- Email: [email protected]
A label-free, G-quadruplex DNAzyme-based fluorescent probe for signal-amplified DNA detection and turn-on assay of endonuclease
Biosensors and Bioelectronics 34 (2012) 100–105
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A label-free, G-quadruplex DNAzyme-based fluorescent probe for signal-amplified DNA detection and turn-on assay of endonuclease Zhixue Zhou a,b , Yan Du a,b , Libing Zhang a,b , Shaojun Dong a,b,∗ a b
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 27 July 2011 Received in revised form 30 November 2011 Accepted 19 January 2012 Available online 28 January 2012 Keywords: DNAzyme Molecular beacon Exonuclease III DNA detection Endonuclease
a b s t r a c t A novel G-quadruplex DNAzyme molecular beacon (G-DNAzymeMB) strategy is developed for assays of target DNA and restriction endonuclease. The detection system consists of G-DNAzymeMB strand and a blocker DNA by using the fluorescence of 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCFDA) catalyzed by G-DNAzymeMB as a signal reporter. G-DNAzymeMB exhibits peroxidase activity in its free hairpin structure, and forms a catalytically inactive hybrid when hybridized with blocker DNA. Upon displacement of blocker DNA by target DNA or cleavage by restriction endonuclease, G-DNAzymeMB is released and two lateral portions of G-DNAzymeMB form a G-quadruplex structure, resulting in the recovery of catalytic activity which acts as a cofactor to catalyze H2 O2 -mediated oxidation of H2 DCFDA. For DNA detection system, exonuclease III (Exo III)-catalyzed amplification strategy is introduced to improve the sensitivity and target DNA could be detected as low as 0.1 pM. With respect to restriction endonuclease detection system, 0.1 U/mL EcoRI endonuclease could be detected and this method could be easily transported to other restriction endonuclease analysis by simply changing the recognition sequence. These results demonstrate that the proposed G-DNAzymeMB strategy could be used as a label-free, simple, sensitive and cost-effective approach in analysis of target DNA and restriction endonuclease. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Molecular beacons (MBs) are specially engineered nucleic acid sequences that fold into a hairpin structure. Since introduction in 1996 (Tyagi and Kramer, 1996), they have shown tremendous use in biochemistry, molecular biology and medical sciences (Broude, 2002; Tan et al., 2004). In the traditional format, MBs act like switches that fluorescing upon hybridization with specific nucleic acid targets. While MBs-based detection systems are one of the most successful separation-free probes (Fang et al., 1999; Sokol et al., 1998), there remain some challenging problems. First, MBs need to be labeled with two non-native moieties (i.e., a donor fluorophore and a quencher), thus suffering from problems associated with double labeling such as high cost, low yield, and singly labeled impurities (Yeh et al., 2010). Second, fluorescence enhancement of MBs is generally limited by background fluorescence, which comes from imperfect quenching of donors and conformational fluctuations of the hairpin structure (Bonnet et al., 1998). Consequently,
∗ Corresponding author at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China. Tel.: +86 431 85262101; fax: +86 431 85689711. E-mail address: [email protected] (S. Dong). 0956-5663/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2012.01.024
the fabrication of a label-free, sensitive, simple, and low cost MBbased system has become highly focusing. DNAzymes are a kind of artificial enzymes that exhibit specific catalytic activities. Compared with natural proteinogenic enzymes, DNAzymes are easier to synthesize and modify, higher thermal stability, and less expensive. Besides, the flexibility in mastering the DNAzyme structures by encoding recognition function into DNAzyme sequences makes DNAzymes ideal candidates for developing bioanalytical platforms. Up to now, they have been widely applied in many biochemical reactions such as DNA, RNA, glycosidic bond cleavage (Burmeister et al., 1997; Carmi et al., 1998; Li et al., 2000a,b; Santoro and Joyce, 1997), porphyrin metalation (Li and Sen, 1996), and DNA self-modification (Li and Breaker, 1999; Li et al., 2000a,b; Sheppard et al., 2000). One kind of DNAzymes formed by hemin and a G-quadruplex aptamer (i.e., G-quadruplex-based DNAzyme) can catalyze H2 O2 -mediated oxidation of 2,2 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), luminol or H2 DCFDA (Li et al., 2007a,b; Qiu et al., 2011; Travascio et al., 1998, 1999, 2001), accompanied by a color change, chemiluminescent or fluorescent emission. Among them, H2 DCFDA is a commercially available fluorone dye which is non-fluorescent itself and can be oxidized by DNAzymes into fluorescent dichlorofluorescein (with excitation and emission wavelengths centered at = 501 and 520 nm, respectively). It has been reported that H2 DCFDA is a superior alternative to ABTS in bioanalytical detection (Nakayama and Sintim, 2010).
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
101
Fig. 1. (A) Schematic illustration of the amplified detection of DNA using label-free G-quadruplex DNAzyme-based molecular beacon. At first, P1 (G-DNAzymeMB1) and B1 (blocker DNA1) formed the catalytically inactive P1/B1 hybrid. T1 (target DNA) hybridized with B1 and released the catalytically active P1 for the generation of fluorescent signals (step (1)); Exonuclease III (Exo III) catalyzed the stepwise removal of mononucleotides from the blunt 3 terminus of B1 (step (2)), liberating T1 (step (3)); the released T1 triggered amplified cycle by hybridizing with another inactive P1/B1 hybrid (step (4)). (B) Schematic illustration of the assay of the restriction endonuclease. P2 represents G-DNAzymeMB2, and B2 represents blocker DNA2. (C) DNA sequences used in the present work. The G-quadruplex portions were shown in red and the recognition sequences of EcoRI were shown in green.For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.
