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Synthesis of hairpin probe using deoxyguanosine as a quencher: Fluorescence and hybridization studies
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 364 (2007) 86–88 www.elsevier.com/locate/yabio
Notes & Tips
Synthesis of hairpin probe using deoxyguanosine as a quencher: Fluorescence and hybridization studies Arvind Misra ¤, P. Kumar, K.C. Gupta Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India NAR Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India Received 28 July 2006 Available online 13 February 2007
Nucleic acids-based detection and quantiWcation methods play an important role in the medical diagnostics and drug discovery. Determining the presence of a speciWc DNA sequence in a homogeneous mixture requires hybridization with Xuorescently labeled oligonucleotides in solution. Recently, hairpin oligonucleotide probes, each of which consists of a Xuorophore at one of the termini and a quencher at the other end, have proven to be eVective in conventional molecular beacons [1]. The molecular beacon possesses a stem–loop-like structure and generally is dual-labeled, with a Xuorophore (a donor dye) at the 5⬘ end and another dye (an acceptor dye that acts as a quencher) at the 3⬘ end of the stem part that may or may not be Xuorescent. The presence of the speciWc target nucleic acid sequences, complementary to the loop part, is detected by a strong increase in the Xuorescence intensity on hybridization, thereby making the homogeneous assay more sensitive and reliable. Thus, the strategy requires labeling at both ends of a single-stranded oligonucleotide probe with speciWc Xuorescent dyes that suVer in overall yield and are expensive. To avoid the limitations that molecular beacons face, hairpin probes have been designed. This kind of probe uses the Xuorescent label at one end and a nonXuorescent molecule (e.g., nucleobase) at the other end and is able to work as an acceptor as well as to quench the Xuorescence intensity of the Xuorophore in the closed-state conformation. For this purpose, a number of unique (rather than traditional) nonXuorescent quenchers, ranging from DNA nucleotides (e.g., guanosine, deazaguanosine) to gold nanoparticles, have already been introduced successfully [2–7]. Furthermore, instead of using interactions between the two extrinsic probes, interactions of Xuorophores with DNA nucleobases or amino acids have potential for the speciWc
*
Corresponding author. Fax: +91 0542 2368175. E-mail address: [email protected] (A. Misra).
0003-2697/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.02.003
detection of DNA or RNA sequences and antibodies at the single molecular level [8–17]. In the current study, in contrast to molecular beacons, only the 5⬘ end-labeled hairpin probe (viz. FAM-C3catcAAAAAAAAAAAAAAAAAAAAgatggg-3⬘) was synthesized. Two extra Gs have been introduced as an overhang in the dangling arm to use guanosine’s characteristic inherent quenching ability as a quencher for homogeneous detection assays that have potential for detection at the single-molecule level. The quenching eYciency of guanosine is due to its low oxidation potential as well as to hydrophobic interaction between the dyes and DNA nucleotide [6,18–23]. Another reasonable possibility for Xuorescence quenching is due to formation of nonXuorescent ground state within close proximity of stacked arrangement of nucleobase G. All of the oligomers were synthesized at the 0.2 M level on an ABI 3800 high-throughput DNA synthesizer using the standard phosphoamidite approach. For the synthesis of hairpin probe, the last coupling was done with amino modiWer (viz. 3-(triXuoroacetylamino)-propyl-2 -(cyanoethyl)-N,N⬘-diisopropyl phosphoramidite [Glen Research]) to get amino functionality at the 5⬘ end. To cleave oligomers from the solid support and to remove base labile-protecting groups, oligomers were treated with 30% aq. NH4OH for 60 °C, 16 h. The ammonical solution was evaporated in a SpeedVac, redissolved in water (200 l), subjected to desalting column (reverse phase C18 silica gel), and eluted with 30% acetonitrile in water. Oligomers were puriWed Wrst on anion exchange fast protein liquid chromatography (FPLC)1 using buVer A (0.1 M NaCl, pH 12.0) 1 Abbreviations used: FPLC, fast protein liquid chromatography; RP– HPLC, reverse-phase high-performance liquid chromatography; 6-FAM, 6-carboxyXuorescein; DMF, dimethyl formamide; Tm, melting temperature; Rf, relative Xuorescence intensity; FRET, Xuorescence resonance energy transfer.
