Biomacromolecules 2004, 5, 49-53
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DNA Hybridization in Nanostructural Molecular Assemblies Enables Detection of Gene Mutations without a Fluorescent Probe Tatsuo Maruyama,† Lian-Chun Park,† Toshimitsu Shinohara,† and Masahiro Goto*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, and PRESTO, JST (Japan Science and Technology Corporation), Fukuoka 812-8581, Japan Received February 12, 2003; Revised Manuscript Received September 23, 2003
We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm. Introduction The great progress in human genome research has revealed that single nucleotide polymorphisms (SNPs) often cause a genetic disease. Exploitation of SNPs has potential for the design of individualized prognostic strategies and therapies.1 For the detection of SNPs, a simple, fast, and high-throughput analysis method employing a small amount of DNA sample is urgently required. Numerous studies have been carried out on the development of novel SNPs detection methods based on DNA hybridization2-9 because the binding energy of a mutated test sequence to the single-stranded probe is lower than that of a complementary strand. Most of the methods developed employ DNA-immobilized chips with fluorescent or electrochemical labels tracking the differential quantity of DNA hybridization.5,7 These methods, however, require DNA immobilization, complicated DNA labeling procedures, and specific apparatuses for the detection of the fluorescent or electrochemical signals. A nanostructural molecular assembly attracted our interest because the nanospace formulated by surfactant molecules in organic solvents has a high potential for the production of novel functions. Here, we have proposed a simple method for SNP detection using the nanostructural molecular assembly (termed reverse micelles), which does not require any DNA labeling or specific apparatus. The method is based on three principles. The first is the conduction of DNA hybridization in nanostructural water pools surrounded by an organic phase (formed in reverse micelles). The reverse * Corresponding author. E-mail:
[email protected]. Tel and Fax: +81-(0)92-642-3575. † Kyushu University. ‡ JST (Japan Science and Technology Corporation).
micelles can dissolve a single- or double-stranded DNA molecule at high concentration in the water pools.10-12 The second is that the DNA hybridization can be monitored by measurement of the UV absorbance at 260 nm. The third is that a mismatched base pair causes a significant change in the DNA hybridization rate. The existence of a mutation in the DNA sequence can be detected as a change in the hybridization rate. In the present report, we adopted a 20-mer oligonucleotide in exon 8 of the p53 gene as a model DNA sequence. The p53 gene is known as a tumor suppressor gene.13-18 The p53 protein is a marker of multiple forms of genotoxic, oncogenic, and nongenotoxic stress. Single nucleotide polymorphisms (SNPs) often exist in exon 8 of the p53 gene, which causes dysfunction of the p53 protein.14 We first examined hybridization of the p53 20-mer oligonucleotide and its complementary oligonucleotide in the nanostructural molecular assembly (reverse micelles). Then mutation detection in the reverse micelles was examined by hybridizing the 20-mer to oligonucleotides containing various mutations. We succeeded in the detection of mutations in the targeted DNA sequence by simply measuring the UV absorbance at 260 nm. Materials and Methods Reagents. Sodium di-2-ethylhexyl sulfosuccinate (AOT) was obtained from Kishida Chemical Co. Ltd., Japan. All other chemicals were obtained from commercial suppliers and were of the highest purity available. Oligonucleotides. Genus-specific and target oligonucleotides were designed around mutation of the p53 “hotspot” (Table 1). These oligonucleotides were synthesized and
10.1021/bm034047z CCC: $27.50 © 2004 American Chemical Society Published on Web 11/14/2003
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Maruyama et al.