In this study, we construct a label-free, G-quadruplex DNAzyme molecular beacon (G-DNAzymeMB)-based strategy for fluorescent detection of DNA and restriction endonuclease. The fluorescence of H2 DCFDA catalyzed by G-DNAzymeMB is used as a signal reporter. In the “off” state, the central loop portion of G-DNAzymeMB is hybridized with a blocker DNA and formation of G-quadruplex is inhibited. Consequently, catalytic activity of DNAzyme is prevented, resulting in low fluorescent signal. Upon addition of target DNA or enzyme cleavage, two lateral portions of G-DNAzymeMB form a G-quadruplex structure. Then in the presence of hemin, they can catalyze oxidation of H2 DCFDA and give high fluorescent signal. To improve the sensitivity of DNA detection using GDNAzymeMB, exonuclease III (Exo III)-catalyzed amplification strategy is introduced. Exonuclease III is sequence-independent and can catalyze the stepwise removal of mononucleotides from 3 -hydroxyl termini of duplex DNA. Its activity on single-stranded DNA is limited and preferred for blunt or recessed 3 termini (Zuo et al., 2010). Thanks to the amplification of Exo III, 0.1 pM target DNA could be distinctly detected. 2. Materials and methods 2.1. Chemicals and materials All oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and their sequences were listed
in Table S1. The oligonucleotides were determined using the 260 nm UV absorbance and the corresponding extinction coefficient. H2 DCFDA was purchased from Sigma (Missouri, USA). Exo III (15 × 104 U/mL), EcoRI and Hind III (both 15 × 103 U/mL) were purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Hemin was purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). The stock solution of hemin (1 mM) was prepared in dimethyl sulfoxide (DMSO), stored in darkness at −20 ◦ C. Tris(hydroxymethyl)aminomethane (Tris) was bought from Shanghai Chemical Reagent Company (Shanghai, China). Other chemicals were of analytical grade and used without further purification. Solutions were prepared with deionized water processed with a Milli-Q ultra-high purity water system (Millipore, Bedford, MA, USA). 2.2. Assay procedures for DNA and endonuclease detection In a typical DNA assay, P1 (G-DNAzymeMB1, 0.5 M) and B1 (blocker DNA1, 0.5 M) were dissolved in Tris–buffer 1 (25 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl2 ). The solution was then heated at 90 ◦ C for 10 min and gradually cooled to room temperature to ensure that nucleic acids were hybridized to each other completely. Afterwards, 2 L T1 (target DNA), 2 L Exo III (20 units) and 4 L 10× Exonuclease III buffer (50 mM Tris–HCl, 5 mM MgCl2 , 1 mM DTT, pH 8.0) were added in succession. Cleavage reaction of Exo III was conducted at 37 ◦ C for 40 min. Then hemin (0.1 M) in Tris–buffer 2 (50 mM Tris–HCl, 20 mM KCl, 150 mM NH4 Ac) was
102
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
added and incubated at room temperature for 30 min to form the hemin–G-DNAzyme. For endonuclease assay, a total volume of 360 L reaction solution containing substrate P2 (G-DNAzymeMB2)/B2 (blocker DNA2) (or P3 (G-DNAzymeMB3)/B3 (blocker DNA3)) (each 0.4 M) and various amounts of EcoRI (or Hind III) were incubated at 30 ◦ C for 1.5 h, and then hemin (0.08 M) was added. 2.3. Fluorescent analysis For the fluorescent measurements, H2 DCFDA (20 M) and H2 O2 (220 M) (H2 DCFDA and H2 O2 must be freshly prepared before use) were added to the product solution with a final volume of 400 L. Fluorescence emission spectra were collected from 510 to 600 nm by using a Fluoromax-4 Spectrofluorometer (HORIBA Jobin Yvon, Inc., NJ, USA) at room temperature. The excitation wavelength was set at 501 nm. For the sensor calibration curve acquisition, F was plotted as sensor signal, where F is the fluorescence intensity of the sensor solution with addition of different concentrations of T1 or restriction endonuclease. All the measurements were performed three times, and the standard deviation was plotted as the error bar. 3. Results and discussion 3.1. Target DNA detection principle The design of the label-free fluorescent sensor for DNA was shown in Fig. 1A. The G-quadruplex oligomer was split into two halves connected with a central loop portion (G-DNAzymeMB: d [G3 T2 G3 C-loop-TG3 TAG3 ]). The formation of G-quadruplex was validated by circular dichroism (CD) spectra of G-DNAzymeMB (P1) in buffer solutions (see Fig. S1 in the Supporting Information). At first, the central loop portion of P1 is hybridized with B1 (blocker DNA) to form a stable duplex (17 bases match). As a result, the two lateral portions of P1 cannot associate with each other to form quadruplex structure and the catalytic activity of P1 is inhibited. Under these conditions, H2 DCFDA cannot be effectively oxidized and only weak fluorescence can be observed. Addition of target DNA (T1), however, can displace P1 from P1/B1 duplex through branch migration since there exist more base pairs (31 bases match) between B1/T1. As a result, the released P1 self-assembles into Gquadruplex structure, and with hemin it can catalyze oxidation of H2 DCFDA, generating a strong fluorescence signal (Fig. 1A step (1)). The hybridization of T1 and B1 strands form a double-stranded structure that contains a blunt 3 terminus and an Exo III-resistant 3 protruding terminus. When Exo III is added, it catalyzes the stepwise removal of mononucleotides from the blunt 3 terminus of
Fig. 2. Fluorescence spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in solutions (a) P1/B1 (each 0.5 M) in the absence of target DNA, (b) P1/B1 (each 0.5 M) in the presence of target DNA (0.1 M), (c) P1/B1 (each 0.5 M) in the presence of Exo III (20U), (d) P1/B1 (each 0.5 M) in the presence of Exo III (20U) and target DNA (0.1 M). Experimental conditions were the optimized conditions shown in Section 2.
B1 (Fig. 1A step (2)), liberating T1 (Fig. 1A step (3)). The released T1 then reacts with another P1/B1 hybrid to initiate a new cycle (Fig. 1A step (4)). Eventually, a single copy of T1 generates many active P1 and thus achieves target assisted enzyme-catalyzed signal amplification. Fluorescence measurements were carried out to serve as a proof of concept to test the principle of our design. As shown in Fig. 2, the fluorescence is very weak in the P1/B1 hybrid system (curve a) and after addition of 50 nM T1 there is a 2-fold increase (curve b). Addition of Exo III, the background fluorescence increased slightly (curve c); however, a significant increase (10-fold) in fluorescence intensity is observed upon addition of 50 nM T1 to the Exo IIIcatalyzed amplification system (curve d). These results indicate that Exo III-catalyzed amplified target DNA detection has been successfully achieved. 3.2. Detection of T1 without Exo III-catalyzed signal amplification Detection of various concentrations of T1 (0–0.5 M) based on P1/B1 hybrid system was first conducted and results were shown in Fig. 3. The significant increase in fluorescence clearly indicates that T1 could hybridize with B1 and replace P1 from P1/B1 duplex (Fig. 3A). It is observed that 5 nM T1 could be significantly detected and a linear correlation (R2 = 0.999) exists between the value of fluorescence and the concentration of T1 over the range of 0–0.5 M (Fig. 3B).