Notes & Tips / Anal. Biochem. 364 (2007) 86–88
and buVer B (1 M NaCl, pH 12.0) with a gradient of 0 to 100% B for 35 min and then analyzed on reverse-phase high-performance liquid chromatography (RP–HPLC) using a gradient of ammonium acetate (0.1 M, pH 7.1) and acetonitrile (0–50% B for 30 min). For covalent attachment of Xuorescent label, 500 l solution containing 0.05 mM hairpin probe, dissolved in 0.1 M NaHCO3, was reacted with 500 l solution containing 5.0 mg/ml N-succinimidyl ester of 6-carboxyXuorescein (6-FAM, Aldrich), dissolved in 100 l dimethyl formamide (DMF), in aliquots at 20-min intervals with continuous stirring for 24 h. Particulate materials were removed by centrifugation. Unreacted dye was removed by passing supernatant over reverse-phase C18 silica gel column and Wnally was puriWed on RP–HPLC (see supplementary material) using the same gradient of the above buVer. Melting temperature (Tm) values and the change in relative Xuorescence intensity (Rf) with fully matched and mismatched targets of base(s)—A, C, and G near the midportion—were recorded in Tris–HCl buVer containing 5.0 mM MgCl2 and 0.1 M NaCl at pH 8.0 on a PerkinElmer Lambda Bio 20 UV–Visible spectrophotometer attached to a PTP1 Peltier temperature programmer and FluoroMax 3 (Spex) spectroXuorometer (Jobin Yvon), respectively. First, the denaturation and change in relative Xuorescence intensity of the hairpin probe (70.0 nM) was recorded in the absence of targets at diVerent temperatures. The temperature was increased from 5 to 80 °C in increments of 10 °C, with each step lasting for 10 min prior to the measurements of Xuorescence. At lower temperature, there was no Xuorescence conWrming the presence of Xuorophore and quencher in close proximity to each other (closed state). However, on increasing the temperature, the helical structure of the stem disrupted to a random coil conformation, separating the Xuorophore and quencher apart from each other and thereby restoring the Xuorescence (56 °C) of hairpin probe (see supplementary material). Similarly, on repeating the experiment in the presence of excess perfectly matched target strand (175 nM) complementary to the loop domain of the probe (wild type), there was a 10- to 11-fold enhancement in Xuorescence signal at lower temperature. As the temperature was slowly raised further, Xuorescence started to diminish. The transition from the helical conformation to a random coil conWguration in the absence of target strand occurred at 56 °C, whereas in the case of the perfectly matched duplex, a transition to the quenched hairpin stem occurred at 51 °C. This is because the stem part of the hairpin probe is less stable than the full matched duplex. As the temperature was raised further, the hairpin probe in the absence and presence of the target strands again started to melt into a Xuorescent random coil, thereby again showing small enhancement in the relative Xuorescence intensity (see supplementary material). The results clearly demonstrated the quenching eYciency of guanosine residue; however, the percentage of signal generated was found to be small in comparison with molecular beacons and TaqMan probes, thereby conWrming that in the current case the quenching
87
or energy transfer is contact mediated rather than via Xuorescence resonance energy transfer (FRET) mode. For the mismatch studies, targets ON2 to ON7 were synthesized as 5⬘- T9NT10-3⬘ and 5⬘-T9NNT9-3⬘, where N D A, C, and G, and the Tm and change in relative Xuorescence intensity of the respective duplexes were recorded at a constant concentration in the same hybridization buVer (see supplementary Fig. 1) According to the Tm and Tm values (see supplementary Table 1), the degree of destabilization of duplexes was found to be in the order of A > G > C when there were both single and double nucleobase mismatches. The diVerences in Tm values and Rf values with a change of single nucleotide, as in the cases of ON3 (A/G) and ON4 (A/A), were ¡0.24 °C and ¡0.115286 £ 106 for the former and ¡1.46 °C and ¡0.071487 £ 106 for the latter with respect to ON2 (A/C). With two mismatches of diVerent nucleobases, as in the cases of ON6 (AA/GG) and ON7 (AA/AA), the diVerences were ¡0.12 °C and ¡0.085676 £ 106 for the former and ¡2.01 °C and ¡0.090746 £ 106 for the latter with respect to ON5 (AA/CC). So, when the number of similar mismatches changed from one to two C ! CC, G ! GG, and A ! AA, the diVerences in the Tm and Rf values were ¡3.62 °C and ¡0.185915 £ 106, ¡3.5 °C and ¡0.156305 £ 106, and ¡4.056 °C and 0.090746 £ 106, respectively. Thus, it can be inferred that with the increase in the number of mismatches of diVerent nucleotides, there was a relatively greater degree of destabilization in probe–target duplexes and, correspondingly, a decrease in the relative Xuorescence intensity. Destabilization was found to be greatest in the case of the mismatch A/A. Moreover, the decrease in the relative Xuorescence intensity of mismatched duplexes was found to be approximately 3 to 5% in comparison with that of the wild-type duplex. Therefore, it can be concluded that aYnity of the hairpin probe toward the targets was in the order of C > G > A. To establish a correlation between change in relative Xuorescence intensity corresponding to the melting temperature, a graph was plotted. It is clear from the sigmoidal graph (inset in supplementary Fig. 1) that as the melting temperature of the duplexes changed, according to the variable mismatches and hence the degree of destabilization, there was a corresponding decrease in the relative Xuorescence intensity. According to Marras and coworkers [19], in the case of standard molecular beacons, quenching eYciency (either by FRET or contact mediated) of quenchers (Xuorescent or nonXuorescent) with respect to donor molecules (Xuorescent) is variable and is in the range of 70 to 99%. Especially with FAM dye, it is in the range of 80 to 92%. However, quenching due to nucleobases was comparatively less than the standard ones. Guanosine and deazaguanosine were found to be the most eYcient quenchers, followed by adenosine > cytosine > thymidine. In general, the Xuorescence in the green and yellow region wavelengths was quenched more eYciently by nucleotides than were the Xuorophores that Xuoresce in the blue and red wavelengths. However, when oligoguanosine was used as a quencher in place of single guanosine moiety, quenching eYciency was
88
Notes & Tips / Anal. Biochem. 364 (2007) 86–88
higher [6,7]. Furthermore, in the case of molecular beacons after hybridization with full matched DNA strand, enhancements in the Xuorescence intensity have been reported to be in the range of 25- to 50-fold, whereas in the case of hairpin probes they are in the range of 10 to 40%, depending on the number of Gs used as an overhang or at neighboring positions for quenching purposes [3,7]. In the current study, approximately 10-to 11-fold enhancement in the Xuorescence intensity was observed with wild-type probe target duplex using two guanosine units as an overhang and one in the complementary stem part, whereas only a 3% enhancement was observed with the target having a mismatch in comparison with Xuorescence signal generated by hairpin probe in the absence of targets. This relatively small enhancement in Xuorescence intensity after hybridization is contributory to background Xuorescence and might be a limiting factor of this strategy. In conclusion, a labeled hairpin probe has been designed and synthesized using the inherent quenching property of the DNA nucleotide deoxyguanosine to study the Xuorescence and thermal behavior, which was found to be sensitive to variations and identities of mismatches in the complementary targets. In contrast to dual-labeled molecular beacons, the synthesis of this kind of probe is highly simple and economical in the sense that it does not require engineered polymer support and stringent puriWcation. The high speciWcity of the structured probe suggests its potential use in a variety of practical applications such as detection of single nucleotide polymorphisms. Acknowledgments Arvind Misra is indebted to Department of Biotechnology, New Delhi, India, for the award of a postdoctoral fellowship and DST, New Delhi for Wnancial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2007.02.003. References [1] S. Tyagi, F.R. Kramer, Molecular beacons: Probes that Xuoresce upon hybridization, Nat. Biotechnol. 14 (1996) 303–308. [2] N.G. Walter, J.M. Bruke, Real-time monitoring of hairpin ribozyme kinetics through base-speciWc quenching of Xuorescein-labeled substrates, RNA 3 (1997) 392–404. [3] J.P. Knemeyer, N. Marme, M. Sauer, Probes for detection of speciWc DNA sequences at the single-molecule level, Anal. Chem. 72 (2000) 3717–3724. [4] B. Dubertret, M. Calame, A.J. Libchaber, Single-mismatch detection using gold-quenched Xuorescent oligonucleotides, Nat. Biotechnol. 19 (2001) 365–370.
