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Letters to Analytical Chemistry Molecular Beacon-Based Junction Probes for Efficient Detection of Nucleic Acids via a True Target-Triggered Enzymatic Recycling Amplification Rong-Mei Kong,† Xiao-Bing Zhang,*,† Liang-Liang Zhang,† Yan Huang,† Dan-Qing Lu,† Weihong Tan,*,†,‡ Guo-Li Shen,† and Ru-Qin Yu† State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China, and Department of Chemistry and Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200, United States This work reports the development of a new molecular beacon-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNAcleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system. Developing methods for highly sensitive detection of sequencespecific oligonucleotides with an equally strong capability to identify single nucleotide polymorphisms (SNPs) is remarkably important for modern life sciences. Insertion/deletion variations or SNPs in oligonucleotides have been associated with several serious diseases, including cancers and some genetic diseases, as well as individual differences in drug metabolism.1 Polymerase chain reaction (PCR)-based commercial methods are typically used for nucleic acids detection in vitro,2 but methods employing endonucleases for isothermal-amplified detection of DNA with * To whom correspondence should be addressed. Phone: +86-731-88821903. Fax: +86-731-88821916. E-mail:
[email protected] (X.-B.Z.);
[email protected] (W.T.). † Hunan University. ‡ University of Florida. (1) (a) Irizarry, K.; Kustanovich, V.; Li, C.; Brown, N.; Nelson, S.; Wong, W.; Lee, C. J. Nat. Genet. 2000, 26, 233–236. (b) Sachidanandam, R.; Weissman, D.; Schmidt, S. C.; Kakol, J. M.; Stein, L. D.; Marth, G. Nature 2001, 409, 928–933. (c) Kim, S.; Misra, A. Annu. Rev. Biomed. Eng. 2007, 9, 289– 320. (2) (a) Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Science 1988, 239, 487–491. (b) Lie, Y. S.; Petropoulos, C. J. Curr. Opin. Biotechnol. 1998, 9, 43–48.
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high sensitivity, yielding added potential for in vivo applications, have also been proposed.3 However, the number of target nucleic acids is limited by the sequence-specific property of endonucleases. Recently, a few universally enzymatic signal amplification strategies for sensitive detection of DNA have been reported.4 However, these schemes, which show high sensitivity to target DNA sequences via the formation of more than 20 nucleotide hybrids, exhibit poor distinguishing capability for single-base mismatch. The Y-shaped junction DNA consists of three complementary oligonucleotide branches and has found applications in the controlled assembly of DNA-based materials and the multiplex detection of DNA.5 Sintim and coauthors have recently reported a so-called “template enhanced hybridization process” (TeHyP) for isothermal-amplified detection of nucleic acids by junction probes with a strong SNPs identifying capability.6 In their scheme, a “Y” junction structure was first formed by the hybridization of a signal probe, an assistant probe, and target DNA. The signal probe was then cleaved by an endonuclease and released, while the assistant probe and the target were regenerated and attended another cleavage cycle to realize the signal amplification. To our surprise, a BfuCI restriction endonuclease, which only cleaved double-stranded DNA, was used in their design,7 and the assistant probe was cleaved together with the signal probe in the TeHyP. Because the overhang of the assistant probe was cleaved and (3) (a) Kiesling, T.; Cox, K.; Davidson, E. A.; Dretchen, K.; Grater, G.; Hibbard, S.; Lasken, R. S.; Leshin, J.; Skowronski, E.; Danielsen, M. Nucleic Acids Res. 2007, 35, e117. (b) Li, J. W. J.; Chu, Y. Z.; Lee, B. Y. H.; Xie, X. L. S. Nucleic Acids Res. 2008, 36, e36. (c) Xu, W.; Xue, X.; Li, T.; Zeng, H.; Liu, X. Angew. Chem., Int. Ed. 2009, 48, 6849–6852. (4) (a) Guo, Q.; Yang, X.; Wang, K.; Tan, W.; Li, W.; Tang, H.; Li, H. Nucleic Acids Res. 2009, 37, e20. (b) Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816–1818. (c) Connolly, A. R.; Trau, M. Angew. Chem., Int. Ed. 2010, 49, 2720–2723. (5) (a) Li, Y.; Tseng, Y. D.; Kwon, S. Y.; D’Espaux, L.; Bunch, J. S.; McEuen, P. L.; Luo, D. Nat. Mater. 2004, 3, 38–42. (b) Li, Y.; Hong, Y. T.; Luo, D. Nat. Biotechnol. 2005, 23, 885–889. (6) Nakayama, S.; Yan, L.; Sintim, H. O. J. Am. Chem. Soc. 2008, 130, 12560– 12561. (7) See http://www.neb.com/nebecomm/products/. 10.1021/ac1025072 2011 American Chemical Society Published on Web 11/30/2010
Figure 1. (a) The Y-shaped junction structure of the proposed sensing system, which contains a two “A” unpaired bulge (in blue) in the assistant probe and an endonuclease recognition sequence (in red) in both the assistant probe and the MB probe. The arrow marked between two bases in the MB probe is the cleavage site for endonuclease Nt.BbvCI. (b) Schematics of a beacon based junction probe system for amplified detection of target DNA.
