Anal. Chem. 2004, 76, 5075-5079
Time-Resolved Total Internal Reflection Fluorescence Study on Hybridization of Complementary Single-Stranded DNAs at a Water/Oil Interface Shoji Ishizaka, Yuichi Ueda, and Noboru Kitamura*
Division of Chemistry, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan
Hybridization of complementary single-stranded DNAs (ssDNA) at a water/CCl4 interface was studied on the basis of picosecond total internal reflection fluorescence spectroscopy. Complementary ssDNAs dissolved in water were shown to produce the relevant double-stranded DNA (dsDNA) at a water/CCl4 interface in the presence of octadecylamine (ODA) in the oil phase, while hybridization between ssDNAs did not proceed in the water phase, as demonstrated by the fluorescence dynamics of ethidium bromide as a probe for the DNA structure. The structures of dsDNA and the roles of ODA in hybridization of ssDNA at the interface were discussed. Duplication of DNA is the most fundamental and important event in life and proceeds in a very sophisticated manner in living organisms. In vitro, on the other hand, hybridization of complementary single-stranded (ss) DNAs does not take place effectively in an aqueous solution, owing to strong electrostatic repulsion between the phosphate groups in DNA, and it proceeds under high ionic-strength conditions. This demonstrates that microenvironments around DNA in vivo play essential roles in hybridization. Recently, Sastry et al. reported hybridization of complementary ssDNA molecules (ssDNA1 and ssDNA2) at a water/ hexane interface in the presence of a cationic surfactant.1 Interestingly, they demonstrated that, although hybridization between ssDNA1 and ssDNA2 took place at the interface, it proceeded neither in an aqueous phase nor in hexane even in the presence of a surfactant. These results indicate that a water/ oil interface provides microenvironments favorable for hybridization of complementary oligonucleotides. However, the work by Sastry et al. does not necessarily provide direct information about hybridization at the interface, since the experiments are conducted for water/hexane emulsion systems. Therefore, factors governing the phenomenon at the interface are still controversial. Clearly, surface-specific experiments are worth exploring to understand hybridization of ssDNAs at a water/oil interface. Previously, we reported experimental approaches to estimate thickness and roughness at water/oil interfaces on the basis of time-resolved total internal (TIR) fluorescence spectroscopy2-4 and * To whom correspondence should be addressed. E-mail: kitamura@ sci.hokudai.ac.jp. Tel: +81 11 706 2697. Fax: +81 11 706 4630. (1) Sastry, M.; Kumar, A.; Pattarkine, M.; Ramakrishnan, V.; Ganesh, K. N. Chem. Commun. 2001, 1434-1435. 10.1021/ac049612i CCC: $27.50 Published on Web 07/30/2004
© 2004 American Chemical Society
demonstrated that the interfacial polarity was dependent on the microscopic structures (i.e., thickness and roughness) of the interface.5 Recently, we also demonstrated directly molecular recognition between a host and a guest mediated by hydrogenbonding interactions at a water/CCl4 interface by means of timeresolved TIR fluorescence spectroscopy and revealed that a water/ oil interface was regarded as a model of molecular recognition in biological systems.6 Therefore, we expected that such techniques would also provide insight into molecular interactions at a water/ oil interface, as in the case for double-helix formation between ssDNAs at the interface. Furthermore, it has been reported that the double-helical structure of DNA in the presence of a cationic surfactant is very sensitive to the water content in an organic solvent7,8 and, thus, to microenvironments around DNA. Therefore, we explored a TIR fluorescence spectroscopic study on hybridization of ssDNAs at a water/oil interface to obtain information about roles of the interface in double-helix formation and double-helical structures of DNA. EXPERIMENTAL SECTION Chemicals. Complementary oligonucleotides with the sequences of GGAAAAAACTTCGTGC (ssDNA1) and GCACGAAGTTTTTTCC (ssDNA2), which were the same as those studied by Sastry et al., were purchased from Invitrogen Corp. (Carlsbad, CA) and used as supplied. For preparation of a doublestranded (ds) DNA sample, an aqueous solution containing equimolar amounts of ssDNA1 and ssDNA2 was heated at 90 °C for 5 min in the presence of NaCl (1 M). After cooling to room temperature, the solution was used as a standard sample for the duplex DNA molecule. Double-helix formation was confirmed by the absorbance of the aqueous dsDNA solution at 260 nm. Octadecylamine (ODA, Wako Pure Chemicals), ethydium bromide (EB, Tokyo Kasei), and NaCl (Kanto Chemicals) were used without further purification. Water was used after deionization and distillation. (2) Ishizaka, S.; Kitamura, N. Bull. Chem. Soc. Jpn. 2001, 74, 1983-1998. (3) Ishizaka, S.; Nakatani, K.; Habuchi, S.; Kitamura, N. Anal. Chem. 1999, 71, 419-426. (4) Ishizaka, S.; Habuchi, S.; Kim, H.-B.; Kitamura, N. Anal. Chem. 1999, 71, 3382-3389. (5) Ishizaka, S.; Kim, H.-B.; Kitamura, N. Anal. Chem. 2001, 73, 2421-2428. (6) Ishizaka, S.; Kinoshita, S.; Nishijima, Y.; Kitamura, N. Anal. Chem. 2003, 75, 6035-6042. (7) Ijiro, K.; Okahata, Y. J. Chem. Soc., Chem. Commun. 1992, 1339-1341. (8) Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679-10683.