[5] M.K. Johansson, H. Fidder, D. Dick, R.M. Cook, Intramolecular dimers: A new strategy to Xuorescence quenching in dual-labeled oligonucleotide probes, J. Am. Chem. Soc. 124 (2002) 6950–6956. [6] C.A.M. Seidel, A. Schulz, M. Sauer, Nucleobase speciWc quenching of Xuorescent dyes: I. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching eYciencies, J. Phys. Chem. 100 (1996) 5541–5553. [7] A.O. Crockett, C.T. Wittwer, Fluorescein-labeled oligonucleotides for real-time PCR using the inherent quenching of deoxyguanosine nucleotides, Anal. Biochem. 290 (2001) 89–97. [8] M. Sauer, Single-molecule-sensitive Xuorescent sensors based on photoinduced intramolecular charge transfer, Angew. Chem. Intl. Ed. 42 (2003) 1790–1793. [9] C. Eggeling, J.R. Fries, L. Brand, R. Gunther, C.A.M. Seidel, Monitoring conformational dynamics of a single molecule by selective Xuorescence spectroscopy, Proc. Natl. Acad. Sci. USA 95 (1998) 1556–1561. [10] J. Widengren, J. Dapprich, R. Rigler, Charge-transfer and stacking reactions between Rh6G and dGTP in water: A Xuorescence correlation spectroscopy study, Chem. Phys. 216 (1997) 417–426. [11] M. Sauer, K.H. Drexhage, U. Lieberwirth, R. Müller, S. Nord, C. Zander, Dynamics of electron-transfer reactions between xanthene dyes and DNA nucleotides monitored on the single molecule level, Chem. Phys. Lett. 284 (1998) 153–163. [12] T. Heinlein, J.P. Knemeyer, O. Piestert, M. Sauer, Photoinduced electron transfer between Xuorescent dyes and guanosine residues in DNA hairpins, J. Phys. Chem. 107 (2003) 7957–7964. [13] U. Lieberwirth, J. Arden-Jacob, K.H. Drexhage, D.P. Herten, R. Müller, M. Neumann, A. Schultz, S. Siebert, G. Sanger, S. Klingel, M. Sauer, J. Wolfrum, Multiplex dye DNA sequencing in capillary gel electrophoresis (CGE) with diode laser based time-resolved Xuorescence detection, Anal. Chem. 70 (1998) 4771–4779. [14] I. Nazarenko, B. Lowe, M. DarXer, P. Ikonomi, D. Schuster, A. Rashtchian, Multiplex quantitative PCR using self-quenched primers labeled with a single Xuorophore, Nucleic Acids Res. 30 (2002) e37. [15] S. Kurata, T. Kanagawa, K. Yamada, M. Torimura, T. Yokomaku, Y. Kamagata, R. Kurane, Fluorescent quenching-based quantitative detection of speciWc DNA/RNA using a BODIPY FL-labeled probe or primer, Nucleic Acids Res. 29 (2001) e34. [16] V.Y. ShaWrovich, S.H. Courtney, N. Ya, N.E. Geacintov, Proton coupled photoinduced electron transfer, deuterium isotope eVects, and Xuorescence quenching in noncovalent benzo-[a]pyrenetetraol–nucleoside complexes in aqueous solutions, J. Am. Chem. Soc. 117 (1995) 4920–4929. [17] F.D. Lewis, R.L. Letsinger, M.R. Wasielewski, Dynamics of photoinduced charge transfer and hole transport in synthetic DNA hairpins, Acc. Chem. Res. 34 (2001) 159–170. [18] S. Steenken, V. Jovanovic, How easily oxidizable is DNA? One-electron reduction potentials of adenosine and guanosine radicals in aqueous solution, J. Am. Chem. Soc. 119 (1997) 617–618. [19] S.A.E. Marras, F.R. Kramer, S. Tyagi, EYciencies of Xuorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes, Nucleic Acids Res. 30 (2002) e122. [20] M.A. O’Neill, J.K. Barton, EVects of strand and directional asymmetry on base–base coupling and charge transfer in double-helical DNA, Proc. Natl. Acad. Sci. USA 99 (2002) 16543–16550. [21] M.A. O’Neill, J.K. Barton, 2-Aminopurine: A probe of structural dynamics and charge transfer in DNA and DNA:RNA hybrids, J. Am. Chem. Soc. 124 (2002) 13053–13066. [22] S.O. Kelley, J.K. Barton, Electron transfer between bases in double helical DNA, Science 283 (1999) 375–381. [23] T. Kimura, K. Kawai, T. Majima, Fluorescence properties of 2- aminopurine-cytidine-7-deazaguanine (5⬘-ApCdzG-3⬘) trimer in B- and Z-DNA, Chem. Commun. (Camb.) 3 (2004) 268–269.