released from the system, the double-stranded sequences formation from the hybridization of the damaged assistant probe with the new signal probe then can no longer be recognized by the restriction endonuclease. For this reason, their proposed regenerating and cycling amplification scheme is not feasibly operational. In contrast to linear probes, molecular beacons (MBs) show a low fluorescent background with a high signal-to-noise (S/N) ratio and improved mismatch distinguishing abilities,8 and they have been widely used in DNA/RNA detections and some protein assays.9 In addition, linear probes require labeling to be located in the midsection of the oligonucleotide,6 while MBs are labeled with a fluorophore and quencher on opposite ends of the oligonucleotide, which should not interfere with the cleavage efficiency of endonuclease when the MB serves as a substrate. In this work, we take advantage of the unique features of the MBs to develop a MB-based junction probe that achieves rapid, isothermal, and highly sensitive detection of sequence-specific oligonucleotides, as well as a strong SNPs identifying capability, via a true target-triggered enzymatic recycling amplification process. Similar to Sintim’s design, our sensing system (Figure 1) also employs a “Y” junction structure that becomes operational via the “target enhanced hybridization process” but with novel modifications. First, an MB, which is complementary to part of a target DNA, serves as a signal probe and contains a nicking endonuclease site. Next, an oligonucleotide that is partially complementary with the signal probe and target DNA acts as an assistant probe. Finally, a nicking endonuclease, Nt.BbvCI, which can recognize a specific sequence of a hybridized double-stranded DNA but hydrolyzes only one specific strand,7 is introduced for beacon cleavage. As a proof-of-concept, an oligonucleotide with 23 bases from the HIV-1 U5 long terminal repeat sequence is (8) (a) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (b) Bonnet, G.; Tyagi, S.; Libchaber, A.; Kramer, F. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6171–6176. (9) (a) Kostrikis, L. G.; Tyagi, S.; Mhlanga, M. M.; Ho, D. D.; Kramer, F. R. Science 1998, 279, 1228–1229. (b) Tan, W.; Fang, X.; Li, J.; Liu, X. Chem.sEur. J. 2000, 6, 1107–1111. (c) Tan, W.; Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8, 547–553. (d) Tyagi, S. Nat. Methods 2009, 6, 331–338. (e) Wang, K.; Tang, Z.; Yang, C. J.; Kim, Y.; Fang, X.; Li, W.; Wu, Y.; Medley, C. D.; Cao, Z.; Li, J.; Colon, P.; Lin, H.; Tan, W. Angew. Chem., Int. Ed. 2009, 48, 856–870.