Analytical Chemistry, Vol. 76, No. 17, September 1, 2004 5075
Figure 2. Fluorescence decay curves of EB (1 × 10-5 M) (a), EB (1 × 10-5 M) + ssDNA1 (1 × 10-6 M) (b), and EB (1 × 10-5 M) + dsDNA (1 × 10-6 M) (c) in aqueous solutions. The upper panels represent the plots of the weighted residuals (Re) for a single- or double-exponential fitting of each decay profile.
Figure 1. Fluorescence decay measurements of water/CCl4 systems under TIR and normal conditions.
Spectroscopic Measurements. Fluorescence dynamic spectroscopy was conducted with a time-correlated single-photoncounting technique.2-6 The fundamental laser pulses from a modelocked Ti:sapphire laser (Coherent, Mira model 900-F), pumped by a diode laser (Verdi), were amplified by a regenerative amplifier (RegA model 9000) pumped by an Ar+ ion laser (Innova 300). Optical parametric amplification (Coherent, model 9400) of the output gave 510-nm pulses as an excitation light source (repetition rate; 100 kHz, fwhm; 200 fs, autocorrelation trace). For TIR experiments, the excitation laser beam, polarized perpendicular to the plane of incidence (s-polarized) by using a Glan laser prism, was irradiated to a water/oil interface through the oil phase as shown in Figure 1a. In the present experiments, the incident angle of the laser beam (θi) was set (80°) larger than the critical total reflection angle at a water/carbon tetrachloride interface (θc ) 66°). The fluorescence from the sample was collected along surface normal, and its polarization was selected with a polarizer (Polaroid, HNP′B). The polarized fluorescence was detected by a microchannel plate photomultiplier (Hamamatsu, R3809U-50) equipped with a monochromator (Jobin Ybon, H-20) and analyzed by a single-photon-counting module (Edinburgh Instruments, SPC300). The monitoring wavelength of the fluorescence was set at 600 nm throughout the study. Fluorescence decay curves were analyzed by a nonlinear least-squares iterative convolution method based on the Marquardt algorithm.9 5076 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
RESULTS AND DISCUSSION Fluorescence Properties of EB in Bulk Aqueous Solution. It is well known that the fluorescence intensity of EB is highly enhanced upon intercalation to dsDNA, and EB has been used widely for a fluorescence assay of DNA. Aqueous solutions of both ss- and dsDNA (1 × 10-6 M) in the presence of EB (1 × 10-5 M) exhibited the fluorescence peaks around 620 nm (excitation wavelength, 480 nm). The difference in the fluorescence maximum wavelength of EB between the two samples was very small, so that the fluorescence spectral band shapes of EB were concluded to not be sensitive enough to discriminate the structural differences between ss- and dsDNA. To discuss hybridization of DNA, therefore, we conducted fluorescence dynamics measurements. Before describing the results on TIR fluorescence of EB at a water/CCl4 interface, we discuss the fluorescence dynamics of EB in a bulk aqueous solution. Figure 2 shows fluorescence decay curves of EB bound to ssand dsDNA, together with that in an aqueous solution. The upper panels in the figure represent relevant weighted residuals (Re) for each single- or double-exponential fit of the observed data and, Table 1 summarizes the fluorescence lifetimes of EB and the fitting parameters. In the absence of DNA (a), the fluorescence decay profile of EB was fitted by a single-exponential function with the decay time of 1.7 ns. In the presence of ssDNA (b), on the other hand, non-single-exponential decay of the fluorescence was evident and the decay profile was best analyzed by a double-exponential function. Since formation of a double-helical structure is not expected for ssDNA, the short- (τ1) and long-lifetime components (τ2) observed are ascribed to EB in the aqueous phase (τ1 ) 1.8 (9) O’Connor, D. V.; Phillips, D. Time-Correlated Single Photon Counting; Academic Press: New York, 1986.
