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DNA Hybridization at the Air-Water Interface Yasuhito Ebara, Katsumi Mizutani, and Yoshio Okahata* Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Midori-ku, Yokohama 226-8501, Japan Received September 8, 1999. In Final Form: December 13, 1999 We observed, by using a quartz crystal microbalance, linear oligonucleotides binding selectively to the planar nucleobase lipid monolayer at the air-water interface and hybridization between two linear oligonucleotides in aqueous solution. Kinetics of DNA hybridization at the interface could be controlled by changing the distance and orientation between nucleobases in the monolayer and compared with those for conventional hybridization in the aqueous solution.
It is well-known that DNA or RNA hybridizes with its complementary nucleobases via hydrogen bonding to form double helical strands in the bulk aqueous phase. Recently, artificial molecules such as PNA,1 polyamides,2 and GNA3 were synthesized and confirmed to form a double or triple helix due to the complementary nucleobase pairing with naturally occurring DNA or RNA molecules. The lipid monolayer at the air-water interface has been widely used as a model surface of biological membranes. Kunitake4 and other workers5 stated that molecular recognition of small molecules based on hydrogen bond formation could be observed at the lipid surface but could not be observed in bulk solution. For example, adenine (A) cannot form A-T base pairs with thymine (T) in bulk solution due to hydration but can form on a lipid membrane at the air-water interface as well as in bulk hydrophobic organic solution. These interactions at the interface have been studied indirectly and qualitatively by changes of the conventional surface pressure (π)-area (A) isotherms and by the analyses of the transferred membrane by X-ray photoelectron spectroscopy (XPS) and FT-IR spectra.4,5 We have reported that the binding amount of guest molecules to the lipid surface could be directly and quantitatively observed from the frequency change of a quartz crystal microbalance (QCM), which attached horizontally to the lipid monolayer from the air phase.6 These experiments suggest that the water surface at the membrane is different from bulk water and close to that in the organic environment.4-6 In this communication, we first report that linear oligonucleotides can bind selectively to the planar nucleobase lipid monolayer at the air-water interface. Binding kinetics could be observed directly by attaching a highly * To whom correspondence should be addressed. Fax: (+81) 45924-5836. E-mail:
[email protected]. (1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500. (2) Dervan, P. B. Science 1986, 232, 464-471. (3) Robert, A.; Goodnow, T., Jr.; Steve, T.; Pruess, D. L.; Warren, W. Tetrahedron Lett. 1997, 38, 3119-3202. (4) Kurihara, K.; Ohto, K.; Tanaka, K.; Aoyama, Y.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 444-450. (b) Sasaki, D. Y.; Kurihara, K.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 9685-9686. (5) Kitano, H.; Ringsdorf, R. Bull. Chem. Soc. Jpn. 1985, 58, 28262828. (b) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 139-140. (c) Cha, X.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 9545-9551. (d) Shimomura, M.; Nakamura, F.; Ijiro, K.; Taketsuna, H.; Tanaka, M.; Nakamura, H.; Hasebe, K. J. Am. Chem. Soc. 1997, 119, 2341-2342. (6) Okahata, Y.; Ebara, Y. J. Chem. Soc., Chem. Commun. 1992, 116-117. (b) Ebara, Y.; Okahata, Y. Langmuir 1993, 9, 574-576. (c) Ebara, Y.; Okahata, Y. J. Am. Chem. Soc. 1994, 116, 11209-11212. (d) Ebara, Y.; Itakura, K.; Okahata, Y. Langmuir 1996, 12, 5165-5170.
