G-quadruplex-Based Fluorescent Assay of S1 Nuclease Activity and

Jan 14, 2013 - G-quadruplex-Based Fluorescent Assay of S1 Nuclease Activity and K+. Zhixue Zhou, Jinbo Zhu, Libing Zhang, Yan Du, Shaojun Dong*, and ...
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G‑quadruplex-Based Fluorescent Assay of S1 Nuclease Activity and K+ Zhixue Zhou, Jinbo Zhu, Libing Zhang, Yan Du, Shaojun Dong,* and Erkang Wang* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China S Supporting Information *

ABSTRACT: Endonuclease plays an important role in many biological processes, and an assay of endonuclease activity is of great significance. However, traditional methods for the assay of endonuclease activity have undesirable limitations, such as high cost, DNA-consuming and laboriousness. In the present work, a G-quadruplex-based, fluorescent assay of endonuclease activity has been developed with protoporphyrin IX (PPIX) as a signal reporter. S1 nuclease, a single strand DNA (ssDNA)-specific endonuclease, is employed as model system. In the “on” state, G-quadruplex DNA can greatly enhance the fluorescence of PPIX. However, if S1 nuclease could cleave G-quadruplex DNA into small fragments, there would be no formation of G-quadruplexes, accompanied by low emission response of PPIX. This fluorescent discrimination before or after digestion by nuclease can be used to monitor the activity of S1 nuclease. This assay is simple in design and offers a convenient protocol for homogeneous, rapid and high-throughput detection. In addition, the proposed strategy avoids complicated covalent modifications or chemical labeling, and thus offers advantages of simplicity and cost efficiency. More importantly, K+ is found to well inhibit the activity of S1 nuclease when using certain G-quadruplex DNA as substrate, and thus this system is further used for turn-on detection of K+. S1 nuclease is critical in the detection of K+ since it helps to reduce the background signal.

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are sensitive to the salt concentration in buffer solutions used for enzymatic reaction, which would limit the application of AuNPs in nuclease assay. It is still desirable to develop efficient, facile, label-free and amenable to high-throughput screening strategies to assay nuclease activity, which would greatly facilitate the identification of nuclease modulators for potential therapeutic applications. Recently, the use of G-quadruplexes as biological sensing elements has received intense research interest. G-rich DNAs are well-known for their conformational polymorphism, and the adopted structures are dependent on several factors, such as base sequence, loop connectivities, or cations present. These aspects endow G-quadruplexes to be utilized for the colorimetric, chemiluminescence, or fluorescent detection of a series of analytes, including metal ions, small molecules, DNA, and proteins.21−27 Herein, we describe a label-free Gquadruplex-based fluorescent assay of endonuclease activity, which is not only sensitive and reliable, but also simple and economical in operation. PPIX is chosen as a signal reporter as its fluorescence can be enhanced greatly via binding to Gquadruplexes. This fluorescent property of PPIX enables it to indicate structural changes of G-quadruplex DNA (such as DNA cleavage in this paper) through modulating fluorescence signal readout by its binding or release. S1 nuclease is chosen as

ndonucleases are a family of nucleases that can hydrolyze the phosphodiester bonds in DNA or RNA. They are involved in many important biological processes, such as DNA replication, recombination, molecular cloning, genotyping, and mapping.1−5 Molecules that inhibit endonucleases activities are therefore considered as candidates for series of drugs.6 Recently, endonucleases are widely used in the fields of nanostructure synthesis and amplified detection of various targets.7−11 On the basis of these intrinsic biological significance, assays of endonuclease activity and inhibitors are of great importance in the fields of clinical diagnostics, drug discovery, biosensing, and nanoscience. Traditional methods for assessing endonuclease activity, have included high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), filter binding, enzyme-linked immunosorbent assay (ELISA), etc.12−15 However, these conventional protocols are time-intensive, DNA-consuming, laborious, and usually require isotope labeling which have limited shelf life. To address these challenges, several other techniques for the assay of nuclease activity have been developed, such as fluorescence-based methods, electrochemical approaches, and gold nanoparticles (AuNPs)-based colorimetric ways.16−20 Although these new approaches partly overcome the limitations of the conventional assays and show promising applications, there still exist undesirable features. For example, some approaches require extortionate chemical label (thiol or dye-modified DNA probe), which makes the assay complex, expensive, and time-consuming. In addition, AuNPs © 2013 American Chemical Society

