Detection of Single-Base Mutations by Fluorogenic Ribonuclease

Moreover, mutation sensing was successfully visualized by a UV transillumination. This simple and rapid mutation sensing method should facilitate a ...
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Anal. Chem. 2005, 77, 7047-7053

Detection of Single-Base Mutations by Fluorogenic Ribonuclease Protection Assay Hirofumi Ichinose,† Momoko Kitaoka,† Nobuko Okamura,† Tatsuo Maruyama,‡,§ Noriho Kamiya,‡,§ and Masahiro Goto*,†,‡,§

Japan Science and Technology Agency, Innovation Plaza Fukuoka, 3-8-34, Momochihama, Fukuoka 814-0001, Japan, and Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, Fukuoka 812-8581 Japan

The ribonuclease protection assay is a generally applicable technique for the detection of known mutations. We have developed a simple and rapid method for mutation detection based on the ribonuclease protection assay using fluorescently labeled oligodeoxyribonucleotide probes. The fluorogenic ribonuclease protection (FRAP) assay uses two differently labeled oligodeoxyribonucleotides, a donor probe and an acceptor probe, to obtain a fluorescence resonance energy transfer (FRET) signal. We have utilized the FRAP assay for the detection of a single-base mutation in the YMDD motif of the hepatic B virus DNA polymerase gene. The occurrence of mismatch-selective RNA cleavage was successfully discriminated by measuring the FRET signal between the donor and acceptor probes. Moreover, mutation sensing was successfully visualized by a UV transillumination. This simple and rapid mutation sensing method should facilitate a highthroughput mutation analysis. Single nucleotide polymorphisms (SNPs) are the most abundant genetic variation among individuals.1,2 Since sequence variations caused by SNPs could sometimes affect the quality or quantity of the correspondent gene products, reliable detection of SNPs in individuals is of great importance in the biological and medical sciences. Genetic variations attributed to single-base substitutions are also found in viruses and other microorganisms. Mutations in pathogenic organisms sometimes give a high resistance to curative medicines for the related disease. One wellknown example is a single-base mutation in the chronic hepatitis B virus (HBV) DNA polymerase gene. The point mutation results in a reduced susceptibility of HBV to inhibition by lamivudine. The emergence of lamivudine-resistant HBV was reported in patients with prolonged lamivudine administration.3 A reliable and * Corresponding author. Phone: +81-92-642-3575. Fax: +81-92-642-3575. E-mail: [email protected]. † Japan Science and Technology Agency. ‡ Department of Applied Chemistry, Graduate School of Engineering, Kyushu University. § Center for Future Chemistry, Kyushu University. (1) Cooper, D. N.; Smith, B. A.; Cooke, H. J.; Niemann, S.; Schmidtke, J. Hum. Genet. 1985, 69, 201-205. (2) Halushka, M. K.; Fan, J. B.; Bentley, K.; Hsie, L.; Shen, N.; Weder, A.; Cooper, R.; Lipshutz, R.; Chakravarti, A. Nat. Genet. 1999, 22, 239-247. (3) Chin, R.; Locarnini, S. Rev. Med. Virol. 2003, 13, 255-272. 10.1021/ac050782k CCC: $30.25 Published on Web 10/05/2005

