts, July 15, 1996. (1) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113-158. (2) Hamilton, A. D. In Advances in Supramolecular Chemistry; Gokel, G. W., Ed.; JAI Press Inc., Greenwich, 1990; pp 1-64. (3) Jorgensen, W. L.; Pranata, J. J. Am. Chem. Soc. 1990, 112, 20082010. (4) Ashman, R. B.; Blanden, R. V.; Ninham, B. W. Immunol. Today 1986, 7A, 278-283. (5) Leckband, D. E.; Israelachvili, J. N.; Schmitt, F.-J.; Knoll, W. Science 1992, 255, 1419-1421; Biochemistry 1994, 33, 4611-4624.
S0743-7463(95)00867-5 CCC: $12.00
nucleic acid bases6-8 have been examined by surface force measurements. However, to the best of our knowledge, interaction forces between complementary and noncomplementary base pairs have not been unequivocally determined by this technique. In previous works, generalization of the nucleic acid base interaction was difficult because of insufficient stability of the base-functionalized surfaces6,7 and the limited conditions of the measurements.8 We have, therefore, undertaken systematic measurements of surface force between apposed amphiphilic monolayers composed of adenine and thymine, adenine and adenine, and thymine and thymine at various pH values from 5.6 to 9.3 and report, for the first time, that interaction between the complementary adeninethymine monolayers is always attractive and stable independently of the surface separation and pH. On the (6) Kurihara, K. In Microchemistry: Spectroscopy and Chemistry in Small Domains; Masuhara, H., et al., Eds.; (Elsevier Science B. V.: Amsterdam, 1994; pp 401-414. (7) Bemdt, P.; Kurihara, K.; Kunitake, T. Langmuir 1995, 11, 30833091. (8) Pincet, F. L.; Perez, E.; Bryant, G.; Lebeau, L.; Misoskowski, C. Phys. Rev. Lett. 1994, 73, 2780-2783.
© 1996 American Chemical Society
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Figure 1. Surface pressure-molecular area isotherms of nucleic acid base functionalized amphiphiles 1 (thymine) on the aqueous phase at pH 9.6 (KOH) and 2 (adenine) on pure water (pH ∼ 5.6) at 20.0 ( 0.1 °C.
other hand, interactions of noncomplementary pairs, adenine and adenine, or thymine and thymine, can be both attractive and repulsive depending on the dissociated state of nucleic acid base layers. This work is also important in connection with so-called “hydrophobic long ranged attraction”, which extends to 100-200 nm and has been measured only between highly hydrophobic surfaces of hydrocarbon and fluorocarbon monolayers previously.9,10 The present work demonstrates, against a generally held view, that a similar attraction can exist between less hydrophobic and nonhydrocarbon surfaces. Nucleic acid base monolayers (Figure 1) were formed on mica surfaces by Langmuir-Blodgett (LB) deposition of amphiphiles 1 (thymine) and 2 (adenine), which had ammonium groups and nucleic acid bases at their opposite terminals.11,12 The surface pressure (π)-molecular area (A) isotherms of monolayers 1 and 2 were measured at various pH’s (adjusted by HNO3 or KOH). The monolayers were transferred from the aqueous subphases at pH 9.6 (KOH) for 1 and from pure water (pH ∼ 5.6) for 2, where the π-A isotherms exhibited inflections. The transfer was performed at a surface pressure of 17 mN/m for 1 and 3.5 mN/m for 2, and at a deposition rate of 10 mm/min in the upstroke mode. At low or high pH’s such as 3 and 11 the nucleic acid bases were ionized; thus, both monolayers 1 and 2 expanded more (data are not shown) compared with those at 5.6 and 9.6 and dissolved gradually into the subphase by compression. The quaternary ammonium groups of 1 and 2, which were cationic at all pH’s, were (9) Kurihara, K.; Kunitake, T. J Am. Chem. Soc. 1992, 114, 1092710933. (10) Claesson, P. M.; Christenson, H. K. J. Phys. Chem. 1988, 92, 1650-1655. (11) Preparations of nucleic acid base functionalized amphiphiles 1 (thymine) and 2 (adenine) will be reported elsewhere.12 A computercontrolled film balance system (USI system, FSD 50) was used for measuring surface pressure as a function of molecular area. Spreading solutions of amphiphiles (1 mg/mL) were prepared in dichloromethane. Water was purified with a Nanopure II and Fi-streem 46D glass still system (Barnstead). (12) Nakashima, N.; Yamauti, Y.; Fukunaga, S. Manuscript in preparation.