Fig. 3. (A) Fluorescent spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the presence of various concentrations of target DNA. (B) Linear relation between the fluorescent intensity at 520 nm and concentrations of target DNA (0–0.5 M). Experimental conditions were the optimized conditions shown in Section 2.
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
103
Fig. 4. (A) Fluorescent spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the presence of various concentrations of target DNA with Exo III-catalyzed amplification system. (B) Plot of the values of fluorescent intensity at 520 nm with respect to the logarithm of the concentrations of T1 (1 pM–5 nM). Experimental conditions were the optimized conditions shown in Section 2.
3.3. Detection of T1 with Exo III-catalyzed signal amplification For Exo III-amplified P1/B1 hybrid system, effect of different amounts of Exo III on the signal amplification was investigated. Fluorescence at 520 nm was recorded in the presence of different amounts of Exo III from 0 to 30 U (see Fig. S2A in the Supporting Information). As can be seen, the amplified effect increases sharply upon increasing the amount of Exo III and is saturated at ≥20 U. Thus, 20 U of Exo III is used in all further experiments. Besides, the Exo III-amplified reaction is rapid and the assay exhibits a nearly saturated signal within 40 min in the presence of 20 U of enzyme (see Fig. S2B in the Supporting Information). With respect to sensitivity and speed, the Exo III reaction time of 40 min was chosen for subsequent study. Under optimized conditions, T1 detection using the Exo III-amplified P1/B1 system was conducted via monitoring the fluorescent change of H2 DCFDA catalyzed by P1/hemin. Fig. 4A showed the fluorescent spectra with various concentrations of T1 from 0 to 100 nM, and apparent fluorescence increases were observed. The fluorescent intensity was linear with the logarithm of the concentration of T1 over the range of 1 pM–5 nM and 0.1 pM T1 could be detected obviously (Fig. 4B). One of the important advantages that use MBs for DNA detection is that they show high selectivity. To test the selectivity of the present system for analysis of target DNA, similar DNA strands with only single (T2), two (T3) and four (T4) nucleotides mismatch were also investigated. As shown in Fig. 5, the fluorescence enhancement value by T2 (curve d) was 20% of that by T1 (curve e), and
Fig. 5. Selectivity of the G-DNAzymeMB in the absence (curve a) and presence of target DNA (T1 50 nM, curve e) over other mismatched DNA (50 nM): T2 (singlebase mismatched target, curve d), T3 (two-base mismatched target, curve c) and T4 (four-base mismatched target, curve b).
there was almost no enhancement for T4 (curve b). These results demonstrated the well selectivity of the proposed approach and even one-nucleotide mismatch could be distinguished. 3.4. Fluorescent detection of restriction endonuclease To address the general applicability of this G-DNAzymeMB in bioanalysis, detection of Type II restriction endonuclease was also explored. Type II restriction endonucleases, among the most important enzymes in molecular biology, recognize short, usually palindromic, sequences of 4–8 bp and cleave DNA within or in close proximity to the recognition sequence (Pingoud and Jeltsch, 2001). They play an important role to protect host genome against foreign DNA and thus assays of these enzymes activities are crucial in many fields ranging from biotechnology to clinical diagnostics and drug discovery. Using EcoRI restriction endonuclease as a model system (Fig. 1B), P2 (G-DNAzymeMB2, 36 mer) was partly hybridized with B2 (blocker DNA2, 18 mer) which contains recognition sequence (5 GAATTC 3 ) located in the intermediate position to form P2/B2 duplex (18 bases matched) that acted as substrate for EcoRI. In the absence of EcoRI, P2 and B2 formed a stable duplex (Tm = 51 ◦ C, calculated according to DNAMelt Server) (Markham and Zuker, 2005, 2008) and the two lateral portions of P2 could not associate with each other to form quadruplex structure. Therefore, the catalytic activity of P2 was inhibited and weak fluorescence was obtained. Upon addition of EcoRI, cleavage reaction was initiated and P2/B2 duplex was cleaved into two parts. Since the newly formed hybrids contained only few base pairs (7 bases matched), they would be separated into short single DNA fragments at 30 ◦ C (Tm values are 12.2 and 13.6 ◦ C, respectively). The separated two lateral portions of P2 were allowed to form quadruplex structure, then the formed G-DNAzyme catalyze the oxidation of H2 DCFDA, resulting in strong fluorescence. In order to validate our approach, firstly, a series of control experiments were performed to ensure that the observed fluorescence enhancement was induced by the presence of EcoRI (Fig. 6A). The fluorescent intensity increased greatly in the presence of EcoRI (Fig. 6A, curve d). However, the emission response was significantly diminished when EcoRI was heat-inactivated before incubation with oligonucleotides (Fig. 6A, curve b). In addition, no significant increase in fluorescence signal was observed upon addition of EcoRI in the absence of the recognition site for EcoRI in oligonucleotides (Fig. 6A, curve c). Then, fluorescence measurements were conducted with various concentrations of EcoRI (0–10 U/mL). Interestingly, the fluorescence intensity increased as increasing the concentration of EcoRI (Fig. 6B). As shown in Fig. 6C, plots of the fluorescent intensity versus nuclease concentration (0.1–5 U/mL) showed good linear relationship (R2 = 0.988).
104
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
Fig. 6. (A) Fluorescence response of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the absence of EcoRI: (a) in the absence of EcoRI; (b) incubated with heat-inactivated EcoRI; (c) incubated with normal EcoRI but lack of EcoRI substrate; (d) incubated with normal EcoRI in the presence of EcoRI substrate. (B) Fluorescence spectra of G-DNAzymecatalyzed oxidation of H2 DCFDA in the presence of P2/B2 (EcoRI substrate, 0.4 M) with various concentrations of EcoRI. (C) Linear relation between the fluorescence intensities at 520 nm and EcoRI concentrations (0.1–5 U/mL). (D) Fluorescence emission spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the presence of P3/B3 (Hind III substrate, 0.4 M) with various concentrations of Hind III (0–5 U/mL).