Received: November 27, 2012 Accepted: January 14, 2013 Published: January 14, 2013 2431

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Sensor Preparation. The sensor solutions were prepared by dissolving G-rich DNA PS2.M (0.4 μM) in 1 × S1 Nuclease Buffer, and then S1 nuclease of various concentrations (0.04− 20 U mL−1) was added and incubated for 0.5 h. Finally, PPIX (0.4 μM) in T-Buffer (10 mM Tris-HNO3, pH 7.0, 100 mM KNO3) was mixed with the prepared solutions and allowed to react for another 0.5 h. The fluorescence of the mixture was then measured at room temperature. As for the detection of K+, K+ of different concentrations (5 × 10−3−15 mM) was first added into the solutions of G-rich DNA PW17 (0.4 μM), and then 20 U mL−1 S1 nuclease was added. Other steps were the same as the assay of S1 nuclease described above. For S1 nuclease sensor calibration curve acquisition, F/F0 was plotted as sensor signal, where F0 is the fluorescence intensity of the sensor solution in the absence of S1 nuclease; F is the fluorescence intensity of the sensor solution with addition of different concentrations of S1 nuclease. As for the assay of K+, F/F0 was plotted as sensor signal, where F0 is the fluorescence intensity of the sensor solution with addition of 0 mM K+ and 20 U mL−1 S1 nuclease; F is the fluorescence intensity of the sensor solution with addition of different concentrations of K+ (5 × 10−3−15 mM) and 20 U mL−1 S1 nuclease. All the measurements were performed three times, and the standard deviation was plotted as the error bar. Fluorescence Spectroscopic Analysis. The fluorescence emission spectra of G-quadruplex DNA/PPIX complex were collected from 570 to 750 nm using a Fluoromax-4 Spectrofluorometer (HORIBA Jobin Yvon, Inc., NJ, USA) at room temperature. The excitation wavelength was set at 410 nm.

a model system to demonstrate the proof-of-concept of our approach. S1 nuclease is a single strand DNA (ssDNA)-specific endonuclease, and can catalyze ssDNA cleavage reactions to yield small mono- or oligonucleotide fragments.28−30 In particular, S1 nuclease can bind with an exposed single strand, such as loops in G-quadruplex structure, i-motif DNA or duplex DNA, and attack on the O-3′-P bond.31−34 In the present work, S1 nuclease is found to destroy some Na+-stabilized Gquadruplexes, and the fluorescent response of PPIX is dependent on the concentration of S1 nuclease. Inspired by these phenomena, we expect that the combination of signalresponsive G-quadruplexes/PPIX complexes and cleavage reactions could offer a unique opportunity to construct robust sensor for S1 nuclease. Moreover, K+ is found to well inhibit the activity of S1 nuclease with certain G-quadruplex DNA as substrate,35 while Na+ can not. This can be utilized to achieve a K+ sensor, in which the presence of S1 nuclease can help to improve the selectivity toward Na+ and reduce the background signal, thereby increasing sensitivity. Significantly, such system does not reqiure sophisticated design or chemical labeling, which reduces complexity, cost and overall assay time. Meanwhile, this strategy might pave the way to apply Gquadruplexes as novel transducers for more sensing systems.

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EXPERIMENTAL SECTION Chemicals and Materials. Oligonucleotides with specific sequences (Scheme 1) were synthesized by Shanghai Sangon Scheme 1. (A, B) Schematic Representation of the Sensing Procedures for the Analysis of S1 Nuclease and K+ and (C) DNA Sequences Used in the Study