Table 1. Fluorescence Decay Parameters of EB in Aqueous Solutions
a b c
EB ssDNA-EB dsDNA-EB
A1
τ1/ns
A2
τ2/ns
χ2 a
DWa
1.00 0.84 0.85
1.7 ( 0.1 1.8 ( 0.1 1.8 ( 0.1
0.16 0.15
13.6 ( 0.2 21.7 ( 0.3
1.05 1.16 1.00
2.09 1.77 2.01
a χ2 and DW represent the χ2 and Durbin-Watson parameters for the fitting, respectively.
ns) and EB bound to DNA with electrostatic interactions (τ2 ) 13.6 ns), respectively. For the dsDNA-EB sample (c in Figure 2), a decay time constant longer than that observed for the ssDNA-EB sample (13.6 ns) was confirmed (τ2 ) 21.7 ns) in addition to a short-lifetime component (τ1 ) 1.8 ns; see also Table 1). It has been reported that radiationless deactivation of the excited state of EB is facilitated by proton transfer to a solvent.10 Therefore, observation of the long-lifetime component for dsDNA indicates that EB is located at more hydrophobic environments as compared to that in ssDNA. On the basis of these discussions, we conclude that the decay component with 21.7 ns is ascribed to EB intercalated to dsDNA. Furthermore, the results demonstrate that the fluorescence lifetime of EB is a suitable parameter to study hybridization between ssDNA1 and ssDNA2 at a water/ oil interface. TIR Fluorescence Decay of EB at Water/CCl4 Interface. To study hybridization of complementary ssDNA molecules at a water/CCl4 interface, an aqueous solution containing ssDNA1, ssDNA2 (both 1 × 10-6 Μ), and EB (1 × 10-5 Μ) was poured carefully onto a CCl4 solution of ODA (1 × 10-4 Μ) in the absence of NaCl. Figure 3 shows the fluorescence decay curve of EB at the water/CCl4 interface observed under the TIR conditions (Figure 1a), together with that in the aqueous phase collected under the normal conditions shown in Figure 1b. The upper panels (a, b) in the figure represent Re for each double-exponential fit. The fluorescence decay profile observed from the water phase (b in Figure 3) was also best analyzed by a double-exponential function. The results on the ssDNA1/ssDNA2-EB sample observed under the TIR and normal conditions are summarized in Table 2. The short- (τ1 ) 1.9 ns) and long-lifetime components (τ2 ) 13.4 ns) observed for the ssDNA1/ssDNA2-EB sample under the normal conditions (see Table 2) agreed very well with those observed for the ssDNA-EB sample in an aqueous homogeneous solution (see Table 1).11 The absence of the fluorescence decay component with ∼21.7 ns demonstrates that hybridization between ssDNA1 and ssDNA2 does not take place in the water phase even in the presence of ODA, which coincides with the report by Sastry (10) Olmsted, J.; Kearns, D. R. Biochemistry 1977, 16, 3647-3654. (11) The difference in the relative amplitude of the long-lifetime component between ssDNA-EB in an aqueous solution (A2 ) 0.16 in Table 1) and ssDNA1/ssDNA2-EB observed under the normal conditions (A2 ) 0.29 in Table 2) is due to that in the total ssDNA concentration between the two samples. Namely, the ssDNA-EB sample contains ssDNA1 (1 × 10-6 M) and EB (1 × 10-5 M), while the ssDNA1/ssDNA2-EB sample involves ssDNA1 (1 × 10-6 M), ssDNA2 (1 × 10-6 M), and EB (1 × 10-5 M). Since the total ssDNA concentration in the water phase for the latter sample is 2 × 10-6 M, the relative amplitude of the long-lifetime component of the ssDNA1/ssDNA2-EB sample (A2 ) 0.29) becomes almost twice of that of the ssDNA-EB sample (A2 ) 0.16).