Notes & Tips
Synthesis of hairpin probe using deoxyguanosine as a quencher: Fluorescence and hybridization studies Arvind Misra ¤, P. Kumar, K.C. Gupta Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India NAR Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India Received 28 July 2006 Available online 13 February 2007
Nucleic acids-based detection and quantiWcation methods play an important role in the medical diagnostics and drug discovery. Determining the presence of a speciWc DNA sequence in a homogeneous mixture requires hybridization with Xuorescently labeled oligonucleotides in solution. Recently, hairpin oligonucleotide probes, each of which consists of a Xuorophore at one of the termini and a quencher at the other end, have proven to be eVective in conventional molecular beacons [1]. The molecular beacon possesses a stem–loop-like structure and generally is dual-labeled, with a Xuorophore (a donor dye) at the 5⬘ end and another dye (an acceptor dye that acts as a quencher) at the 3⬘ end of the stem part that may or may not be Xuorescent. The presence of the speciWc target nucleic acid sequences, complementary to the loop part, is detected by a strong increase in the Xuorescence intensity on hybridization, thereby making the homogeneous assay more sensitive and reliable. Thus, the strategy requires labeling at both ends of a single-stranded oligonucleotide probe with speciWc Xuorescent dyes that suVer in overall yield and are expensive. To avoid the limitations that molecular beacons face, hairpin probes have been designed. This kind of probe uses the Xuorescent label at one end and a nonXuorescent molecule (e.g., nucleobase) at the other end and is able to work as an acceptor as well as to quench the Xuorescence intensity of the Xuorophore in the closed-state conformation. For this purpose, a number of unique (rather than traditional) nonXuorescent quenchers, ranging from DNA nucleotides (e.g., guanosine, deazaguanosine) to gold nanoparticles, have already been introduced successfully [2–7]. Furthermore, instead of using interactions between the two extrinsic probes, interactions of Xuorophores with DNA nucleobases or amino acids have potential for the speciWc
*
Corresponding author. Fax: +91 0542 2368175. E-mail address: [email protected] (A. Misra).
0003-2697/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.02.003
detection of DNA or RNA sequences and antibodies at the single molecular level [8–17]. In the current study, in contrast to molecular beacons, only the 5⬘ end-labeled hairpin probe (viz. FAM-C3catcAAAAAAAAAAAAAAAAAAAAgatggg-3⬘) was synthesized. Two extra Gs have been introduced as an overhang in the dangling arm to use guanosine’s characteristic inherent quenching ability as a quencher for homogeneous detection assays that have potential for detection at the single-molecule level. The quenching eYciency of guanosine is due to its low oxidation potential as well as to hydrophobic interaction between the dyes and DNA nucleotide [6,18–23]. Another reasonable possibility for Xuorescence quenching is due to formation of nonXuorescent ground state within close proximity of stacked arrangement of nucleobase G. All of the oligomers were synthesized at the 0.2 M level on an ABI 3800 high-throughput DNA synthesizer using the standard phosphoamidite approach. For the synthesis of hairpin probe, the last coupling was done with amino modiWer (viz. 3-(triXuoroacetylamino)-propyl-2 -(cyanoethyl)-N,N⬘-diisopropyl phosphoramidite [Glen Research]) to get amino functionality at the 5⬘ end. To cleave oligomers from the solid support and to remove base labile-protecting groups, oligomers were treated with 30% aq. NH4OH for 60 °C, 16 h. The ammonical solution was evaporated in a SpeedVac, redissolved in water (200 l), subjected to desalting column (reverse phase C18 silica gel), and eluted with 30% acetonitrile in water. Oligomers were puriWed Wrst on anion exchange fast protein liquid chromatography (FPLC)1 using buVer A (0.1 M NaCl, pH 12.0) 1 Abbreviations used: FPLC, fast protein liquid chromatography; RP– HPLC, reverse-phase high-performance liquid chromatography; 6-FAM, 6-carboxyXuorescein; DMF, dimethyl formamide; Tm, melting temperature; Rf, relative Xuorescence intensity; FRET, Xuorescence resonance energy transfer.