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likely oriented toward the aqueous phase when the nucleic acid bases were in the electrically neutral or near neutral state (∼pH 5.6 and 9.6). They anchored the monolayers on a negatively charged mica surface and thereby formed stable nucleic acid base monolayers, as shown in Figures 3 and 4. The transfer ratios were 1.1 ( 0.1 for 1 and 1.0 ( 0.1 for 2, providing the molecular areas of 1 and 2 on mica to be 0.25 ( 0.02 and 0.24 ( 0.02 nm2/molecule, respectively. The thicknesses of the nucleic acid base monolayers on mica were determined from the contact positions of unmodified and monolayer-covered mica surfaces during the force measurements. They were 2.2 ( 0.6 nm for 1 and 3.4 ( 0.9 nm for 2, close to the lengths of these amphiphiles in the stretched forms, 3.5 ( 0.3 nm for 1 and 4.2 ( 0.3 nm for 2, which were estimated using a CPK molecular model. These density and length data of the monolayers indicate the formation of stable monolayers on mica. Atomic force microscope (AFM) images of transferred monolayers 1 and 2 (Figure 2) demonstrated further that regularly packed and dense monolayers were indeed formed on mica. Similar images were observed at all positions of the LB films studied, and no noticeable defects were found. Interestingly, a ridgelike pattern was seen frequently in the images of 2, which probably reflected the high tendency of base stacking between the adenine groups. Force profiles measured between adenine-thymine monolayers (complementary pair) are shown in Figure 3.13 In pure water (pH ∼ 5.6), the long-range attraction appears at a separation of 150 nm and increases with decreasing distance. When the slope of this attraction exceeds the spring constant (dF/dD g K), the surface jumps into contact at 33 nm ((3 nm). Phenomenologically, this long-range attraction is described by F/R ) -0.61 exp(-D/50) mN/m. This force is similar in range and magnitude to the long-range attraction found for hydrophobic surfaces in water.9,10 However, the pull-off (adhesive) force15 from the contact is 65 ( 13 mN/m, which is considerably smaller than those of hydrophobic surfaces (190-470 mN/m, depending on preparation).9 At a higher pH of 8.5 (KOH), the attraction increases. The long-range component follows the form F/R ) -1.35 exp(-D/60) mN/ m. The jump-in distance and the pull-off force are 42 nm (13) Forces measurement was performed by using the Surface Forces Apparatus Mark 4 (ANUTECH). Atomically flat mica surfaces were used as substrates and glued with an epoxy resin (Epikot 1004, Shell) onto cylindrical silica lenses (radius, R ∼ 20 mm). Subsequent to LB deposition of amphiphiles 1 and 2, the lenses were mounted as crossed cylinders in the apparatus, which was filled with pure water. The pH of the aqueous medium was adjusted by adding an appropriate amount of KOH. The surface separation D (nm) was measured by use of multiplebeam interferometry. The distance zero was set at a position in contact. The force F was determined from deflection of a double-cantilever spring (spring constant, K ∼ 100 N/m) on which one surface was mounted. The measured force was normalized by the mean radius R of the surface curvature. This quantity is proportional to the free energy of interaction of flat surfaces Gf according to the formula F/R ) 2πGf. When the gradient force (dF/dD) is positive and exceeds the stiffness of a spring (K), dF/dD g K, instability occurs, leading the surface to jump into the contact. In the surface separation process, the spring is elongated up to the tension which equals adhesive forces. The surface will then jump apart to a distance D′. The pull-off force F′, which indicates the intensity of adhesion between two surfaces, was obtained to be F′ ) KD′. Detailed procedures of the measurement can be found in refs 9 and 14. The force data presented here were obtained during the first compression-decompression cycle. In most cases, the second run gave the same or similar curves, though the maximum decrease of about 20% in these forces was seen in some cases. Reproducibility of pull-off forces was good during the repeating compression cycles (five cycles were tested). (14) Israelachvili J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1 1978, 74, 975-1001. (15) The pull-off force can be converted to the interfacial energy γ using the formula F/R ) 3πγ.9 For example, the pull-off force of 65 ( 13 mN/m corresponds to an interfacial energy γ of 6.9 ( 1.4 mJ/m2. (16) Kurihara, K.; Kunitake, T.; Higashi, N.; Niwa, M. Langmuir 1992, 8, 2087-2089.
