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Immobilization of DNA through Intercalation at Self-Assembled Monolayers on Gold Nobuyuki Higashi,* Minoru Takahashi, and Masazo Niwa* Department of Molecular Science & Technology, Doshisha University, Kyokanabe, Kyoto 610-0321, Japan Received March 26, 1998. In Final Form: June 19, 1998 A novel acridine derivative (1) connected with a disulfide bond through a long methylene spacer has been synthesized, and its interactions with DNA in solutions and at a self-assembled monolayer surface on gold have been studied. Spectroscopic and thermal data indicated that 1 intercalated successfully to DNA in buffer solutions. The mixed monolayers of 1 and 2, which has a similar molecular structure to that of 1 except for the lack of the acridine moiety, on gold substrates were then prepared. When DNA binding properties onto these monolayers were examined by means of a quartz crystal microbalance, there was a monolayer composition (mole fraction of 1 in the mixed monolayer, 0.04) at which the most effective binding of DNA took place. The interaction of this DNA-immobilized monolayer with polylysines (D, L) was examined by comparing circular dichroism spectra before and after binding of polylysines, and such a two-dimensionally immobilized DNA was found to have an ability to bind poly-L-lysine predominately.
Introduction In a previous study, we demonstrated that a cationic self-assembled monolayer film can be used to immobilize calf thymus DNA on the basis of the interaction of the quaternary ammonium group at the monolayer surface with the phosphate group of DNA through electrostatic interaction and that the immobilized DNA retained a double-stranded structure.1 The strategy employed in this study was to prepare, on a gold substrate, a well-ordered monolayer assembly that contains the acridine derivative 1, which is a typical DNA
intercalator,2 and to immobilize DNA onto the monolayer surface by intercalation of the acridine moiety. In particular, it should be important to elucidate the composition effect of the mixed monolayers composed of 1 and 2 with and without the acridine moiety, respectively, on DNA binding because of the existence of a suitable drug-toDNA nucleotide ratio. The immobilized DNA on monolayers would also be useful for studying molecular recognition because many experimental procedures can be readily applied to examine interactions with guest molecules. In the present study, we chose polylysine as a guest molecule, and its interaction with the immobilized DNA was examined. In DNA replication processes, Lproteins have been demonstrated to bind enantiomerically to nucleic acids and inhibit the replication.3,4 There has (1) Higashi, N.; Inoue, T.; Niwa, M. Chem. Commun. 1997, 1507. (2) Lerman, L. S. J. Mol. Biol. 1961, 3, 18. (3) Joyce, G. F.; Visser, G. M.; van Boom, J. H.; Orgel, L. E.; van Westrenen, J. Nature 1984, 310, 602. (4) Joyce, G. F.; Schwartz, A. W.; Miller, S. L.; Orgel, L. E. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4398.
been, however, no satisfactory experimental evidence to prove such an enantiomeric binding. Results and Discussion DNA Binding Properties of 1 in Solutions. To find out if the acridine derivative carrying a long alkyl chain (1) also has the ability to intercalate with DNA, a binding experiment was performed in the solution phase. The binding was carried out by first dissolving 1 in DMSO and then mixing with DNA in an aqueous buffer (50 mM HEPES; buffer/DMSO ) 95/5 v/v) at pH 7.3, and it was monitored by means of UV-vis absorption spectroscopy. Upon addition of DNA, the spectra exhibit hypochromism and a small (4 nm) red shift of the absorption maximum. These observations are explained by a stacking interaction of the intercalated acridine unit with nucleic acid base pairs in DNA and electrostatic interaction with phosphate anions of DNA, respectively.5 At pH 7.3 the acridine ring should be free, since the pKa value of acridine is presumed to be around 6. Thus, the electrostatic interaction with phosphate anions of DNA should be a minor contribution, which is the reason for the small red shift. The fluorescence studies also support intercalation of 1 to DNA; with excitation at 350 nm, addition of DNA led to a drastic fluorescence decrease due to quenching based on formation of an acridine-nucleic acid base complex.5 Intercalation is also known to affect the thermal denaturation of DNA. Figure 1 displays the plots of absorbance at 260 nm (A260) versus temperature. The thermal denaturation curve for calf thymus DNA shows a drastic increase in A260 at around 73 °C, which corresponds to the melting or helix-to-coil transition temperature (Tm). When 1 is incorporated into DNA, the Tm is found to shift to higher temperature, giving ∆Tm ()Tm of the complex - Tm of the free DNA) ) 6 °C, which is well consistent with those (∆Tm ) 4-9 °C) for acridine derivatives reported previously,6 implying that 1 can definitely intercalate into calf thymus DNA, despite the presence of a long alkyl chain. (5) Porumb, H. Prog. Biophys. Mol. Biol. 1978, 34, 175. (6) Wilson, W. D.; Ratmeyer, L.; Zhao, M.; Strekowski, L.; Boykin, D. Biochemistry 1993, 32, 4098.