Table 1. Sequences of Probe and Target DNAs Tested in This Study abbreviation
sequences of sample DNAs
probe target 1 target 2 target 3 target 4 target 5 target 6 target 7 target 8
5′- GCTTTGAGGTGCGTGTTTGT-3′ 3′- CGAAACTCCACGCACAAACA-5′ 3′- CGAAACTCCACGAACAAACA-5′ 3′- CGAAACTCCACGTACAAACA-5′ 3′- CGAAACTCCACGGACAAACA-5′ 3′- TGAAACTCCACGCACAAACA-5′ 3′- CGAAACCCCACGCACAAACA-5′ 3′- CGAAACTCCATTCACAAACA-5′ 3′- CGAAACTCCTTTCACAAACA-5′
purified (more than 90%) using reversed-phase chromatography by Hokkaido System Science Co. Ltd (Sapporo, Japan). Each oligonucleotide was dissolved in a 10 mM TrisHCl buffer (pH 7.0) containing 1 mM EDTA (2Na). The concentrations of these oligonucleotides (198 µM) were determined by the absorbance at 260 nm using their extinction coefficients (http://paris.chem.yale.edu/extinct.frames.html). The extinction coefficient of the probe DNA in reverse micelles was almost the same as that in an aqueous solution (data not shown). DNA Hybridization in the Reverse Micelles. A reverse micellar solution containing a single-stranded oligonucleotide was prepared by direct injection. An aqueous probe solution (9 µL, 198 µM oligonucleotide) was simply added to the isooctane solution (1 mL) containing 50 mM AOT using a micropipet. The solution was vigorously shaken to prepare the homogeneous reverse micellar solution. The targeted oligonucleotide solution (9 µL) was then injected into the reverse micellar solution in the same way and was shaken several times to initiate the DNA hybridization at 15 °C. The final Wo ([H2O]/[surfactant]) was set at 20. The size of reverse micelles was analyzed by a dynamic light scattering method and was found to be 6 nm. The apparent concentration of each oligonucleotide in the reverse micellar solution was 1.75 µM, based on the whole volume. The length of the oligonucleotide is theoretically 6.4 nm, that is, close to the size of reverse micelles. The number of the oligonucleotide molecules in the water pool is one-fifth under the typical experimental conditions. DNA hybridization in the reverse micelles was monitored by measuring the absorbance at 260 nm at 15 °C using a UV-vis spectrophotometer (JASCO Vbest-570). All of the experiments were conducted in triplicate, and the reproducibility was confirmed. DNA Hybridization in an Aqueous Solution as a Control Experiment. A probe solution (containing 9 µL of 198 µM oligonucleotide, 10 mM Tris-HCl, and 1 mM EDTA(2Na), pH 7) and a target 1 solution (9 µL, 198 µM oligonucleotide, 10 mM Tris-HCl, pH 7) were added using a micropipet to 1 mL of a 20× SSC buffer (containing 3.0 M NaCl plus 0.3 M sodium citrate). The 20× SSC buffer is known to be an appropriate buffer for DNA hybridization. The total amount (1.8 nmol) of an oligonucleotide used was the same as that of the reverse micellar solution. The measurement of the absorbance at 260 nm of the aqueous solution was started immediately to monitor the DNA hybridization process. Measurement of the Melting Temperature of Duplex DNAs. The melting temperature of the perfectly matched
perfectly matched mismatch at position 13 mismatch at position 13 mismatch at position 13 terminal mismatch mismatch at position 7 mismatches at positions 11 and 12 mismatches at positions 10, 11, and 12
DNA was measured by the absorbance at 260 nm using the spectrophotometer. A ramp rate of 1 °C/min was used. DNA melting was carried out in the reverse micellar solution described above. Results and Discussion DNA Hybridization in Nanostructural Molecular Assemblies (Reverse Micelles). When a pair of single-stranded DNAs forms a duplex DNA strand, the UV absorbance decreases (hypochromic effect). The DNA hybridization can therefore be readily monitored by measuring the UV absorbance at 260 nm. The existence of a mismatched base pair in the target DNA sequence reduces the hybridization rate and the thermal stability of the double-stranded DNA. The hybridization rate in an aqueous solution is, however, too fast for correct determination.19 The hybridization of DNA oligonucleotides has been usually studied by stopped-flow absorption measurements. In fact, Christensen et al. reported that synthetic oligonucleotides formed the duplex within 1 s.19 And the change in the thermal stability caused by one mismatched base pair should be quite small, corresponding to as little as 0.5 °C in the melting temperatures.20,21 These conditions make it difficult to observe a difference between the perfectly matched and mismatched oligonucleotides. To observe the DNA hybridization with a considerable duplex formation ratio, a relatively high DNA concentration (sometimes several hundred micromolar) is required to shift the equilibrium of the DNA hybridization, while the amount of DNA sample available is severely limited. Furthermore, the UV absorption coefficient of a DNA molecule is too high to measure the absorbance of such concentrated DNA aqueous solutions using a regular spectrophotometer. To overcome these problems, we have utilized the nanospace that is formed by the nanostructural molecular assembly called reverse micelles (Figure 1). The reverse micelles provide water pools surrounded by an organic solvent, in which a small amount of DNA molecules can be dissolved at a high concentration.10 The volume percentage of the water pools is usually 1% or 2% based on the total volume. The highly concentrated single-stranded DNAs will be likely to hybridize with the complementary strands. The reverse micelles are expected to provide a suitable field for DNA hybridization. We first investigated DNA hybridization in AOT/isooctane reverse micelles. The DNA hybridization in the reverse micelles could be monitored by measuring the UV absorbance at 260 nm in the reverse micellar solution containing
DNA Hybridization in Nanostructural Assemblies
Biomacromolecules, Vol. 5, No. 1, 2004 51
Figure 1. Illustration of DNA hybridization in nanostructural molecular assemblies (reverse micelles).