Therefore, the nuclease activities could be quantitatively analyzed by using the plot of fluorescent intensity versus enzyme concentrations in the present G-DNAzymeMB-based enzyme detection system. Compared with traditional enzyme detection systems such as enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) and fluorescence resonance energy transfer (FRET) (Hiramatsu et al., 1998; Jeltsch et al., 1993; Li et al., 2007a,b; McLaughlin et al., 1987; Spitzer and Eckstein, 1988), our method is simple, time-saving and there is no need for isotope or DNA labeling. To address the general approach for restriction endonucleases, detection of Hind III restriction endonuclease was also studied. The specific substrates P3 (G-DNAzymeMB3)/B3 (blocker DNA 3) for Hind III were obtained by simply replacing the recognition sequence with 5 -AAGCTT-3 within the central loop portion. Upon addition of Hind III (0–5 U/mL), similar fluorescent enhancement was observed (Fig. 6D). These results demonstrate its potential to be a general method for the detection of restriction endonucleases.
endonuclease by simply replacing the recognition portions. Using EcoRI as a model analyte, 0.1 U/mL EcoRI could be detected. The new approach is expected to promote the exploitation of GDNAzymeMB-based biosensors for target assays in biochemical and biomedical studies.
4. Conclusions
Bonnet, G., Krichevsky, O., Libchaber, A., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (15), 8602–8606. Broude, N.E., 2002. Trends Biotechnol. 20 (6), 249–256. Burmeister, J., vonKiedrowski, G., Ellington, A.D., 1997. Angew. Chem. Int. Ed. 36 (12), 1321–1324. Carmi, N., Balkhi, S.R., Breaker, R.R., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (5), 2233–2237. Fang, X.H., Liu, X.J., Schuster, S., Tan, W.H., 1999. J. Am. Chem. Soc. 121 (12), 2921–2922. Hiramatsu, K., Miura, H., Kamei, S., Iwasaki, K., Kawakita, M., 1998. J. Biochem. 124 (1), 231–236. Jeltsch, A., Fritz, A., Alves, J., Wolfes, H., Pingoud, A., 1993. Anal. Biochem. 213 (2), 234–240. Li, D., Shlyahovsky, B., Elbaz, J., Willner, I., 2007a. J. Am. Chem. Soc. 129 (18), 5804–5805. Li, J., Zheng, W.C., Kwon, A.H., Lu, Y., 2000a. Nucleic Acids Res. 28 (2), 481–488. Li, J., Yan, H.F., Wang, K.M., Tan, W.H., Zhou, X.W., 2007b. Anal. Chem. 79 (3), 1050–1056.
In conclusion, a simple, sensitive and label-free G-quadruplex DNAzyme molecular beacon-based biosensor has been constructed for monitoring target DNA and restriction endonuclease, using fluorescent emission of H2 DCFDA catalyzed by G-quadruplex DNAzyme molecular beacon as readout signal. The present G-DNAzymeMB method is a turn-on model and does not require any chemical modification of DNA, which shows advantages of simplicity in operation, cost and time efficiency, and sensitivity. For target DNA detection, Exo III was introduced to initiate the amplified strategy and 0.1 pM target DNA could be detected, which is 4-fold than the unamplified strategy. More importantly, this G-DNAzymeMB was generally applicable and could be easily adaptable to assay of restriction
Acknowledgments This work was supported by the National Natural Science Foundation of China (nos. 20935003, 21075116), and 973 project (nos. 2011CB911002, 2010CB933603). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2012.01.024. References
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105 Li, Y.F., Sen, D., 1996. Nat. Struct. Biol. 3 (9), 743–747. Li, Y.F., Breaker, R.R., 1999. Proc. Natl. Acad. Sci. U.S.A. 96 (6), 2746–2751. Li, Y.F., Liu, Y., Breaker, R.R., 2000b. Biochemistry 39 (11), 3106–3114. Markham, N.R., Zuker, M., 2005. Nucleic Acids Res. 33, W577–W581. Markham, N.R., Zuker, M., 2008. In: Keith, J.M. (Ed.), Bioinformatics, Volume II. Structure, Function and Applications, number 453 in Methods in Molecular Biology, chapter 1. Humana Press, Totowa, pp. 3–31, NJ. ISBN 978-1-60327-428-9. McLaughlin, L.W., Benseler, F., Graeser, E., Piel, N., Scholtissek, S., 1987. Biochemistry 26 (23), 7238–7245. Nakayama, S., Sintim, H.O., 2010. Mol. Biosyst. 6 (1), 95–97. Pingoud, A., Jeltsch, A., 2001. Nucleic Acids Res. 29 (18), 3705–3727. Qiu, B., Zheng, Z.Z., Lu, Y.J., Lin, Z.Y., Wong, K.Y., Chen, G.N., 2011. Chem. Commun. 47 (5), 1437–1439. Santoro, S.W., Joyce, G.F., 1997. Proc. Natl. Acad. Sci. U.S.A. 94 (9), 4262–4266.
105
Sheppard, T.L., Ordoukhanian, P., Joyce, G.F., 2000. Proc. Natl. Acad. Sci. U.S.A. 97 (14), 7802–7807. Sokol, D.L., Zhang, X.L., Lu, P.Z., Gewitz, A.M., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (20), 11538–11543. Spitzer, S., Eckstein, F., 1988. Nucleic Acids Res. 16 (24), 11691–11704. Tan, W.H., Wang, K.M., Drake, T.J., 2004. Curr. Opin. Chem. Biol. 8 (5), 547–553. Travascio, P., Li, Y.F., Sen, D., 1998. Chem. Biol. 5 (9), 505–517. Travascio, P., Bennet, A.J., Wang, D.Y., Sen, D., 1999. Chem. Biol. 6 (11), 779–787. Travascio, P., Witting, P.K., Mauk, A.G., Sen, D., 2001. J. Am. Chem. Soc. 123 (7), 1337–1348. Tyagi, S., Kramer, F.R., 1996. Nat. Biotechnol. 14 (3), 303–308. Yeh, H.C., Sharma, J., Han, J.J., Martinez, J.S., Werner, J.H., 2010. Nano Lett. 10 (8), 3106–3110. Zuo, X.L., Xia, F., Xiao, Y., Plaxco, K.W., 2010. J. Am. Chem. Soc. 132 (6), 1816–1817.