© 2005 American Chemical Society

rapid detection of single-base mutation has, therefore, considerable significance in clinical diagnosis. Nucleases are important and useful analytical enzymes for research into several nucleic acids. A series of application studies have focused on the utilization of various nucleases for SNPs discovery and mutation detection.4-10 Among a number of nuclease-based techniques, ribonuclease-catalyzed cleavage of mismatched RNA/RNA or RNA/DNA duplexes is one of the most attractive approaches. Mutation detection using ribonucleases was originally described by Myers et al. (1985) and subsequently developed by several researchers.8-10 Ribonuclease-based mutation detection has several advantages over the nuclease-based techniques because (i) large amounts of single-stranded RNA samples can be readily derived from PCR products that were amplified with promoter-tailed primers, (ii) ribonuclease exhibits a high sensitivity and specificity against mismatched ribonucleobases, and (iii) ribonuclease is a stable and robust enzyme. Recent studies have made an effort to develop a simple and rapid procedure to discriminate mismatch cleavage events without sizeseparation steps. Fluorescence techniques have been widely used in biological research fields. The fluorescence measurements promise to provide a rapid and simple procedure, with a high sensitivity for molecular analysis. Fluorescence resonance energy transfer (FRET) is based on an interaction between the electronic excited states of two dye molecules. The excitation energy is transferred from one dye molecule to another dye molecule without emission of a photon. Since the energy transfer reaction is significantly dependent on the distance between a donor and an acceptor molecule, FRET is useful for increasing our understanding of biological phenomena that produce a change in molecular proximity. (4) Taylor, G. R.; Deeble, J. Genet. Anal. 1999, 14, 181-186. (5) Taylor, G. R. Electrophoresis 1999, 20, 1125-1130. (6) Till, B. J.; Burtner, C.; Comai, L.; Henikoff, S. Nucleic Acids Res. 2004, 32, 2632-2641. (7) Shagin, D. A.; Rebrikov, D. V.; Kozhemyako, V. B.; Altshuler, I. M.; Shcheglov, A. S.; Zhulidov, P. A.; Bogdanova, E. A.; Staroverov, D. B.; Rasskazov, V. A.; Lukyanov, S. Genome Res. 2002, 12, 1935-1942. (8) Krebs, S.; Medugorac, I.; Seichter, D.; Fo ¨rster, M. Nucleic Acids Res. 2003, 31, e37. (9) Grange, D. K.; Gottesman, G. S.; Lewis, M. B.; Marini, J. C. Nucleic Acids Res. 1990, 18, 4227-4236. (10) Myers, R. M.; Larin, Z.; Maniatis, T. Science 1985, 230, 1242-1246.

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Herein, we report a simple and rapid method for mutation detection using ribonucleases and fluorescently labeled oligodeoxyribonucleotide probes. The key feature of the fluorogenic ribonuclease protection (FRAP) assay is to use two differently labeled probes, which can act as FRET probes. The two-probe system is simple enough to be designed easily. We validated the FRAP assay with single-base substitution within the YMDD motif of the HBV DNA polymerase gene. The single-base mutation was successfully discriminated by measuring the fluorescence intensity after ribonuclease digestion. Moreover, the mutation sensing based on the FRAP assay was successfully achieved by a simple UV transillumination. This approach should facilitate the detection of various single-base mutations without expensive instruments. EXPERIMENTS Oligonucleotide. Synthetic oligonucleotide probes and PCR primers were custom-synthesized by Hokkaido System Science Co. Ltd. Oligonucleotides labeled with 5-carboxytetramethylrhodamine (TAMRA), fluorescein-5-isothiocyanate (FITC), and black hole quencher (BHQ1 or BHQ2) were dissolved in a TE buffer (pH 7.0) and stored at -20 °C under dark. Preparation of DNA Templates for in Vitro Transcription. The liner gene fragments attached with an additional T7 promoter sequence was PCR-amplified from the HBV gene cloned in pUC18 plasmid DNA. The reaction mixture (100 µL) for PCR amplification of the HBV contained 0.1 µg of plasmid DNA, 2.5 U of Pyrobest DNA polymerase (TaKaRa), 1× Pyrobest Buffer II, 100 pmol each of ymdd-T7 (5′-ATGATCACTAATACGACTCACTATAGGGCTTTCCCCCACTGTTTGGC-3′) and ymdd-r80 (5′-GGACTCAAGATGTTGTACAGACT-3′), and dNTPs with a final concentration of 200 µM. The PCR temperature was programmed as follows: 94 °C for 3 min, 60 °C for 20 s, and 72 °C for 20 s for 30 cycles. The PCR products were used for in vitro transcription of the HBV gene (708-786 site) (Figure S1 of the Supporting Information). The double-stranded DNA template for the purine-rich model sequence was obtained from synthetic oligonucleotide, F-oligo and R-oligo, by treating with Pyrobest DNA polymerase (Figure S2 of the Supporting Information). The reaction mixture (100 µL), containing Pyrobest DNA polymerase (2.5 U), 1× Pyrobest Buffer II, 100 pmol each of F-oligo and R-oligo, and dNTPs (200 µM), was incubated for 5-min at 72 °C. The resultant double-stranded DNA fragment was used as the DNA template for in vitro transcription. RNA Synthesis by in Vitro Transcription Reaction. In vitro transcription reactions were performed by a T7 RiboMAX Express Large Scale RNA Production System (Promega). The resultant RNA samples were purified by a Dr. GenTLE Precipitation Carrier (TaKaRa) and dissolved in nuclease-free H2O (100 µL). A typical yield of in vitro RNA synthesis was around 200 µg of RNA samples. Mutation Detection in the HBV Gene by FRAP Assay. The RNA samples were heat-denatured at 95°C for 3 min and chilled on ice prior to use. The heat-denatured RNA sample (50 µg) was incubated with 20 pmol of donor probe (5′-CCATATAACTGAAAGCCAAA-3′; modified with TAMRA at the 5′ end) and 200 pmol of acceptor probe (5′-GGCCCCCAATACCACATCAT-3′; modified with BHQ2 at the 3′ end) in the hybridization buffer (10 mM Tris-HCl containing 50 mM MgCl2, pH 7.4) at 25 °C. The spectral change of fluorescence emissions at 590 nm with excitation at 555 nm was monitored by a luminescence spectrometer, LS 50B (Perkin-Elmer), equipped with a cutoff filter (560 7048