chosen as the target DNA.10 The melting temperature for the hybrid of the signal probe with the assistant probe is estimated to be 34 °C.11 Thus, in the absence of a target, the MB fails to hybridize with the assistant probe to form a double-stranded recognition sequence under the reaction temperature (37 °C). As a consequence, since Nt.BbvCI is disabled and cannot cleave the MB, no remarkable fluorescent signal is triggered. However, in the presence of a target, the assistant probe, together with the target, can hybridize with the MB and open its hairpin structure to form a “Y” junction structure, as well as form the doublestranded recognition sequence for Nt.BbvCI. Once the MB is cleaved, it is dissociated from the sensing system, and fluorescence is restored. The released hybrid of the assistant probe with the target can then hybridize with another MB and trigger the second cycle of cleavage. Eventually, each hybrid of the assistant probe with the target can undergo many cycles to trigger the cleavage of many MBs, providing an amplified detection signal for target DNA. In order to achieve the best sensing performance, the sequence of the assistant probe and MB and the concentration for both endonuclease and MB were optimized. An assistant probe containing 9 base pairs complementary with the MB signal probe showed a larger S/N ratio than the assistant probes containing 7, 8, and 10 base pairs (see the Supporting Information, Figure S1a). However, too many complementary base pairs will lead to strong hybridization between the assistant probe and the MB signal probe, which can be recognized and cleaved by the Nt.BbvCI in the absence of target DNA, resulting in high background signal and low sensitivity. On the other hand, an assistant probe with two “A” bases in the middle region would form an unpaired nucleotide bulge in the Y junction probe system (Figure 1a), thus providing better sensitivity toward the target DNA than a junction probe lacking such a bulge because of its higher enzymatic cleaving efficiency from the thermodynamically improved stability.12 Therefore, we chose the assistant probe contains nine base pairs complementary to MB with two “A” bases in the middle (10) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785–4787. (11) Zuker, M. Nucleic Acids Res. 2003, 31, 3406–3415. (12) (a) Wang, D. Y.; Sen, D. J. Mol. Biol. 2001, 310, 723–734. (b) Wang, D. Y.; Lai, B. H.Y.; Sen, D. J. Mol. Biol. 2002, 318, 33–43.
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Figure 2. Time-dependent fluorescence responses of the sensing system to 5 nM of target DNA in the presence (a) and absence of endonuclease (b), with corresponding backgrounds (c and d), respectively. Reactions were performed in 100 µL 1× NEB buffer 4 with 4 U Nt.BbVCI (for curves a and c), 50 nM MB, and 50 nM assisted probe at 37 °C.
region as our optimized assistant probe. The stem length of MB is also critical for the sensing performance. Our experimental results showed that a MB containing a stem of five base pairs could provide obviously better sensing performance than that of MBs with a stem of four and six base pairs (see the Supporting Information, Figure S1b). Experimental results also showed that a concentration of 0.04 U/µL of endonuclease and 50 nM MB could provide a maximum S/N ratio for the sensing system (see the Supporting Information, Figure S2a,b). To estimate the amplification function of the proposed sensing system, we incubated 50 nM assistant probe and 50 nM MB signal probe with target DNA (0 or 5 nM) and then monitored the kinetics of the fluorescence enhancement for the mixed solution in the presence and absence of 4 U of Nt.BbVCI, respectively. Under these conditions, the endonuclease also cleaves some of the MBs which may be partially hybridized to the assistant probe in the absence of target DNA, resulting in increased background fluorescence (Figure 2, curve c). Fortunately, the target-induced signal enhancement is much larger than the background, assuring a high sensitivity for detection of target DNA. As shown in Figure 2, by introducing endonuclease amplification, we observed a 404 ± 31% signal increase upon addition of 5 nM target DNA within 30 min. The corresponding signal enhancement in the absence of endonuclease was only 78 ± 11%. These results indicate that our developed MB-based junction probe can feasibly provide an amplified signal. Figure 3a shows the time-dependent fluorescence enhancement of the sensing system upon introduction of different concentrations of target DNA. The rate of fluorescence enhancement was increased with the increase of target DNA concentration. A 32-fold fluorescence enhancement was observed when the target DNA concentration reached 100 nM (Figure 3a). The large fluorescence enhancement (high S/N ratio) together with the multiple turnover capability of the endonuclease could improve the sensitivity of the new sensing system. Figure 3b depicts the fluorescence enhancement of the system after 30 min of reaction initiation induced by different concentrations of target DNA. It shows a dynamic range from 5 pM to 100 nM. The amplified 16
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Figure 3. Sensitivity of the amplified fluorescence sensing system for target HIV1 DNA. (a) Time-dependent fluorescence responses in the presence of different concentrations of target DNA. Inset: Responses of the sensing system to target DNA at low concentrations. (b) The relationship of the fluorescence enhancement (at 30 min) with the target concentration. Inset shows the responses of the sensing system to target DNA at low concentration. Reactions were performed in 100 µL of 1× NEB buffer 4, with 4 U Nt.BbVCI, 50 nM MB, and 50 nM assisted probe at 37 °C.