Figure 3. Fluorescence decay curves of EB at the water/CCl4 interface observed under the TIR conditions (a) and that in the aqueous phase collected under the normal conditions (b) (ssDNA1/ ssDNA2-EB, see also main text). The upper panels (a and b) represent the plots of Re for each double-exponential fit. The upper panel (c) represents the plots of Re for the double-exponential fit of the decay data (a) with the long-lifetime component being fixed at 13.6 ns (see also main text). Table 2. Flourescence Decay Parameters of EB Observed for the Water/CCl4 Systems A1
τ1/ns
ssDNA1normal 0.71 1.9 ( 0.1 ssDNA2-EB TIR 0.28 2.2 ( 0.2 ssDNA-EB normal 0.85 1.8 ( 0.1 TIR 0.49 3.2 ( 0.2 dsDNA-EB normal 0.87 1.7 ( 0.1 TIR 0.58 2.3 ( 0.1
A2
τ2/ns
χ2 a
DWa
0.29 0.72 0.15 0.51 0.13 0.42
13.4 ( 0.1 14.6 ( 0.1 13.5 ( 0.2 12.8 ( 0.1 21.0 ( 0.2 15.2 ( 0.2
1.05 1.08 1.10 1.09 1.01 1.04
1.74 1.92 1.78 1.77 1.95 1.73
a χ2 and DW represent the χ2 and Durbin-Watson parameters for the fitting, respectively.
et al.1 The role of ODA is worth discussing here. Sastry et al. reported that ODA acted as a cationic surfactant and facilitated hybridization between ssDNAs by reducing the mutual electrostatic repulsion. Since the pKb value of ODA is 10.8,1 ODA should be protonated if it is solublized in the water phase (pH ∼7) and acts as a surfactant. However, ODA is poorly soluble in water. Therefore, the absence of hybridization between ssDNA1 and ssDNA2 in the water phase is a reasonable consequence. The fluorescence decay profile of the ssDNA1/ssDNA2-EB sample observed from the interface (a in Figure 3) was also best analyzed by a double-exponential function (see Table 2). It is worth noting that the relative amplitude of the long-lifetime component (τ2 ) 14.6 ns, A2 ) 0.72) observed at the interface is much larger than that in the water phase (τ2 ) 13.4 ns, A2 ) 0.29). Since ODA molecules at the water/CCl4 interface are likely to be protonated and will adsorb strongly on the interface owing to the long alkyl chain in ODA, ODA+ (i.e., protonated form of ODA) facilitates Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
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interfacial adsorption of ssDNA1 and ssDNA2 through electrostatic attraction between ODA+ and DNAs at the interface. This was confirmed by the fact that the fluorescence from the interface could not be observed without ODA in the CCl4 phase. Therefore, ODA (i.e., ODA+) acts certainly as a cationic surfactant and facilitates hybridization between ssDNA1 and ssDNA2. In practice, the fluorescence lifetime of ssDNA1/ssDNA2-EB sample observed under the TIR conditions (τ2 ) 14.6 ns) was slightly longer than that observed for EB bound to DNA by electrostatic interactions (τ2 ) 13.6 ns) in the water phase. To check the accuracy of the curve fitting, we analyzed the decay curve in (a) in Figure 3 by a double-exponential function with the long-lifetime component being fixed at 13.6 ns. The relevant weighted residuals (Re) for the fitting are shown in the upper panel (c) in Figure 3. Clearly, Re exhibited nonrandom distributions from those predicted by the best fit, in particular, during 25-50 ns after excitation. Furthermore, the χ2 (χ2 ) 2.21) and Durbin-Watson parameters (DW ) 0.89) for the fitting were worse as compared with the relevant values listed in Table 2: χ2 ) 1.08 and DW ) 1.92 for ssDNA1/ssDNA2-EB. Therefore, the difference in the τ2 value between those at the interface and in the water phase is meaningful and worth discussing here to gain information about hybridization of ssDNAs at the interface. To understand the physical meaning of the lifetime component of 14.6 ns observed for the ssDNA1/ssDNA2-EB sample at the interface, we conducted TIR fluorescence measurements under several conditions. When an aqueous solution of EB (1 × 10-5 M) and ssDNA1 (1 × 10-6 M) made contact with a CCl4 solution of ODA (1 × 10-4 M), the TIR fluorescence decay curve observed from the interface was fitted reasonably by a double-exponential function, as the results were included in Table 2 (ssDNA-EB). The long-lifetime component observed (τ2 ) 12.8 ns) was slightly shorter than that for EB bound to ssDNA by electrostatic interactions (τ2 ) 13.6 ns) in water. It is worth noting that the value of 12.8 ns is clearly shorter than the value observed for ssDNA1/ssDNA2-EB at the interface: 14.6 ns. Therefore, the fluorescence lifetime component of 14.6 ns observed for ssDNA1/ ssDNA2-EB under the TIR conditions is not ascribed to EB bound electrostatically to ssDNA at the interface. We also studied TIR fluorescence dynamics of a dsDNA-EB sample. When