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A label-free, G-quadruplex DNAzyme-based fluorescent probe for signal-amplified DNA detection and turn-on assay of endonuclease Zhixue Zhou a,b , Yan Du a,b , Libing Zhang a,b , Shaojun Dong a,b,∗ a b
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 27 July 2011 Received in revised form 30 November 2011 Accepted 19 January 2012 Available online 28 January 2012 Keywords: DNAzyme Molecular beacon Exonuclease III DNA detection Endonuclease
a b s t r a c t A novel G-quadruplex DNAzyme molecular beacon (G-DNAzymeMB) strategy is developed for assays of target DNA and restriction endonuclease. The detection system consists of G-DNAzymeMB strand and a blocker DNA by using the fluorescence of 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCFDA) catalyzed by G-DNAzymeMB as a signal reporter. G-DNAzymeMB exhibits peroxidase activity in its free hairpin structure, and forms a catalytically inactive hybrid when hybridized with blocker DNA. Upon displacement of blocker DNA by target DNA or cleavage by restriction endonuclease, G-DNAzymeMB is released and two lateral portions of G-DNAzymeMB form a G-quadruplex structure, resulting in the recovery of catalytic activity which acts as a cofactor to catalyze H2 O2 -mediated oxidation of H2 DCFDA. For DNA detection system, exonuclease III (Exo III)-catalyzed amplification strategy is introduced to improve the sensitivity and target DNA could be detected as low as 0.1 pM. With respect to restriction endonuclease detection system, 0.1 U/mL EcoRI endonuclease could be detected and this method could be easily transported to other restriction endonuclease analysis by simply changing the recognition sequence. These results demonstrate that the proposed G-DNAzymeMB strategy could be used as a label-free, simple, sensitive and cost-effective approach in analysis of target DNA and restriction endonuclease. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Molecular beacons (MBs) are specially engineered nucleic acid sequences that fold into a hairpin structure. Since introduction in 1996 (Tyagi and Kramer, 1996), they have shown tremendous use in biochemistry, molecular biology and medical sciences (Broude, 2002; Tan et al., 2004). In the traditional format, MBs act like switches that fluorescing upon hybridization with specific nucleic acid targets. While MBs-based detection systems are one of the most successful separation-free probes (Fang et al., 1999; Sokol et al., 1998), there remain some challenging problems. First, MBs need to be labeled with two non-native moieties (i.e., a donor fluorophore and a quencher), thus suffering from problems associated with double labeling such as high cost, low yield, and singly labeled impurities (Yeh et al., 2010). Second, fluorescence enhancement of MBs is generally limited by background fluorescence, which comes from imperfect quenching of donors and conformational fluctuations of the hairpin structure (Bonnet et al., 1998). Consequently,
∗ Corresponding author at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China. Tel.: +86 431 85262101; fax: +86 431 85689711. E-mail address: [email protected] (S. Dong). 0956-5663/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2012.01.024
the fabrication of a label-free, sensitive, simple, and low cost MBbased system has become highly focusing. DNAzymes are a kind of artificial enzymes that exhibit specific catalytic activities. Compared with natural proteinogenic enzymes, DNAzymes are easier to synthesize and modify, higher thermal stability, and less expensive. Besides, the flexibility in mastering the DNAzyme structures by encoding recognition function into DNAzyme sequences makes DNAzymes ideal candidates for developing bioanalytical platforms. Up to now, they have been widely applied in many biochemical reactions such as DNA, RNA, glycosidic bond cleavage (Burmeister et al., 1997; Carmi et al., 1998; Li et al., 2000a,b; Santoro and Joyce, 1997), porphyrin metalation (Li and Sen, 1996), and DNA self-modification (Li and Breaker, 1999; Li et al., 2000a,b; Sheppard et al., 2000). One kind of DNAzymes formed by hemin and a G-quadruplex aptamer (i.e., G-quadruplex-based DNAzyme) can catalyze H2 O2 -mediated oxidation of 2,2 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), luminol or H2 DCFDA (Li et al., 2007a,b; Qiu et al., 2011; Travascio et al., 1998, 1999, 2001), accompanied by a color change, chemiluminescent or fluorescent emission. Among them, H2 DCFDA is a commercially available fluorone dye which is non-fluorescent itself and can be oxidized by DNAzymes into fluorescent dichlorofluorescein (with excitation and emission wavelengths centered at = 501 and 520 nm, respectively). It has been reported that H2 DCFDA is a superior alternative to ABTS in bioanalytical detection (Nakayama and Sintim, 2010).
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
101
Fig. 1. (A) Schematic illustration of the amplified detection of DNA using label-free G-quadruplex DNAzyme-based molecular beacon. At first, P1 (G-DNAzymeMB1) and B1 (blocker DNA1) formed the catalytically inactive P1/B1 hybrid. T1 (target DNA) hybridized with B1 and released the catalytically active P1 for the generation of fluorescent signals (step (1)); Exonuclease III (Exo III) catalyzed the stepwise removal of mononucleotides from the blunt 3 terminus of B1 (step (2)), liberating T1 (step (3)); the released T1 triggered amplified cycle by hybridizing with another inactive P1/B1 hybrid (step (4)). (B) Schematic illustration of the assay of the restriction endonuclease. P2 represents G-DNAzymeMB2, and B2 represents blocker DNA2. (C) DNA sequences used in the present work. The G-quadruplex portions were shown in red and the recognition sequences of EcoRI were shown in green.For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.