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Figure 2. Atomic force microscope (AFM) images of monolayers 1 (a) and 2 (b) transferred on mica substrates. The AFM images were obtained using a SEIKO SPI 3700 atomic force microscope in the contact mode with a pyramid-shaped cantilever (silicone nitride, Si3N4) with a force constant of 0.021 N m-1. The force applied to the sample was kept as low as possible and was expected to be less than 1 nN.
Figure 3. Force profiles measured between a complementary pair of adenine-thymine (1-2) surfaces in pure water (pH ∼ 5.6) (0 and 9) and at pH 8.5 (KOH) (O).
and 106 ( 3 mN/m, respectively. At these pH’s, nucleic acid base monolayers are neutral (hydrophobic) or very weakly dissociated (negatively charged thymine and positively charged adenine, see below). Therefore, these attractions can be accounted for in terms of the sum of a long-range hydrophobic attraction9 and an attractive double-layer force, although we do not yet know the exact fraction of each component. At higher pH’s beyond 9.5, the thymine monolayer is not stable and dissolves into the aqueous solution.
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Figure 4. (a, top) Force profiles between a noncomplementary pair of thymine-thymine (1-1) surfaces in pure water (pH ∼ 5.6) (0), at pH 9.1 (4), and at pH 9.3 (O). (b, bottom) Profiles between adenine-adenine surfaces in pure water (pH ∼ 5.6) (0), at pH 9.1 (4), and at pH 9.3 (O). Note that the pKa values of nucleic acid bases written in the text are values in aqueous solutions and can be shifted about 1-2 pH units on the monolayer surfaces due to the concentration effect of functional groups.16
On the other hand, long-range forces between thyminethymine surfaces (a noncomplementary pair) change from attraction to repulsion depending on pH (Figure 4a). In pure water (pH ∼ 5.6), a long-range attraction appears at a separation of 180 nm and the surface jumps into contact at a separation of 46 nm ((3 nm). The absence of electric double-layer repulsion supports that the thymine groups, not the ammonium ones, are exposed to water. The longrange component of attraction is described by F/R ) -1.1 exp(-D/50) mN/m, which is similar to the formula for adenine-thymine surfaces in pure water. The pull-off force of 144 ( 10 mN/m is larger than that for adeninethymine in pure water and is close to the pull-off forces of hydrophobic surfaces. These characteristics reveal that, in pure water (pH ∼ 5.6), where the thymine group is electrically neutral, the monolayers interact with each other similarly to hydrophobic surfaces. The contact angle of pure water to the thymine monolayer surface of 72 ( 2°, though, indicates that this surface is not highly hydrophobic such as a surface exhibiting a contact angle of water higher than 90°. This point will be discussed later. At pH 9.1 (KOH), where the thymine group starts to dissociate, the attraction decreases, as shown in Figure 4a. The force profile fits F/R ) -0.2 exp(-D/50) mN/m, and the jump-in distance is 24 nm. The pull-off force decreases also to 62 ( 3 mN/m, which should reflect the increased charges of the surface. At pH 9.3, the longrange interaction changes to a repulsion. The decay length of this repulsion, 70 ( 5 nm, is in good agreement with
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Figure 5. Plots of pull-off forces of adenine-thymine (1-2) (4), thymine-thymine (1-1) (0), and adenine-adenine surfaces (2-2) (O), as a function of the pH of the medium. The pH value of the medium was adjusted by adding an appropriate amount of KOH.