sensing process is also fast with the fluorescent enhancement reaching a plateau within 30 min upon the addition of 100 nM target DNA, which is remarkably improved when compared with the first-generation junction probe system. It shows a very slow response to target DNA and obtains a detection limit of 50 pM under a long reaction time (9 h).6 Moreover, control experiments with target DNA at various concentrations in the absence of endonuclease were also carried out (see the Supporting Information, Figure S3), and a detection limit of only 1.6 nM was obtained. Its sensitivity is almost 3 orders of magnitude poorer than that of the Nt.BbVCI-amplified method. These results reveal that the endonuclease does indeed play an important role in the recycling amplification. The proposed sensing system also shows remarkably high sequence specificity for DNA detection. In order to investigate its capability of identifying SNPs, we compared the fluorescence response induced by DNA strands containing single-base- and twobase-mismatches with that of target DNA (Figure 4a). It could be observed that even the fluorescence intensity changes triggered by a single-base mismatched DNA were similar to those of the target-free reaction, which is obviously better than that of the single MB probe-based exonuclease-amplified sensing system.4b
Figure 4. (a) Fluorescence response of the sensing system toward perfectly matched and mismatched DNA targets. Reactions were performed in 100 µL of 1× NEB buffer 4 with 4 U Nt.BbVCI, 50 nM MB, 50 nM assisted probe, and 50 nM DNA target. (b) Gel image of the sensing system. Lane 1, target alone; lane 2, MB alone; lanes 3 and 4, 2 h after addition of 0 and 0.2 µM of target DNA into the mixture of 1 µM assistant probe, 1 µM MB signal probe, and 4 U Nt.BbvCI; lane 5, 2 h after addition of 1 µM of single base mismatch DNA1 into the mixture.
Gel electrophoresis experiments provided similar results. When single-base mismatch DNA was used, no obvious cleavage product was observed (Figure 4b, lane 5), while the matched target provided a significant product band on a PAGE gel (Figure 4b, lane 4). The sequence specificity of this sensing system is attributed to the Y-shaped junction structure which needs an assistant probe for stabilization, and the use of MBs, which possess unique stem-loop structures. To verify if the target DNA can indeed act as a catalyst to induce multiple turnover catalyzing cleavage of MBs and realize the recycling signal amplification, the turnover number (TON) for different concentrations of target DNA was estimated by a reported method.6 The yield for the enzymatic cleavage reaction in 30 min was 212% with the addition of 2 nM (0.04 equiv) of target DNA, corresponding to a TON of 2.12 (see the Supporting Information, Figure S4). A lower concentration of target DNA could provide larger TON. The TON for 0.1 nM (0.002 equiv) of (13) (a) Cho, E. J.; Lee, J.-W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241–264. (b) Fang, X.; Tan, W. Acc. Chem. Res. 2010, 43, 48–57.
target DNA was estimated to be 15.21. These results indicate that the target DNA could be regenerated after catalyzing the cleavage of MBs and that one target molecule could cleave several MBs molecules. These results indicated that target DNA has indeed triggered enzymatic recycling cleavage of MBs and afforded amplified signal for target DNA detection. In summary, we have developed a novel molecular beaconbased junction sensing system for highly efficient detection of DNA. In the first-generation junction sensing systems, linear signal probes were labeled in the midsection of oligonucleotide. In contrast, we use a hairpin-structured MB with labels on the each end of oligonucleotide, a design which is completely capable of maintaining the cleaving activity of endonuclease. An ssDNAcleaved endonuclease, Nt.BbvCI, rather than dsDNA-cleaved endonuclease, was also introduced in our sensing system. These design improvements could guarantee a true and efficient targettriggered enzymatic recycling amplification process in our sensing system and afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system. Furthermore, with the combination of the Y junction-structured binary probe strategy and the hairpin-structured MB probe with its ability to distinguish mismatches, our proposed sensing system shows a strong SNPs identifying capability, thus creating a tool with potential for the clinical diagnosis of genetic diseases. With the incorporation of aptamers into the junction probes, the proposed strategy is expected to hold an even greater potential in the re-engineering of our sensing system for the fast and amplified detection of a broad range of targets beyond nucleic acids.13 ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grants J0830415, 20975034), The National Key Scientific Program of China (Grant 2011CB911000), ″973″ NationalKeyBasicResearchProgramofChina(Grant2007CB310500), Program for Changjiang Scholars and Innovative Research Team in University, and Ministry of Education of China (Grant NCET07-0272). We also thank NIH and China National Grand Program on Key Infectious Disease (2009ZX10004-312) for partial support. SUPPORTING INFORMATION AVAILABLE Apparatus, experimental procedures, and supplementary spectra data. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 21, 2010. Accepted November 23, 2010. AC1025072
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