Figure 2. DNA hybridization in (a) an aqueous solution and (b) an AOT/isooctane reverse micellar solution.
DNA. Figure 2 shows the spectral changes of the aqueous solution and the reverse micellar solution containing a singlestranded DNA (probe, 20-mer) and the complementary strand (target 1) at 15 °C. The UV absorbance (at around 260 nm) of the reverse micellar solution markedly decreased with increasing hybridization time (Figure 2c), although there was
no change in that of the aqueous solution even within several seconds (Figure 2a,b). The DNA hybridization in the aqueous solutions was too fast (finished within a few seconds) to monitor the absorbance changes along with the DNA hybridization. The high absorbance (around 0.6) of the oligonucleotides in an aqueous solution means a low ratio of the duplex formation, which might also prevent monitoring the DNA hybridization. Although higher DNA concentrations shift the equilibrium of the DNA hybridization to improve the degree of the duplex formation, the absorbance of the oligonucleotides in the aqueous solution will be too high to be measured using a conventional spectrophotometer. In a control experiment, we confirmed no change in the UV absorbance of the reverse micellar solution containing the probe in the absence of the complementary strand (nonhybridizing condition, data not shown). These results demonstrate that DNA hybridization occurred in the reverse micelles and was easily monitored. It should be noted that the hybridization in the reverse micelles reached equilibrium at more than 4 h (data not shown). The DNA hybridization in the reverse micelles was initiated by the fusion of the water pools. The frequency of the fusion of the water pools generally increases as temperature increases. The DNA hybridization is, however, highly temperature-sensitive, and at high temperature, the oligonucleotides do not hybridize each other. The Wo value and surfactant concentration are also significant factors affecting the fusion of the water pools. In the preliminary investigation, these factors were optimized for the DNA hybridization in the reverse micelles. Porschke reported that the short DNA oligonucleotide less than 50 nm behaves as a rigid rod. In the present study, we employed 20-mer oligonucleotides (approximately 6.8 nm length) in the nanoscale water pools.22 The diameter of the reverse micelles (Wo ) 20) was approximately 6 nm.
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Figure 3. Tm measurement of the perfectly matched probe in reverse micelles.
Therefore, the 20-mer oligonucleotides might be slightly folded in the water pools because of the restricted nanospace or produce relatively large water pools to dissolve them in a rigid-rod form. The size effect of the water pools on the DNA hybridization is currently studied. After the probe hybridized to the target in the reverse micelles for 4 h, the melting temperature (Tm) of the duplex DNA in the reverse micelles was measured (Figure 3). The absorbance at 260 nm increased up to approximately 0.7, which was almost the same as the initial absorbance of the DNA in the reverse micellar solution. The Tm of the perfectly matched probe was determined to be 33 °C. It should be noted that there was no change in the absorbance of a reverse micellar solution containing a nonhybridizing oligonucleotide, for example, containing only the probe (data not shown). These results confirmed that the single-stranded DNAs hybridized to each other to form duplex DNA strands in the reverse micelles and that the DNA hybridization could be simply monitored by measuring the UV absorbance of the reverse micellar solution. The DNA molecules were concentrated in the nanosized water pools to a high concentration (198 µM based on the aqueous volume), but the DNA concentration based on the total reverse micellar volume was about 2 µM, which reduced the UV absorbance to a detectable range. In contrast, the Tm was 47 °C in a Tris-HCl buffer (10 mM, pH 7.0, 1 mM EDTA). This means that the reverse micelles contributed to destabilizing the duplex DNA. Single-Base Mismatches Influence the Hybridization Rate. Okahata et al. reported that the presence of a mismatch in the target oligonucleotide reduced the hybridization rate and hybridization ratio, probably related to the stability of the duplex with a mismatch.23 Our present study demonstrated that the DNA hybridization in the reverse micelles was slow enough to be monitored using a conventional spectrophotometer and that the reverse micellar solution decreased the stability of the DNA duplex. These findings indicate that the reverse micellar solution also destabilizes the mismatched duplex and induces the detectable change in the hybridization rate between the perfectly matched and mismatched oligonucleotides. The hybridization of a probe oligonucleotide mismatched at a single base in the reverse micelles was compared to that of the perfectly matched probe. Figure 4 shows the considerable differences in hybridization behavior between the mismatched and perfectly matched oligonucleotides. As expected, a single-base mis-
Maruyama et al.