In this study, we construct a label-free, G-quadruplex DNAzyme molecular beacon (G-DNAzymeMB)-based strategy for fluorescent detection of DNA and restriction endonuclease. The fluorescence of H2 DCFDA catalyzed by G-DNAzymeMB is used as a signal reporter. In the “off” state, the central loop portion of G-DNAzymeMB is hybridized with a blocker DNA and formation of G-quadruplex is inhibited. Consequently, catalytic activity of DNAzyme is prevented, resulting in low fluorescent signal. Upon addition of target DNA or enzyme cleavage, two lateral portions of G-DNAzymeMB form a G-quadruplex structure. Then in the presence of hemin, they can catalyze oxidation of H2 DCFDA and give high fluorescent signal. To improve the sensitivity of DNA detection using GDNAzymeMB, exonuclease III (Exo III)-catalyzed amplification strategy is introduced. Exonuclease III is sequence-independent and can catalyze the stepwise removal of mononucleotides from 3 -hydroxyl termini of duplex DNA. Its activity on single-stranded DNA is limited and preferred for blunt or recessed 3 termini (Zuo et al., 2010). Thanks to the amplification of Exo III, 0.1 pM target DNA could be distinctly detected. 2. Materials and methods 2.1. Chemicals and materials All oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and their sequences were listed
in Table S1. The oligonucleotides were determined using the 260 nm UV absorbance and the corresponding extinction coefficient. H2 DCFDA was purchased from Sigma (Missouri, USA). Exo III (15 × 104 U/mL), EcoRI and Hind III (both 15 × 103 U/mL) were purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Hemin was purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). The stock solution of hemin (1 mM) was prepared in dimethyl sulfoxide (DMSO), stored in darkness at −20 ◦ C. Tris(hydroxymethyl)aminomethane (Tris) was bought from Shanghai Chemical Reagent Company (Shanghai, China). Other chemicals were of analytical grade and used without further purification. Solutions were prepared with deionized water processed with a Milli-Q ultra-high purity water system (Millipore, Bedford, MA, USA). 2.2. Assay procedures for DNA and endonuclease detection In a typical DNA assay, P1 (G-DNAzymeMB1, 0.5 M) and B1 (blocker DNA1, 0.5 M) were dissolved in Tris–buffer 1 (25 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl2 ). The solution was then heated at 90 ◦ C for 10 min and gradually cooled to room temperature to ensure that nucleic acids were hybridized to each other completely. Afterwards, 2 L T1 (target DNA), 2 L Exo III (20 units) and 4 L 10× Exonuclease III buffer (50 mM Tris–HCl, 5 mM MgCl2 , 1 mM DTT, pH 8.0) were added in succession. Cleavage reaction of Exo III was conducted at 37 ◦ C for 40 min. Then hemin (0.1 M) in Tris–buffer 2 (50 mM Tris–HCl, 20 mM KCl, 150 mM NH4 Ac) was
102
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
added and incubated at room temperature for 30 min to form the hemin–G-DNAzyme. For endonuclease assay, a total volume of 360 L reaction solution containing substrate P2 (G-DNAzymeMB2)/B2 (blocker DNA2) (or P3 (G-DNAzymeMB3)/B3 (blocker DNA3)) (each 0.4 M) and various amounts of EcoRI (or Hind III) were incubated at 30 ◦ C for 1.5 h, and then hemin (0.08 M) was added. 2.3. Fluorescent analysis For the fluorescent measurements, H2 DCFDA (20 M) and H2 O2 (220 M) (H2 DCFDA and H2 O2 must be freshly prepared before use) were added to the product solution with a final volume of 400 L. Fluorescence emission spectra were collected from 510 to 600 nm by using a Fluoromax-4 Spectrofluorometer (HORIBA Jobin Yvon, Inc., NJ, USA) at room temperature. The excitation wavelength was set at 501 nm. For the sensor calibration curve acquisition, F was plotted as sensor signal, where F is the fluorescence intensity of the sensor solution with addition of different concentrations of T1 or restriction endonuclease. All the measurements were performed three times, and the standard deviation was plotted as the error bar. 3. Results and discussion 3.1. Target DNA detection principle The design of the label-free fluorescent sensor for DNA was shown in Fig. 1A. The G-quadruplex oligomer was split into two halves connected with a central loop portion (G-DNAzymeMB: d [G3 T2 G3 C-loop-TG3 TAG3 ]). The formation of G-quadruplex was validated by circular dichroism (CD) spectra of G-DNAzymeMB (P1) in buffer solutions (see Fig. S1 in the Supporting Information). At first, the central loop portion of P1 is hybridized with B1 (blocker DNA) to form a stable duplex (17 bases match). As a result, the two lateral portions of P1 cannot associate with each other to form quadruplex structure and the catalytic activity of P1 is inhibited. Under these conditions, H2 DCFDA cannot be effectively oxidized and only weak fluorescence can be observed. Addition of target DNA (T1), however, can displace P1 from P1/B1 duplex through branch migration since there exist more base pairs (31 bases match) between B1/T1. As a result, the released P1 self-assembles into Gquadruplex structure, and with hemin it can catalyze oxidation of H2 DCFDA, generating a strong fluorescence signal (Fig. 1A step (1)). The hybridization of T1 and B1 strands form a double-stranded structure that contains a blunt 3 terminus and an Exo III-resistant 3 protruding terminus. When Exo III is added, it catalyzes the stepwise removal of mononucleotides from the blunt 3 terminus of
Fig. 2. Fluorescence spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in solutions (a) P1/B1 (each 0.5 M) in the absence of target DNA, (b) P1/B1 (each 0.5 M) in the presence of target DNA (0.1 M), (c) P1/B1 (each 0.5 M) in the presence of Exo III (20U), (d) P1/B1 (each 0.5 M) in the presence of Exo III (20U) and target DNA (0.1 M). Experimental conditions were the optimized conditions shown in Section 2.
B1 (Fig. 1A step (2)), liberating T1 (Fig. 1A step (3)). The released T1 then reacts with another P1/B1 hybrid to initiate a new cycle (Fig. 1A step (4)). Eventually, a single copy of T1 generates many active P1 and thus achieves target assisted enzyme-catalyzed signal amplification. Fluorescence measurements were carried out to serve as a proof of concept to test the principle of our design. As shown in Fig. 2, the fluorescence is very weak in the P1/B1 hybrid system (curve a) and after addition of 50 nM T1 there is a 2-fold increase (curve b). Addition of Exo III, the background fluorescence increased slightly (curve c); however, a significant increase (10-fold) in fluorescence intensity is observed upon addition of 50 nM T1 to the Exo IIIcatalyzed amplification system (curve d). These results indicate that Exo III-catalyzed amplified target DNA detection has been successfully achieved. 3.2. Detection of T1 without Exo III-catalyzed signal amplification Detection of various concentrations of T1 (0–0.5 M) based on P1/B1 hybrid system was first conducted and results were shown in Fig. 3. The significant increase in fluorescence clearly indicates that T1 could hybridize with B1 and replace P1 from P1/B1 duplex (Fig. 3A). It is observed that 5 nM T1 could be significantly detected and a linear correlation (R2 = 0.999) exists between the value of fluorescence and the concentration of T1 over the range of 0–0.5 M (Fig. 3B).