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nm). After hybridization, nuclease digestion was initiated by an exogenous addition of 0.5 µg of ribonuclease A (Nippon Gene). The reaction mixture was then subjected to fluorescence measurement. The hybridization reaction and subsequent nuclease digestion were carried out in a quartz cell at 25 °C. Mutation Sensing on UV Transilluminator. The RNA sample (100 µg) was heat-treated (95°C, 5 min) with 20 pmol of donor probe (5′-CCATATAACTGAAAGCCAAA-3′; modified with FITC at the 5′ end) and 200 pmol of acceptor probe (5′GGCCCCCAATACCACATCAT-3′; modified with BHQ1 at the 3′ end) in 20 µL of hybridization buffer (10 mM Tris-HCl containing 50 mM MgCl2, pH 7.4) and incubated for 30 min at 16 °C. The reaction mixture was then treated with RNase A (0.05 µg) and further incubated for 1 h at 16 °C. After additional incubation, the resultant reaction mixture was photographed with a UV transilluminator (ATTO, AE-6911FXFD) under UV light (312 nm). Kinetic Analysis for Mismatch Cleavage by RNase A and RNase I. The initial rate of mismatch cleavages was determined by measuring the fluorescence quenching elimination. To normalize the differences in quenching rates for each duplex, the recovery of fluorescence emission (Fr) was defined as follows:

Fr ) (F2 - F1)/(F0 - F1) where F0 indicates the fluorescence intensity of the donor probe before hybridization, F1 is the fluorescence intensity of the donor probe after hybridization, and F2 is the fluorescence intensity at the measured point. The heat-denatured RNA sample (50 µg) was incubated with 25 pmol of donor probe modified with TAMRA at the 5′ end and 250 pmol of acceptor probe modified with BHQ2 at the 3′ end in the hybridization buffer (10 mM Tris-HCl containing 50 mM MgCl2, pH 7.4). After hybridization, a nuclease digestion reaction was initiated with the addition of 20 U of RNase I (RNase ONE from Promega) or 2 µg RNase A (Nippon Gene). The change in the fluorescence emission at 590 nm with excitation at 555 nm was recorded on a luminescence spectrometer, LS 50B, equipped with a cutoff filter (560 nm). The hybridization reaction and subsequent nuclease digestion was carried out in a quartz cell at 25 °C. Mutation Discrimination Based on Fluorescence Quenching. A FRAP assay was performed using RNase A with the combination of a donor probe (723-742) and acceptor probe (743-762). The quenching rate (Qr) of fluorescence intensity was defined as follows:

Qr ) (F0 - F1)/F0 × 100 [%] where F0 indicates the fluorescence intensity of the donor probe before hybridization and F1 indicates the fluorescence intensity after RNase A digestion. The change in the fluorescence intensity was measured after 10-min incubation with RNase A. Excitation and emission wavelengths were 555 and 590 nm, respectively. Kinetic Analysis of Mismatch Cleavage for Purine-Rich Sequences. The artificial RNA sample was transcribed from synthetic DNA templates via in vitro transcription reaction (Figure S2, Supporting Information). The heat-denatured RNA sample (50 µg) was incubated with 25 pmol of donor probe (5′-TATCCCTTTTCGTACGTAGG-3′; modified with FITC at the 5′ end) and

Scheme 1. Principle of Fluorogenic Ribonuclease Protection Assay for Mutation Detection

250 pmol of acceptor probe (5′-TGTCTCTTTCCCAGATGT-3′; modified with BHQ1 at the 3′ end) in the hybridization buffer (10 mM Tris-HCl containing 50 mM MgCl2, pH 7.4). After hybridization, the nuclease digestion reaction was initiated with the addition of 20 U of RNase I or 20 U of RNase A. The change in the fluorescence emission at 540 nm with excitation at 490 nm was recorded on a luminescence spectrometer, LS 50B, equipped with a cutoff filter (500 nm). The hybridization reaction and subsequent nuclease digestion were carried out in a quartz cell at 25 °C. One unit of enzymes was defined as the amount of enzyme required to cleave the rC-rC bond in a chimeric oligonucleotide (5′-GTTTGGCTTTCAGTTATATTCCACATCTGGGAAAGAGACA3′; underline indicates the ribonucleotides) hybridized to complementary oligodeoxyribonucleotides (FITC-modified 5′-AATATAACTGAAAGCCAAAC-3′ and BHQ1-modified 5′-TGTCTCTTTCCCAGATGT-3′) at the initial rate of 100 pmol/min at 25 °C in 10 mM Tris-HCl (pH 7.5), 50 mM MgCl2. The rC-rC cleavage was determined by a FRET signal between FITC and BHQ1. Quantitative Detection of HBV Mutant Levels in Mixed Virus Populations. For the determination of the mutant HBV level, the DNA templates for in vitro transcription reaction were amplified from the premixed plasmid samples of the wild-type and mutant HBV gene. The RNA samples were synthesized from the resultant PCR products, a mixture of wild-type and mutant HBV gene. FRAP assay was carried out using RNase I with the combination of donor probe (5′-TCCATATAACTGAAAGCCAA3′; modified with TAMRA at the 5′ end) and acceptor probe (5′TGGCCCCCAATACCACATCA-3′; modified with BHQ2 at the 3′ end). The mutant HBV level was determined by a measuring of the quenching rate. The quenching rate (Qr) was defined as the following:

Qr ) (F3 - F0)/(F0)