10.1021/la9803427 CCC: $18.00 © 1999 American Chemical Society Published on Web 12/11/1998
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Higashi et al.
Figure 1. Thermal denaturation of DNA in the absence (a) and in the presence (b) of 1 in 50 mM HEPES buffer/DMSO (95/5 v/v) at pH 7.3: [1] ) 6.1 × 10-6 M; [DNA(phosphate unit)] ) 6.8 × 10-5 M.
Monolayer Formation and Immobilization of DNA. Disulfide compounds similar to those used in this study have been described to form well-ordered self-assembled monolayers on gold substrates, and the assembling processes have been successfully monitored in situ by means of a quartz crystal microbalance (QCM) technique.7 Thus the adsorption processes of the mixtures of 1 and 2 with various compositions, denoted as molar fraction of 1 in the feed solution (x1), onto gold-coated QCMs in THF were first of all examined in the same way. The mass increase due to adsorption can be estimated from the QCM frequency shift by using the Sauerbrey equation.8 After the frequency of the QCM became unchanged with time in pure THF, the THF solution containing 1 and 2 was added and the frequency shift was recorded. The total concentrations of 1 and 2 were held constant at 1.5 × 10-4 M after the addition. The frequency decreased gradually upon the addition, reflecting adsorption of disulfide compounds (1 and 2) on the gold surface of the QCM. The frequency was found to become constant within 4 h for all mixtures. To obtain information about the resulting monolayer composition (X1), XPS spectra were measured for three typical monolayers of x1 ) 0.1, 0.2, and 0.5. From the elemental ratios of nitrogen and bromine referred to total number of carbon atoms, which were estimated on the basis of N 1s, Br 3d, and C1s spectra, respectively, the monolayer composition (X1) was evaluated and plotted against x1 in Figure 2. The X1 values display positive deviation from the linear correlation between x1 and X1, meaning nonideal adsorption behavior with adsorption of 1 being favored under mixed composition conditions. Subsequently, the properties of binding of DNA onto these monolayers having various compositions were examined on the basis of frequency changes of the QCM. After the frequency became unchanged with time in 8 mL of 5 mM HEPES buffer (pH 7.3), in which the QCM sensor covered with a binary monolayer of 1 and 2 (X1 ) 0.04) was immersed, 0.5 mL of buffer solution of DNA was added and the frequency shift was recorded as shown in Figure 3. The concentration of DNA (phosphate unit) was 7 × 10-4 M after the addition. Figure 3B displays the result for the single-component monolayer of 2 (X1 ) 0). Upon addition of the DNA solution, the frequency shows no (7) Niwa, M.; Date, M.; Higashi, N. Macromolecules 1996, 29, 3681. (8) Sauerbrey, G. Z. Phys. 1959, 155, 206.
Figure 2. XPS spectrum of the 1 monolayer (x1 ) 1.0) at the takeoff angle 46° (top), and mole fraction of 1 in the 1/2 mixed monolayer (X1) as a function of x1 (bottom).
Figure 3. Resonance frequency changes of QCMs modified with 1/2 mixed monolayers upon addition of DNA at pH 7.3; X1 ) 0.04 (a) and 0 (b). Arrows indicate the addition of DNA ([DNA(phosphate unit)] ) 7.0 × 10-4 M).
change. On the other hand, for the binary monolayer containing 1 (Figure 3A), the frequency decreased steeply, reflecting adsorption of DNA on the binary monolayer surface of the QCM. The frequency became constant within about 15 min after DNA addition. The same experiment was performed for the other binary monolayers. Figure 4 shows a total frequency shift during adsorption of DNA (∆∆F) as a function of the monolayer composition (X1). It is clearly seen from the figure that the monolayers having higher X1 values (X1) 0.2-1.0) do not interact with DNA.
Intercalation of DNA at SAMs on Gold
Figure 4. Relationship between X1 and the resonance frequency shifts (∆∆F). Experimental conditions are the same as those of Figure 3.
Figure 5. Phosphorus (A) and gold (B) signals of XPS for a bare gold plate (c), a 1/2 mixed monolayer (X1 ) 0.04) (b and d), and its DNA hybrid (a and e) at the takeoff angle 90°.
This result suggests that the lateral density of the acridine moiety on the two-dimensional monolayer surface is too high for it to intercalate with DNA. In the lower X1 region (