a CCl4 solution of ODA (1 × 10-4 M) make contact with an aqueous EB solution (1 × 10-5 M) of preformed dsDNA (1 × 10-6 M) in the presence of NaCl (1 M), the part of the dsDNA molecules intercalated with EB in the water phase would adsorb on the interface. Figure 4 shows the fluorescence decay curves of dsDNA-EB at the water/CCl4 interface observed under the TIR (a) and normal conditions (b). The upper panels of the figure represent Re for each double-exponential fit. Both fluorescence decay curves were fitted reasonably by double-exponential functions, as the results were shown in Table 2 (dsDNA-EB). The short- (τ1 ) 1.7 ns) and long-lifetime components (τ2 ) 21.0 ns) observed from the water phase (b in Figure 4) agreed very well with those observed for the dsDNA-EB sample in an aqueous homogeneous solution (1.8 and 21.7 ns; see Table 1). Nonetheless, it is worth emphasizing that the long-lifetime component observed for dsDNA-EB from the interface (τ2 ) 15.2 ns) is much shorter than that for dsDNA-EB observed under the normal conditions (21.0 ns, Table 2) as well as for an aqueous dsDNA-EB solution 5078 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
Figure 4. Fluorescence decay curves of EB at the water/CCl4 interface observed under the TIR conditions (a) and that in the aqueous phase collected under the normal conditions (b) (dsDNAEB, see also main text). The upper panels represent the plots of the weighted residuals (Re) for each double-exponential fit.
(21.7 ns, Table 1). To clarify the issue, we conducted analogous experiments at several ODA concentrations (1 × 10-6-1 × 10-4 M) in the CCl4 phase (the data are not shown here). The fluorescence decay time constants of EB were independent of the concentration of ODA in the CCl4 phase, while the relative amplitude of τ2 increased with increase in the ODA concentration. This indicates that quenching of the excited state of EB by ODA is not the reason for the lifetime of EB in the dsDNA-EB system at the interface (τ2 ) 15.2 ns). Therefore, it is suggested that the excited-state lifetime of EB intercalated to dsDNA adsorbed on the water/CCl4 interface is intrinsically shorter than that in the bulk aqueous phase, as discussed later again. The experimental results and discussions described above indicate that the long-lifetime component observed for ssDNA1/ ssDNA2-EB at the interface (14.6 ns) is close to that for the dsDNA-EB system (15.2 ns) rather than that for the ssDNAEB system (12.8 ns) at the interface. Therefore, we concluded that the long-lifetime component observed for ssDNA1/ssDNA2EB at the interface could be assigned to the excited-state lifetime of EB intercalated to dsDNA, which was hybridized in situ at the interface. Complementary ssDNA1 and ssDNA2 dissolved in water were shown to produce relevant dsDNA at the water/CCl4 interface in the presence of ODA, while hybridization between ssDNAs did not proceed in the water phase. Characteristics of Hybridization of ssDNAs and Structures of dsDNA at the Water/CCl4 Interface. As discussed in the previous section, radiationless deactivation of the excited state of EB is facilitated by proton transfer to a solvent, so that the excited lifetime of EB is in general longer in a nonpolar or aprotic medium as compared to that in a polar or protic solvent.10 Therefore, the fluorescence lifetime of EB intercalated to dsDNA (21.7 ns) becomes longer than that in an aqueous solution (1.7 ns). Nonetheless, the fluorescence lifetime of EB intercalated to dsDNA at the interface (15.2 ns) is shorter than that of EB
intercalated to dsDNA prepared in a bulk aqueous solution in the presence of NaCl: 21.7 ns. These results suggest that the structures of dsDNA adsorbed on the water/CCl4 interface are influenced to some extent by the interfacial structure itself, the presence of ODA as a cationic surfactant, or both. Tanaka and Okahata reported that the internal conformation of a DNA-lipid complex in a bulk organic solution (CHCl3:EtOH ) 4:1) changed from a B- to C-form with decrease in the water content in the solution, as revealed by the CD spectra of the complex.8 In the B-form of DNA, water molecules interact with the oxygen atoms of the ribose and phosphate groups as well as with the minor/major grooves of DNA. On the other hand, since the C-form has a large winding angle and the minor groove is shorter than that in the B-form, the inclination of the bases to the helical axis makes the interaction of EB with DNA weaker, and consequently, water molecules can access easily to EB in the DNA. Previously, we reported that a water/CCl4 interface was molecularly sharp (