where F0 and F3 indicate the fluorescence intensity of the donor probe before hybridization and after the nuclease digestion, respectively. RESULTS AND DISCUSSION Principles of FRAP Assay. Nuclease protection assays are extremely sensitive methods widely used for detection and quantitation of specific RNAs.11,12 Mutation detection techniques based on the catalytic properties of ribonucleases to cleave mismatched RNA/RNA or RNA/DNA duplexes while leaving perfectly matched duplexes intact have also been described.8-10 Although it has been commonly accepted that the ribonuclease protection assay has beneficial approaches for sensitive detection of mutations, only a few techniques, such as gel electrophoresis or mass spectrometry, have been developed for sensing of mismatch cleavage events.8-10 In the present study, we have attempted to develop a simple procedure for the sensing of mismatch cleavage events based on FRET phenomena. The principle of the FRAP assay is illustrated in Scheme 1. The FRAP assay uses two differently labeled oligodeoxyribonucleotides, a donor probe and an acceptor probe, which can act as a FRET probe. The two-probe system is simple enough to be easily designed and useful for various applications.13-16 We employed a combination of a 5′-end-modifed donor probe and 3′(11) Rottman, J. B. Vet. Pathol. 2002, 39, 2-9. (12) Gilman, M. Ribonuclease protection assay. In Current Protocols in Molecular Biology; Ausubel, E. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Stuhl, K., Eds.; John Wiley and Sons: New York, 1993; p 4.7.1-4.7.8. (13) Sei-Iida, Y.; Koshimoto, H.; Kondo, S.; Tsuji, A. Nucleic Acids Res. 2000, 28, e59. (14) Cardullo, R. A.; Agrawal, S.; Flores, C.; Zamecnik, P. C.; Wolf, D. E. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 8790-8794. (15) Mergny, J.-L.; Boutorine, A. S.; Garestier, T.; Belloc, F.; Rougee, M.; Bulychev, N. V.; Koshkin, A. A.; Bourson, J.; Lebedev, A. V.; Valeur, B.; Thung, N. T.; Helene, C. Nucleic Acids Res. 1994, 22, 920-928.

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Figure 2. Mutation sensing on an UV transilluminator.

Figure 1. Fluorescence spectral change after hybridization (A) and ribonuclease digestion (B). (A) The wild-type (dotted line) and G741Tmutant (broken line) RNA samples were incubated with donor probe (5′-CCATATAACTGAAAGCCAAA-3′; modified with TAMRA at the 5′ end) and acceptor probe (5′-GGCCCCCAATACCACATCAT-3′; modified with BHQ2 at the 3′ end) for 30 min at 20° C. The solid line indicates the fluorescence spectrum of the donor probe before hybridization. The emission spectra (λex ) 555 nm) were recorded on the spectrometer equipped with a cutoff filter (560 nm). (B) Fluorescence spectra were obtained after 10-min incubation with RNase A. Further incubation caused no change in fluorescence spectra.

end-modified acceptor probe for the FRAP assay. The acceptor probes were modified with a nonfluorescent black hole quencher (BHQ) to reduce the background fluorescence. Validation of FRAP Assay for Mutation Detection. The single-base substitution of G741 f (T741/A741/C741) within the YMDD motif of the HBV DNA polymerase gene was chosen as the mutation model. The donor probe was designed to perfectly match against the wild-type sequence but to allow a mismatch at the penultimate position against mutants (723-742 site) (Figure S1, Supporting Information). The acceptor probe was positioned in the adjacent region of the mutation site (743-762 site) (Figure S1, Supporting Information). As shown in Figure 1A, the fluorescence emission from the donor probe was effectively diminished (16) Tsuji, A.; Sato, Y.; Hirano, M.; Suga, T.; Koshimoto, H.; Taguchi, T.; Ohsuka, S. Biophys. J. 2001, 81, 501-515.

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upon hybridization to complementary RNA samples. Significant fluorescence quenching phenomena were only observed when the donor and acceptor probes coexisted with RNAs, indicating that the change in fluorescence spectra could be attributed to the FRET phenomena. Furthermore, the fluorescence intensity decreased more strongly when probes were hybridized to the wild-type RNA than the mutant RNA (Figure 1A). The differences in the quenching magnitudes could be attributed to the change in the FRET efficiency, which is significantly dependent on the proximity between donor and acceptor molecules. These results suggest that the penultimate mismatch induced the dissociation of the ultimate nucleobase from the mutant RNA strand,17,18 which may have increased the distance between FRET probes. We further performed the nuclease digestion reactions by an exogenous addition of RNase A. Figure 1B shows the fluorescence spectra of RNA/DNA duplexes after the nuclease digestion. It was clearly demonstrated that the fluorescence quenching in the mutant duplex was completely eliminated by nuclease digestion, whereas the spectral change in the wild-type duplex was negligible (Figure 1B). These results implied that mismatched duplex was selectively and site-specifically cleaved by RNase A. The fluorescence intensity of the mutant duplex after nuclease digestion was almost identical to that of nonhybridized donor probe. Consequently, we succeeded in a mutation detection based on differences in fluorescence intensity. The marked change in fluorescence intensity enabled us to detect a mutation on the UV transilluminator. As shown in Figure 2, the fluorescence signal for mismatch cleavage was successfully visualized by UV transillumination. Rapid and simple mutation detection by the FRAP assay can facilitate the development of an automated analytical procedure and also be compatible with a commonly used microtiter plate reader. (17) Abravaya, K.; Carrino, J. J.; Muldoon, S.; Lee, H. H. Nucleic Acids Res. 1995, 23, 675-682. (18) Peyret, N. P.; Seneviratne, A.; Allawi, H. T.; SantaLucia, A., Jr. Biochemistry 1999, 38, 3468-3477.