Fig. 3. (A) Fluorescent spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the presence of various concentrations of target DNA. (B) Linear relation between the fluorescent intensity at 520 nm and concentrations of target DNA (0–0.5 M). Experimental conditions were the optimized conditions shown in Section 2.
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
103
Fig. 4. (A) Fluorescent spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the presence of various concentrations of target DNA with Exo III-catalyzed amplification system. (B) Plot of the values of fluorescent intensity at 520 nm with respect to the logarithm of the concentrations of T1 (1 pM–5 nM). Experimental conditions were the optimized conditions shown in Section 2.
3.3. Detection of T1 with Exo III-catalyzed signal amplification For Exo III-amplified P1/B1 hybrid system, effect of different amounts of Exo III on the signal amplification was investigated. Fluorescence at 520 nm was recorded in the presence of different amounts of Exo III from 0 to 30 U (see Fig. S2A in the Supporting Information). As can be seen, the amplified effect increases sharply upon increasing the amount of Exo III and is saturated at ≥20 U. Thus, 20 U of Exo III is used in all further experiments. Besides, the Exo III-amplified reaction is rapid and the assay exhibits a nearly saturated signal within 40 min in the presence of 20 U of enzyme (see Fig. S2B in the Supporting Information). With respect to sensitivity and speed, the Exo III reaction time of 40 min was chosen for subsequent study. Under optimized conditions, T1 detection using the Exo III-amplified P1/B1 system was conducted via monitoring the fluorescent change of H2 DCFDA catalyzed by P1/hemin. Fig. 4A showed the fluorescent spectra with various concentrations of T1 from 0 to 100 nM, and apparent fluorescence increases were observed. The fluorescent intensity was linear with the logarithm of the concentration of T1 over the range of 1 pM–5 nM and 0.1 pM T1 could be detected obviously (Fig. 4B). One of the important advantages that use MBs for DNA detection is that they show high selectivity. To test the selectivity of the present system for analysis of target DNA, similar DNA strands with only single (T2), two (T3) and four (T4) nucleotides mismatch were also investigated. As shown in Fig. 5, the fluorescence enhancement value by T2 (curve d) was 20% of that by T1 (curve e), and
Fig. 5. Selectivity of the G-DNAzymeMB in the absence (curve a) and presence of target DNA (T1 50 nM, curve e) over other mismatched DNA (50 nM): T2 (singlebase mismatched target, curve d), T3 (two-base mismatched target, curve c) and T4 (four-base mismatched target, curve b).
there was almost no enhancement for T4 (curve b). These results demonstrated the well selectivity of the proposed approach and even one-nucleotide mismatch could be distinguished. 3.4. Fluorescent detection of restriction endonuclease To address the general applicability of this G-DNAzymeMB in bioanalysis, detection of Type II restriction endonuclease was also explored. Type II restriction endonucleases, among the most important enzymes in molecular biology, recognize short, usually palindromic, sequences of 4–8 bp and cleave DNA within or in close proximity to the recognition sequence (Pingoud and Jeltsch, 2001). They play an important role to protect host genome against foreign DNA and thus assays of these enzymes activities are crucial in many fields ranging from biotechnology to clinical diagnostics and drug discovery. Using EcoRI restriction endonuclease as a model system (Fig. 1B), P2 (G-DNAzymeMB2, 36 mer) was partly hybridized with B2 (blocker DNA2, 18 mer) which contains recognition sequence (5 GAATTC 3 ) located in the intermediate position to form P2/B2 duplex (18 bases matched) that acted as substrate for EcoRI. In the absence of EcoRI, P2 and B2 formed a stable duplex (Tm = 51 ◦ C, calculated according to DNAMelt Server) (Markham and Zuker, 2005, 2008) and the two lateral portions of P2 could not associate with each other to form quadruplex structure. Therefore, the catalytic activity of P2 was inhibited and weak fluorescence was obtained. Upon addition of EcoRI, cleavage reaction was initiated and P2/B2 duplex was cleaved into two parts. Since the newly formed hybrids contained only few base pairs (7 bases matched), they would be separated into short single DNA fragments at 30 ◦ C (Tm values are 12.2 and 13.6 ◦ C, respectively). The separated two lateral portions of P2 were allowed to form quadruplex structure, then the formed G-DNAzyme catalyze the oxidation of H2 DCFDA, resulting in strong fluorescence. In order to validate our approach, firstly, a series of control experiments were performed to ensure that the observed fluorescence enhancement was induced by the presence of EcoRI (Fig. 6A). The fluorescent intensity increased greatly in the presence of EcoRI (Fig. 6A, curve d). However, the emission response was significantly diminished when EcoRI was heat-inactivated before incubation with oligonucleotides (Fig. 6A, curve b). In addition, no significant increase in fluorescence signal was observed upon addition of EcoRI in the absence of the recognition site for EcoRI in oligonucleotides (Fig. 6A, curve c). Then, fluorescence measurements were conducted with various concentrations of EcoRI (0–10 U/mL). Interestingly, the fluorescence intensity increased as increasing the concentration of EcoRI (Fig. 6B). As shown in Fig. 6C, plots of the fluorescent intensity versus nuclease concentration (0.1–5 U/mL) showed good linear relationship (R2 = 0.988).
104
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105
Fig. 6. (A) Fluorescence response of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the absence of EcoRI: (a) in the absence of EcoRI; (b) incubated with heat-inactivated EcoRI; (c) incubated with normal EcoRI but lack of EcoRI substrate; (d) incubated with normal EcoRI in the presence of EcoRI substrate. (B) Fluorescence spectra of G-DNAzymecatalyzed oxidation of H2 DCFDA in the presence of P2/B2 (EcoRI substrate, 0.4 M) with various concentrations of EcoRI. (C) Linear relation between the fluorescence intensities at 520 nm and EcoRI concentrations (0.1–5 U/mL). (D) Fluorescence emission spectra of G-DNAzyme-catalyzed oxidation of H2 DCFDA in the presence of P3/B3 (Hind III substrate, 0.4 M) with various concentrations of Hind III (0–5 U/mL).