Figure 4. Hybridization of a probe with targets in reverse micelles. The data were from single experiments. Values in parentheses represent the relative hybridization rate. The experimental errors were within 7%.
match markedly lowered the initial hybridization rate. Interestingly, the hybridization rate varied with each target. In Figure 4, values in parentheses represent the relative initial hybridization rate of the perfectly matched and single-, double-, or triple-base mismatched probes. The relative decrease in the UV absorbance within 60 min was taken as the initial hybridization rate. The relative value makes the obtained results qualitative. A G-G or G-A mismatch between the probe and the target (probe vs targets 2 or 4) reduced the hybridization rate, although the distinction of these mismatches from the perfect match has generally been difficult by the conventional mutation detection methods.24 The change induced by the G-G mismatch was much smaller than that of the G-T mismatch. A single-base mismatch located in the end of the sequence (probe vs target 5) induced a small but detectable change in the hybridization rate. In contrast, a single-base mismatch located in the middle of the sequence (probe vs targets 3 or 6) caused a drastic change in the hybridization rate. An increase in the number of mismatched base-pairs (targets 7 and 8) caused a further decrease in the hybridization rate. We succeeded in the detection of all of the types of singlebase mismatches and, moreover, observed differences in the hybridization rate among various mismatch patterns. The Tm’s of the mismatched duplexes (probe vs targets 2, 5, and 6) in the Tris-HCl buffer were 44, 47, and 42 °C, respectively. The thermal stability of the perfectly matched and mismatched DNA duplexes in the aqueous buffer was very similar. The hybridization rates of the duplexes in the reverse micelles reflected their thermal stability in the aqueous buffer, and the differences in the hybridization rate between the perfect match and the mismatches were enhanced. These enhanced differences are thought to be related to the stability of the hybridized or hybridizing DNA molecules in the nanostructural reverse micelles. The nanosized space containing the DNA molecules probably amplified the instability of the mismatched DNA molecules. We have demonstrated for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (reverse micelles) and that the DNA hybridization in the reverse micelles can be utilized as a simple and useful technique for the detection of mutations in a DNA sequence. This technique does not require any fluorescent probes, DNA immobilization, or special apparatus. We are currently
DNA Hybridization in Nanostructural Assemblies
studying mismatch detection in longer oligonucleotides, such as 50-mer and 100-mer, in terms of practical nucleic acid analysis. Acknowledgment. This work was supported by the research project of PRESTO (JST). References and Notes (1) Pettipher, R.; Cardon, L. R. Pharmacogenomics 2002, 3, 257. (2) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (3) Ross, P. L.; Lee, K.; Belgrader, P. Anal. Chem. 1997, 69, 4197. (4) Gilles, P. N.; Wu, D. J.; Foster, C. B.; Dillon, P. J.; Chanock, S. J. Nat. Biotechnol. 1999, 17, 365. (5) Shalon, D.; Smith, S. J.; Brown, P. O. Genome Res. 1996, 6, 639. (6) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365-370. (7) Schena, M. DNA Microarrays; Oxford University Press: New York, 1999. (8) Guo, Z.; Liu, Q. H.; Smith, L. M. Nat. Biotechnol. 1997, 15, 331. (9) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096.
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