Figure 3. Effects of the mismatch type and position on mismatch cleavage by RNase. FRAP assay was performed using RNase A (A) or RNase I (B) with the combination of donor probe (722-741) and acceptor probe (742-761) for the ultimate (n ) 1) mismatch, donor probe (723-742) and acceptor probe (743-762) for the penultimate (n ) 2) mismatch, donor probe (724-743) and acceptor probe (747766) for the internal (n ) 3) mismatch, donor probe (725-744) and acceptor probe (748-767) for the internal (n ) 4) mismatch, and donor probe (726-744) and acceptor probe (748-767) for the internal (n ) 5) mismatch (see Figure S1, Supporting Information). Relative mismatch cleavage activity was expressed as the ratio of enzyme activity of RNase A against penultimate dC‚rA mismatch or RNase I against the penultimate dC‚rC mismatch. The mismatch position from the 5′ end of the donor probe was indicated by “n”.

Scheme 2. Proposed Duplex Structures of Penultimate Mismatch

Effects of Type and Position of a Mismatch in HBV Gene on FRAP Assay. Mutation detection by the FRAP assay was examined using several combinations of donor and acceptor probes. As shown in Figure 3, the mismatch cleavage reaction for the penultimate mismatch (n ) 2) was significantly faster than that for the ultimate (n ) 1) or internal (n ) 3, 4, or 5) mismatch. Similar reaction profiles were observed with both RNase A and ribonuclease I (RNase I). These results mean that the selection of suitable mismatch positions is crucial for the FRAP assay. It has been known that the penultimate mismatches often behave as terminal mismatches instead of internal mismatches, since duplexes fold into their thermodynamically lowest energy structures.18 The thermodynamic properties of the penultimate mismatches would suggest that the mismatched duplex forms the structures found in Scheme 2. The fact that the efficiency of FRET phenomena for the mismatched duplex was lower than that for the perfectly matched duplex when a single-base mismatch was positioned at the penultimate site (Figure 1A) also suggests the dissociation of the ultimate nucleobase from the RNA strand. Therefore, the penultimate mismatch can shift the equilibrium from the matched to