Notes & Tips / Anal. Biochem. 364 (2007) 86–88
and buVer B (1 M NaCl, pH 12.0) with a gradient of 0 to 100% B for 35 min and then analyzed on reverse-phase high-performance liquid chromatography (RP–HPLC) using a gradient of ammonium acetate (0.1 M, pH 7.1) and acetonitrile (0–50% B for 30 min). For covalent attachment of Xuorescent label, 500 l solution containing 0.05 mM hairpin probe, dissolved in 0.1 M NaHCO3, was reacted with 500 l solution containing 5.0 mg/ml N-succinimidyl ester of 6-carboxyXuorescein (6-FAM, Aldrich), dissolved in 100 l dimethyl formamide (DMF), in aliquots at 20-min intervals with continuous stirring for 24 h. Particulate materials were removed by centrifugation. Unreacted dye was removed by passing supernatant over reverse-phase C18 silica gel column and Wnally was puriWed on RP–HPLC (see supplementary material) using the same gradient of the above buVer. Melting temperature (Tm) values and the change in relative Xuorescence intensity (Rf) with fully matched and mismatched targets of base(s)—A, C, and G near the midportion—were recorded in Tris–HCl buVer containing 5.0 mM MgCl2 and 0.1 M NaCl at pH 8.0 on a PerkinElmer Lambda Bio 20 UV–Visible spectrophotometer attached to a PTP1 Peltier temperature programmer and FluoroMax 3 (Spex) spectroXuorometer (Jobin Yvon), respectively. First, the denaturation and change in relative Xuorescence intensity of the hairpin probe (70.0 nM) was recorded in the absence of targets at diVerent temperatures. The temperature was increased from 5 to 80 °C in increments of 10 °C, with each step lasting for 10 min prior to the measurements of Xuorescence. At lower temperature, there was no Xuorescence conWrming the presence of Xuorophore and quencher in close proximity to each other (closed state). However, on increasing the temperature, the helical structure of the stem disrupted to a random coil conformation, separating the Xuorophore and quencher apart from each other and thereby restoring the Xuorescence (56 °C) of hairpin probe (see supplementary material). Similarly, on repeating the experiment in the presence of excess perfectly matched target strand (175 nM) complementary to the loop domain of the probe (wild type), there was a 10- to 11-fold enhancement in Xuorescence signal at lower temperature. As the temperature was slowly raised further, Xuorescence started to diminish. The transition from the helical conformation to a random coil conWguration in the absence of target strand occurred at 56 °C, whereas in the case of the perfectly matched duplex, a transition to the quenched hairpin stem occurred at 51 °C. This is because the stem part of the hairpin probe is less stable than the full matched duplex. As the temperature was raised further, the hairpin probe in the absence and presence of the target strands again started to melt into a Xuorescent random coil, thereby again showing small enhancement in the relative Xuorescence intensity (see supplementary material). The results clearly demonstrated the quenching eYciency of guanosine residue; however, the percentage of signal generated was found to be small in comparison with molecular beacons and TaqMan probes, thereby conWrming that in the current case the quenching
87
or energy transfer is contact mediated rather than via Xuorescence resonance energy transfer (FRET) mode. For the mismatch studies, targets ON2 to ON7 were synthesized as 5⬘- T9NT10-3⬘ and 5⬘-T9NNT9-3⬘, where N D A, C, and G, and the Tm and change in relative Xuorescence intensity of the respective duplexes were recorded at a constant concentration in the same hybridization buVer (see supplementary Fig. 1) According to the Tm and Tm values (see supplementary Table 1), the degree of destabilization of duplexes was found to be in the order of A > G > C when there were both single and double nucleobase mismatches. The diVerences in Tm values and Rf values with a change of single nucleotide, as in the cases of ON3 (A/G) and ON4 (A/A), were ¡0.24 °C and ¡0.115286 £ 106 for the former and ¡1.46 °C and ¡0.071487 £ 106 for the latter with respect to ON2 (A/C). With two mismatches of diVerent nucleobases, as in the cases of ON6 (AA/GG) and ON7 (AA/AA), the diVerences were ¡0.12 °C and ¡0.085676 £ 106 for the former and ¡2.01 °C and ¡0.090746 £ 106 for the latter with respect to ON5 (AA/CC). So, when the number of similar mismatches changed from one to two C ! CC, G ! GG, and A ! AA, the diVerences in the Tm and Rf values were ¡3.62 °C and ¡0.185915 £ 