sensitive 27 MHz QCM on the lipid monolayer (see Figure 1). Binding behavior could be controlled by changing the distance and orientation between nucleobases in the monolayer. QCMs are known as a very sensitive mass measuring device both in the gas phase7 and in aqueous solution,8 in which resonance frequency decreases with increase of mass on the electrode. Our calibration of a 27 MHz QCM both in gas phase and aqueous solution shows that 1 Hz of frequency decrease corresponds to the mass increase of 0.62 ng cm-2 on the electrode.6c,d,8d-f We have prepared various lipid molecules having a nucleobase as a hydrophilic headgroup as shown in Figure 1: the dialkyl lipids having adenine, thymine, guanine, and cytosine (2C18-A, 2C18-T, 2C18-G, and 2C18-C); the adenine lipids having single, double, and triple alkyl chains (C18-A, 2C18-A, and 3C18-A); the dialkyl lipids having adenine, deoxyadenosine, and adenosine-5′-monophosphate as a headgroup (2C18-A, 2C18-dA, and 2C18-P-dA). Chemical structures and purities were confirmed by NMR and FT-IR spectra and elemental analyses of C, N, and H. These lipids were dissolved in chloroform and spread on buffer subphase (25 °C, 0.01 M Tris-HCl, pH 7.8, 1.0 M NaCl). The π-A isotherms indicated that the molecular area of each lipid at 30 mN m-1 was independent of kinds of nucleobase headgroups but dependent on the number of alkyl chains: 0.26, 0.43, and 0.55 nm2 per molecule for single-, double-, and triple-chain lipids, respectively. Figure 2 shows typical frequency changes as a function of time of 2C18-A and 2C18-T monolayers responding to the addition of various oligonucleotides in the subphase (25 nM). dT30, dA30, dG30, and dC30 show 30-mer oligonucleotides of 2′-deoxythymidine-5′-phosphate, 2′-deoxyadenosine-5′-phosphate, 2′-deoxyguanosine-5′-phosphate, and 2′-deoxycytidine-5′-phosphate, respectively. As expected, to the adenine monolayer of 2C18-A, only the complementary thymidine oligomer (dT30) was bound selectively, but not adenosine (dA30), cytidine (dC30), and guanosine (dG30) oligomers (Figure 2A). For the 2C18-T monolayer, only the dA30 expectedly bound with high selectivity (Figure 2B). Similarly, dG30 and dC30 were (7) Schierbaum, K. D.; Weiss, T.; Thoden von Velzen E. U.; Engbersen, J. F. J.; Reinhoudt, D. N.; Go¨pel, W. Science 1994, 265, 1413-1415. (b) Guilbault, G. G. Anal. Chem. 1983, 55, 1682-1684. (c) K. Matsuura, Y.; Ebara, Y.; Okahata, Langmuir 1997, 13, 814-820. (8) Muramatsu, H.; Dicks, J. M.; Tamiya, E.; Karube, I. Anal. Chem. 1987, 59, 2760-2763. (b) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 110, 8623-8628. (c) Yamaguchi, S.; Shimomura, T.; Tatsuma, T.; Oyama, N. Anal. Chem. 1993, 65, 1925-1928. (d) Niikura, K.; Matsuno, H.; Okahata, Y. J. Am. Chem. Soc. 1998, 120, 8537-8538. (e) Okahata, Y.; Niikura, K.; Sugiura, Y.; Sawada, M.; Morii, T. Biochemistry 1998, 37, 5666-5672. (f) Okahata, Y.; Kawase, M.; Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288-1296.
10.1021/la991189z CCC: $19.00 © 2000 American Chemical Society Published on Web 02/05/2000
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Figure 2. Time courses of frequency decreases (mass increases) responding to the addition of 30-mer oligonucleotides (dT30, dA30, dG30, and dC30 at 25 nM) to (A) 2C18-A and (B) 2C18-T monolayers (25 °C, 10 mM Tris-HCl, pH 7.8, 1.0 M NaCl).
Figure 3. (A) Time dependence of binding behaviors of oligo deoxythymidine dT30 and (B) saturation behaviors of binding amounts (∆m) to various adenine monolayers (25 °C, 10 mM Tris-HCl, pH 7.8, 1.0 M NaCl): (a) 2C18-A, (b) 2C18-dA, (c) 3C18A, (d) C18-A, and (e) 2C18-P-dA. Figure 1. A schematic illustration of DNA hybridization between the nucleobase monolayer and oligonucleotides at the interface, and chemical structures of the monolayer-forming nucleobase lipids.
bound selectively to the 2C18-C and 2C18-G monolayer, respectively (data not shown). These results indicate clearly that the nucleobase monolayer can bind only the complementary oligonucleotides selectively even at the interface. Figure 3 shows binding behaviors of thymidine oligomer dT30 to various adenine monolayers depending on the
number of alkyl chains of C18-A, 2C18-A, and 3C18-A (distance between nucleobases) and the structure of headgroups (2C18-A, 2C18-dA, and 2C18-P-dA). When the concentration of added dT30 in the subphase was increased (1.0-25 nM), the binding amount for the each membrane showed a simple saturation curve (Figure 2B). Association constant (Ka) and maximum binding amount (∆mmax) could be obtained from the slope and intercept of the reciprocal plots of [dT30]/∆m against [dT30]. Results are summarized in Table 1. Comparing the number of alkyl chains of nucleobase lipids (curves a, c, and d in Figure 3), dT30 largely bound
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Table 1. Association Constants (Ka) and Maximum Binding Amounts (∆mmax) of dT30 to the Adenine Lipid Monolayersa
a
lipid
Ka/106 M-1
∆mmax/ng cm-2
C18-A 2C18-A 3C18-A 2C18-dA 2C18-P-dA