the mismatched duplex structure (on the right in Scheme 2), which makes it a better substrate for ribonucleases than other mismatched duplexes. The fact that ribonucleases bind to duplexes exhibiting helix-unwinding activity also suggests the relationship between thermodynamic stability and mismatch cleavage efficiency.19,20 Although further investigations are required to better understand the relationship between the position of mismatch and nuclease activity, the differences in reaction efficiencies could presumably be a result of changes in the thermodynamic stability of the mismatched duplexes. In addition, the efficiency of the mismatch cleavage reaction was also influenced by the type of mismatches. When the FRAP assay was carried out using RNase A, the cleavage of dC‚rA mismatch was faster than that of dC‚rC and dC‚rT mismatches. It has been demonstrated that RNase A possesses three base binding domains: B1, B2, and B3. Although the B1 domain exhibits significant specificity against pyrimidine bases, the B2 and B3 domains can bind to all bases, with a base preference of B2 domain against adenine bases and of B3 domain against purine bases.21-23 The kinetic data for reaction efficiencies of the mismatch cleavage was dC‚rA . dC‚rC > dC • rT and was compatible with the base preferences of the B2 and B3 domains. Consequently, the differences in reaction efficiencies could be due to the base preference of RNase A. On the other hand, the dC‚rA mismatch was cleaved slightly more slowly than dC‚rC and dC‚ rT mismatches when ribonuclease I (RNase I) was employed for the FRAP assay. Since RNase I has basically no base preferences,24,25 the change in the reaction efficiency could be attributed to the thermodynamic stability of mismatched duplexes.26 The mismatched C‚A pair can form a hydrogen-bonded structure, resulting in a thermodynamically more stable duplex, as compared to the other types of duplexes.26,27 Although the efficiency of the mismatch cleavage reaction could be influenced by several aspects, both enzymes were found to be practically useful for the validation of a single-base mutation within the HBV gene in the FRAP assay. In practical terms, mutations in HBV, G741T, G741C ,and G741A were perfectly discriminated by the FRAP assay with both donor and acceptor probes adjusted to the penultimate mismatch position (Figure 4). Generalization of FRAP Assay by Alteration of Ribonucleases. Sensitivity of the ribonuclease protection assay is particularly dependent on the substrate specificity of ribonucleases. The choice of enzymes is significantly important for sensitive detection of the mutation site. RNase A catalyzes the phosphodiester bond cleavage on the 3′-side of a pyrimidine residue.28 RNase A exhibits poor sensitivity against a purine (19) Felsenfeld, G.; Sandeen, G.; von Hippel, P. H. Proc. Natl. Acad. Sci. 1963, 50, 644-651. (20) Nenci, A.; Gotte, G.; Bertoldi, M.; Libonati M. Protein Sci. 2001, 10, 20172027. (21) Katoh, H.; Yoshinaga, M.; Yanagita, T.; Ohgi, K.; Irie, M.; Beintema, J. J.; Meinsma, D. Biochem. Biophys. Acta 1986, 873, 367-371. (22) Rushizky, G. W.; Knight, C. A.; Sober, H. A. J. Biol. Chem. 1961, 236, 27322737. (23) Irie, M.; Watanabe, H.; Ohgi, K.; Tobe, M.; Matsumura, G.; Arata, Y.; Hirose, T.; Inayama, S. J. Biochem. 1984, 95, 751-759. (24) Meador, J., III; Kennel, D. Gene 1990, 95, 1-7. (25) Meador, J., III; Cannon, B.; Cannistraro, V. J.; Kennell, D. Eur. J. Biochem. 1990, 187, 549-553. (26) Sugimoto, N.; Nakano, M.; Nakano, S. Biochemistry 2000, 39, 11270-11281. (27) Allawi, H. T.; SantaLucia, J., Jr. Biochemistry 1998, 37, 9435-9444. (28) Raines, R. T. Chem. Rev. 1998, 98, 1045-1065.

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Figure 4. FRAP-based mutation detection of YMDD motif in the HBV DNA polymerase gene. Error bars represent the standard deviation calculated from at least three independent experiments.

Figure 6. Quantitative detection of HBV mutant by FRAP assay. The mixture of mutant and wild-type HBV was analyzed by the FRAP assay using RNase I with a combination of donor probe (5′TCCATATAACTGAAAGCCAA-3′; modified with TAMRA at the 5′ end) and acceptor probe (5′-TGGCCCCCAATACCACATCA-3′; modified with BHQ2 at the 3′ end), which was the most effective pair as a FRET probe. Error bars represent the standard deviation calculated from at least three independent experiments.

Figure 5. Catalytic sensitivity of RNase I and RNase A against the purine-rich sequence. The mismatched duplex (see Figure S2, Supporting Information) was digested by RNase I (A) or RNase A (B). The time courses of change in fluorescence (∆F) concomitant with the mismatch cleavage were followed at 540 nm. Inset: Time course analysis of mismatch cleavage of HBV gene by RNase I (A) or RNase A (B) under the same conditions.