the Debye length, 68 nm, for a corresponding salt (KOH) concentration of 10-4.7 M. This indicates that the repulsion can be attributed to a double-layer interaction. At short distances the attraction remains and leads the surfaces to jump into contact at 17 nm. A similar short-range attraction has been reported for weakly charged hydrophobic surfaces.9 This indicates that the thymine surfaces are still only weakly charged and hydrophobic. The contact angle of water to the thymine monolayer at pH 9.1 is 71 ( 2°, the same as that at pH ∼ 5.6, which supports this explanation. Profiles of forces between adenine-adenine surfaces are similar to those between thymine layers, although the dependence on pH is opposite (Figure 4b). In pure water (pH ∼ 5.6), where adenine is in the weakly protonated and charged state (basic pKa of adenine is 4.3), the interaction is repulsive at longer distances. Shortrange attraction also remains, and the surface jumps into the contact at 21 nm. At the higher pH of 9.1, where adenine is deprotonated to form the neutral state, no longrange force appears and the jump-in distance is 37 nm. At pH 9.3, attraction appears at 150 nm and increases with decreasing distance. This long-range attraction is described by F/R ) -0.83 exp(-D/50) mN/m. The surface jumps into the contact at a separation of 39 nm. In the electrically neutral form, adenine monolayers seem to interact also as hydrophobic surfaces. The contact angle of pure water to the surface of the adenine monolayer has been measured to be 76.6 ( 3.1°, which is the same as the value for thymine. The unexpected difference is small pull-off forces: 20 ( 6 mN/m at pH 5.6, 26 ( 2 mN/m at pH 9.1, and 34 ( 2 mN/m at pH 9.3. The pull-off forces of various nucleic acid base pairs are plotted as a function of pH in Figure 5. It is clear that the pull-off force for the complementary pair is always stable and large regardless of the pH values of the medium. Hydrogen bonding should be, at least partly, responsible for this stable pull-off force. On the other hand, that between thymine-thymine changes drastically depending on pH. One may note that the values for adenine-adenine are considerably smaller at all pH’s than those for thymine-thymine, although the contact angle of pure
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water to these surfaces is practically identical, around 70°. The adenine groups are known to tend to stack in an aligned state. The small pull-off forces for adenine may be attributed to the surface molecular roughness or the small local hydrophobicity, which is due to the NH groups oriented toward water when the adenine groups are stacking on each other. These results suggest that pull-off forces (adhesive) are affected more by different molecular structures in the contact region than the shortrange (D < 20 nm) noncontact attraction. Recently, the pull-off forces are often discussed as a measure of interaction energies in hydrogen bonding8 and receptorligand complexation.17 Our results show that considerable care has to be taken to account for the origins of the pulloff forces. Whatever the origins are, it is important to note that the complementary pair presents the most stable pull-off forces. The origin of the very long range attraction between hydrophobic surfaces (extending to 200 nm) is not presently understood and is under active investigation.9,10,18,19 This long-range attraction was previously measured between hydrocarbon or fluorocarbon surfaces and is discussed in connection with the strength of the surface hydrophobicity. Generally it has been believed that the attraction requires high hydrophobicity, which causes the contact angle of pure water to surfaces to be larger than 90°. Our systems are different in two aspects: (1) the contact angle is considerably lower than 90°, and (2) the surface is not composed of only hydrocarbon chains. It is necessary to re-examine the physical conditions required for the appearance of very long range attraction besides “hydrophobicity”, although we use the term “hydrophobic long-range attraction” in this paper. The advantage of our system is that we can change the charge and the hydrophobicity of surfaces by simply varying the pH of the medium. Demonstration of nucleic acid base pair complementarity by the direct measurement of surface forces is the most significant result of the present work. Apparently, nature has effectively manipulated pKa’s and hydrophobicities to accomplish its purpose. This work also presents clearly several factors determining interactions between nucleic acid base pairs. Surface force measurements between monolayers, functionalized to mimic given biochemical functions, are continuing in our laboratories. Acknowledgment. This work was supported in part by the PRESTO program of Research Development Corporation of Japan and by a Grant-in-Aid (7241106) from the Ministry of Education, Science and Culture, Japan. LA950867O (17) Moy, V. T.; Florn, E.-T.; Gaub, H. E. Science 1994, 266, 257259. (18) Yaminsky, V. V.; Ninham, B. W. Langmuir 1993, 9, 3618-3624. (19) Tsao, Y.-H.; Evans, D. F.; Wennerstro¨m, H. Science 1993, 262, 547-550.