Therefore, the nuclease activities could be quantitatively analyzed by using the plot of fluorescent intensity versus enzyme concentrations in the present G-DNAzymeMB-based enzyme detection system. Compared with traditional enzyme detection systems such as enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) and fluorescence resonance energy transfer (FRET) (Hiramatsu et al., 1998; Jeltsch et al., 1993; Li et al., 2007a,b; McLaughlin et al., 1987; Spitzer and Eckstein, 1988), our method is simple, time-saving and there is no need for isotope or DNA labeling. To address the general approach for restriction endonucleases, detection of Hind III restriction endonuclease was also studied. The specific substrates P3 (G-DNAzymeMB3)/B3 (blocker DNA 3) for Hind III were obtained by simply replacing the recognition sequence with 5 -AAGCTT-3 within the central loop portion. Upon addition of Hind III (0–5 U/mL), similar fluorescent enhancement was observed (Fig. 6D). These results demonstrate its potential to be a general method for the detection of restriction endonucleases.
endonuclease by simply replacing the recognition portions. Using EcoRI as a model analyte, 0.1 U/mL EcoRI could be detected. The new approach is expected to promote the exploitation of GDNAzymeMB-based biosensors for target assays in biochemical and biomedical studies.
4. Conclusions
Bonnet, G., Krichevsky, O., Libchaber, A., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (15), 8602–8606. Broude, N.E., 2002. Trends Biotechnol. 20 (6), 249–256. Burmeister, J., vonKiedrowski, G., Ellington, A.D., 1997. Angew. Chem. Int. Ed. 36 (12), 1321–1324. Carmi, N., Balkhi, S.R., Breaker, R.R., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (5), 2233–2237. Fang, X.H., Liu, X.J., Schuster, S., Tan, W.H., 1999. J. Am. Chem. Soc. 121 (12), 2921–2922. Hiramatsu, K., Miura, H., Kamei, S., Iwasaki, K., Kawakita, M., 1998. J. Biochem. 124 (1), 231–236. Jeltsch, A., Fritz, A., Alves, J., Wolfes, H., Pingoud, A., 1993. Anal. Biochem. 213 (2), 234–240. Li, D., Shlyahovsky, B., Elbaz, J., Willner, I., 2007a. J. Am. Chem. Soc. 129 (18), 5804–5805. Li, J., Zheng, W.C., Kwon, A.H., Lu, Y., 2000a. Nucleic Acids Res. 28 (2), 481–488. Li, J., Yan, H.F., Wang, K.M., Tan, W.H., Zhou, X.W., 2007b. Anal. Chem. 79 (3), 1050–1056.
In conclusion, a simple, sensitive and label-free G-quadruplex DNAzyme molecular beacon-based biosensor has been constructed for monitoring target DNA and restriction endonuclease, using fluorescent emission of H2 DCFDA catalyzed by G-quadruplex DNAzyme molecular beacon as readout signal. The present G-DNAzymeMB method is a turn-on model and does not require any chemical modification of DNA, which shows advantages of simplicity in operation, cost and time efficiency, and sensitivity. For target DNA detection, Exo III was introduced to initiate the amplified strategy and 0.1 pM target DNA could be detected, which is 4-fold than the unamplified strategy. More importantly, this G-DNAzymeMB was generally applicable and could be easily adaptable to assay of restriction
Acknowledgments This work was supported by the National Natural Science Foundation of China (nos. 20935003, 21075116), and 973 project (nos. 2011CB911002, 2010CB933603). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2012.01.024. References
Z. Zhou et al. / Biosensors and Bioelectronics 34 (2012) 100–105 Li, Y.F., Sen, D., 1996. Nat. Struct. Biol. 3 (9), 743–747. Li, Y.F., Breaker, R.R., 1999. Proc. Natl. Acad. Sci. U.S.A. 96 (6), 2746–2751. Li, Y.F., Liu, Y., Breaker, R.R., 2000b. Biochemistry 39 (11), 3106–3114. Markham, N.R., Zuker, M., 2005. Nucleic Acids Res. 33, W577–W581. Markham, N.R., Zuker, M., 2008. In: Keith, J.M. (Ed.), Bioinformatics, Volume II. Structure, Function and Applications, number 453 in Methods in Molecular Biology, chapter 1. Humana Press, Totowa, pp. 3–31, NJ. ISBN 978-1-60327-428-9. McLaughlin, L.W., Benseler, F., Graeser, E., Piel, N., Scholtissek, S., 1987. Biochemistry 26 (23), 7238–7245. Nakayama, S., Sintim, H.O., 2010. Mol. Biosyst. 6 (1), 95–97. Pingoud, A., Jeltsch, A., 2001. Nucleic Acids Res. 29 (18), 3705–3727. Qiu, B., Zheng, Z.Z., Lu, Y.J., Lin, Z.Y., Wong, K.Y., Chen, G.N., 2011. Chem. Commun. 47 (5), 1437–1439. Santoro, S.W., Joyce, G.F., 1997. Proc. Natl. Acad. Sci. U.S.A. 94 (9), 4262–4266.
105
Sheppard, T.L., Ordoukhanian, P., Joyce, G.F., 2000. Proc. Natl. Acad. Sci. U.S.A. 97 (14), 7802–7807. Sokol, D.L., Zhang, X.L., Lu, P.Z., Gewitz, A.M., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (20), 11538–11543. Spitzer, S., Eckstein, F., 1988. Nucleic Acids Res. 16 (24), 11691–11704. Tan, W.H., Wang, K.M., Drake, T.J., 2004. Curr. Opin. Chem. Biol. 8 (5), 547–553. Travascio, P., Li, Y.F., Sen, D., 1998. Chem. Biol. 5 (9), 505–517. Travascio, P., Bennet, A.J., Wang, D.Y., Sen, D., 1999. Chem. Biol. 6 (11), 779–787. Travascio, P., Witting, P.K., Mauk, A.G., Sen, D., 2001. J. Am. Chem. Soc. 123 (7), 1337–1348. Tyagi, S., Kramer, F.R., 1996. Nat. Biotechnol. 14 (3), 303–308. Yeh, H.C., Sharma, J., Han, J.J., Martinez, J.S., Werner, J.H., 2010. Nano Lett. 10 (8), 3106–3110. Zuo, X.L., Xia, F., Xiao, Y., Plaxco, K.W., 2010. J. Am. Chem. Soc. 132 (6), 1816–1817.