residue, as compared to a pyrimidine residue, because of the base preference of the pyrimidine-specific B1 domain.29,30 On the other hand, RNase I from Escherichia coli is one of the few known nucleases to cleave phosphodiester bonds of all 4 ribonucleotides without base preference.24,25 The superior sensitivity of RNase I would be advantageous for mutation detection in purine-rich sequences over other ribonucleases. To confirm the generality of the FRAP assay, we examined the mismatch cleavage in a purine-rich sequence using RNase A and RNase I. The tentative purine-rich sequence for the FRAP assay was originally designed (Figure S2, Supporting Information). Figure 5 shows a comparison of the FRAP assay against a purine-rich sequence performed by RNase I and RNase A. It clearly demonstrates that RNase I exhibited superior performance for mutation detection in the purine-rich sequence over RNase A; however, both enzymes (29) Gutte, B.; Merrifield, R. B. J. Am. Chem. Soc. 1969, 91, 501-502. (30) Kunitz, M.; McDonald, M. R. Biochem. Prep. 1953, 3, 9-19.

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exhibited similar reaction profiles against the HBV gene under similar conditions (Figure 5, inset). Although various ribonucleases can be useful for the FRAP assay, the use of RNase I and its combination with other nucleases could extend the usefulness of the FRAP assay for SNPs detection. Determination of HBV Mutant Levels by FRAP Assay. The FRAP assay is useful for quantification of the mutant gene in the samples with mixed virus populations containing wild-type and mutant DNA. As shown in Figure 6, we have successfully determined the mutant HBV level in the entire virus population on the basis of the fluorescence quenching rate in FRAP assay. There was a good correlation between quenching rate and heterogeneity, even when the mutant HBV made up a small fraction (10%) of the entire virus population. These results strongly suggest that the FRAP assay should be sensitive enough to detect various types of mutation, including heterozygous SNPs. We finally validated that the FRAP assay is practically useful for the real HBV samples isolated from sera of patients with chromic hepatitis B. Although only a single HBV variant with the G741T mutation was found in chronic hepatitis B patients with prolonged lamivudine administration in the Kyushu University Hospital, we have successfully discriminated the mutant HBV in four patients and the wild-type HBV in six patients; however, it was likely that each serum sample consisted of a single population of wild-type or mutant HBV (Figure 7). In closing, we have described a novel method for mutation detection using fluorescently labeled oligonucleotide probes and ribonucleases, the FRAP assay. The FRAP assay is potentially useful for the discrimination of various types of single-base mutants and has several useful features for practical applications, such as (i) easily designable fluorescent probes; (ii) compatibility

methods proposed here should be further applicable to highthroughput mutation detection analysis.

Figure 7. Mutation detection of real HBV samples by FRAP assay. HBV DNA was extracted from patient serum using the QIAamp DNA mini kit (Qiagen). The HBV gene fragment for in vitro transcription was amplified from 0.5-µg DNA extracts. FRAP assay was carried out using RNase I with the combination of donor probe (5′-TCCATATAACTGAAAGCCAA-3′; modified with TAMRA at the 5′ end) and acceptor probe (5′-TGGCCCCCAATACCACATCA-3′; modified with BHQ2 at the 3′ end). The nucleotide sequence was determined by direct sequencing.

with inexpensive instrumentation, such as an UV-transilluminator; (iii) capability of quantitative mutant detection; and (iv) no requirement of size-separation steps. The simple and rapid

ACKNOWLEDGMENT We thank Naotaka Hamasaki and Nahoko Nakano (Graduate School of Medical Sciences, Kyushu University),and Hinako Hatae and Hiroyuki Morita (Ninelab Co. Ltd.) for providing serum samples, excellent technical assistance, and valuable advice. This research was supported by the GOTO Project of the “Science and Technology Incubation Program in Advanced Region” of Innovation Plaza Fukuoka under the Japan Science and Technology (JST) Agency (to H.I., M.K., N.O., and M.G.) and a Grant-in-Aid for the 21st Century COE Program, “Functional Innovation of Molecular Informatics”, from the Ministry of Education, Culture, Science, Sports and Technology of Japan (to M.G.). SUPPORTING INFORMATION AVAILABLE Supporting Information as mentioned in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 6, 2005. Accepted August 12, 2005. AC050782K

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