106, ¡3.5 °C and ¡0.156305 £ 106, and ¡4.056 °C and 0.090746 £ 106, respectively. Thus, it can be inferred that with the increase in the number of mismatches of diVerent nucleotides, there was a relatively greater degree of destabilization in probe–target duplexes and, correspondingly, a decrease in the relative Xuorescence intensity. Destabilization was found to be greatest in the case of the mismatch A/A. Moreover, the decrease in the relative Xuorescence intensity of mismatched duplexes was found to be approximately 3 to 5% in comparison with that of the wild-type duplex. Therefore, it can be concluded that aYnity of the hairpin probe toward the targets was in the order of C > G > A. To establish a correlation between change in relative Xuorescence intensity corresponding to the melting temperature, a graph was plotted. It is clear from the sigmoidal graph (inset in supplementary Fig. 1) that as the melting temperature of the duplexes changed, according to the variable mismatches and hence the degree of destabilization, there was a corresponding decrease in the relative Xuorescence intensity. According to Marras and coworkers [19], in the case of standard molecular beacons, quenching eYciency (either by FRET or contact mediated) of quenchers (Xuorescent or nonXuorescent) with respect to donor molecules (Xuorescent) is variable and is in the range of 70 to 99%. Especially with FAM dye, it is in the range of 80 to 92%. However, quenching due to nucleobases was comparatively less than the standard ones. Guanosine and deazaguanosine were found to be the most eYcient quenchers, followed by adenosine > cytosine > thymidine. In general, the Xuorescence in the green and yellow region wavelengths was quenched more eYciently by nucleotides than were the Xuorophores that Xuoresce in the blue and red wavelengths. However, when oligoguanosine was used as a quencher in place of single guanosine moiety, quenching eYciency was
88
Notes & Tips / Anal. Biochem. 364 (2007) 86–88
higher [6,7]. Furthermore, in the case of molecular beacons after hybridization with full matched DNA strand, enhancements in the Xuorescence intensity have been reported to be in the range of 25- to 50-fold, whereas in the case of hairpin probes they are in the range of 10 to 40%, depending on the number of Gs used as an overhang or at neighboring positions for quenching purposes [3,7]. In the current study, approximately 10-to 11-fold enhancement in the Xuorescence intensity was observed with wild-type probe target duplex using two guanosine units as an overhang and one in the complementary stem part, whereas only a 3% enhancement was observed with the target having a mismatch in comparison with Xuorescence signal generated by hairpin probe in the absence of targets. This relatively small enhancement in Xuorescence intensity after hybridization is contributory to background Xuorescence and might be a limiting factor of this strategy. In conclusion, a labeled hairpin probe has been designed and synthesized using the inherent quenching property of the DNA nucleotide deoxyguanosine to study the Xuorescence and thermal behavior, which was found to be sensitive to variations and identities of mismatches in the complementary targets. In contrast to dual-labeled molecular beacons, the synthesis of this kind of probe is highly simple and economical in the sense that it does not require engineered polymer support and stringent puriWcation. The high speciWcity of the structured probe suggests its potential use in a variety of practical applications such as detection of single nucleotide polymorphisms. Acknowledgments Arvind Misra is indebted to Department of Biotechnology, New Delhi, India, for the award of a postdoctoral fellowship and DST, New Delhi for Wnancial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2007.02.003. References [1] S. Tyagi, F.R. Kramer, Molecular beacons: Probes that Xuoresce upon hybridization, Nat. Biotechnol. 14 (1996) 303–308. [2] N.G. Walter, J.M. Bruke, Real-time monitoring of hairpin ribozyme kinetics through base-speciWc quenching of Xuorescein-labeled substrates, RNA 3 (1997) 392–404. [3] J.P. Knemeyer, N. Marme, M. Sauer, Probes for detection of speciWc DNA sequences at the single-molecule level, Anal. Chem. 72 (2000) 3717–3724. [4] B. Dubertret, M. Calame, A.J. Libchaber, Single-mismatch detection using gold-quenched Xuorescent oligonucleotides, Nat. Biotechnol. 19 (2001) 365–370.
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