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RESULTS AND DISCUSSION Sensor Design. The design of the label-free fluorescent assay of S1 nuclease activity is shown in Scheme 1A. The fluorescence of PPIX can be enhanced greatly upon binding with G-quadruplex DNA. In the presence of S1 nuclease, however, G-quadruplex DNA can be hydrolyzed by S1 nuclease, resulting in no formation of G-quadruplexes and low fluorescent response of PPIX (Scheme 1A). Therefore, the cleavage of G-quadruplex DNA can be monitored through observing the emission changes of PPIX. Furthermore, K+ is known to stabilize the G-quadruplex structure,36 and thus can well inhibit the activity of S1 nuclease.35 As illustrated in Scheme 1B, after enzymatic digestion, small fragments are generated and the fluorescent response of PPIX is low. Whereas, if the cleavage process is conducted in the presence of K+, G-quadruplex DNA would remain intact due to the reduced activity of S1 nuclease, and the fluorescence of PPIX can be enhanced greatly. This K+-induced inhibition of S1 nuclease activity can be utilized to develop a sensitive and selective sensor for K+. Assay of S1 Nuclease Activity. To improve the sensitivity of the nuclease assay, three G-quadruplex DNAs (i.e., PS2.M, PW17 and T30695) are tested to optimize the digestion substrate. These three G-rich DNAs can form Na+-stabilized Gquadruplex structure in 1 × S1 Nuclease Buffer (30 mM NaAc, pH 4.6, 280 mM NaCl, 1 mM ZnSO4), which can be validated by their circular dichroism (CD) spectra in Figure S1. As shown in Figure 1, the fluorescence of PPIX itself is low (Figure 1, curve a), and these three DNAs all can greatly enhance the fluorescence of PPIX (Figure 1, curves b−d). However, after enzymatic digestion by 10 U mL−1 S1 nuclease, only the T30695/PPIX system remains its high fluorescence (Figure 1,

Biotechnology Co. Ltd. (Shanghai, China). The concentrations of oligonucleotides were determined using the 260 nm UV absorbance with the corresponding extinction coefficient. S1 nuclease (1.6 × 105 U mL−1) and 10 × S1 Nuclease Buffer (300 mM NaAc, pH 4.6, 2800 mM NaCl, 10 mM ZnSO4) were purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Protoporphyrin IX (PPIX) and tris (hydroxymethyl) aminomethane (Tris) were obtained from Alfa Aster. All other chemicals were of analytical grade and used without further treatment. All stock and buffer solutions were prepared using ultrapure water (>18 MΩ) from a Milli-Q Plus system (Millipore). 2432

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system. As the concentration of S1 nuclease increases from 0.04 to 20 U mL−1, the fluorescent intensity is continually decreased. (Figure 2A) Since PPIX is specific for the G-quadruplexes formed by PS2.M, the fluorescent decrease indicates a gradual cleavage of PS2.M. Figure 2B outlines the relationship between the relative fluorescence intensity and S1 nuclease concentration, indicating a saturation point near 10 U mL−1 S1 nuclease. The inset in Figure 2B shows the linear relationship (R2 = 0.959) in the concentration range from 0.04 to 0.4 U mL−1. Besides, it can be observed that 0.04 U mL−1 S1 nuclease can be sensitively detected (Figure 2B), which is better than the previous reported data obtained by gel electrophoresis, chromatography, and Au NPs-based assay.37 In general, increasing the amount of enzyme or extending the reaction time can improve the digestion efficiency. Therefore, we expect that more sensitive detection could be achieved by increasing the concentration of substrate or prolonging the cleavage time. Performance of the K+ Sensor. It is well-known that K+ plays an important role in living organisms, such as reducing the risk of high blood pressure and stoke, maintaining muscular strength, balancing the pH, etc.38 Therefore, a variety of studies that focus on the assay of K+ have been reported.38−45 Despite the notable progress, it is still difficult to selectively determine K+ due to the excess of Na+ and other cations present in the physiological conditions. Another challenge for the detection of K+ is the unsatisfactory sensitivity. With these thoughts in mind, we are committed to build a label-free and turn-on sensor for the detection of K+ by combining the cleavage reaction and G-quadruplexes. As shown in Figure 3, in the presence of 20 U mL−1 S1 nuclease, the fluorescence of PS2.M/PPIX and PW17/PPIX systems is unobservable (Figure 3A, curves a and b). However,

Figure 1. Fluorescence emission spectra of various solutions: (a) PPIX, (b) PW17 + PPIX, (c) PS2.M + PPIX, (d) T30695 + PPIX, (e) T30695 + S1 nuclease + PPIX, (f) PS2.M + S1 nuclease + PPIX, and (g) PW17 + S1 nuclease + PPIX.

curve e). The fluorescence of PS2.M/PPIX and PW17/PPIX systems demonstrates significantly decrease after the cleavage process (Figure 1, curves f and g). Particularly, PS2.M can even be hydrolyzed completely within 15 min, and almost no fluorescent response of PPIX can be observed (Figure 1, curve f). Based on the fact that PS2.M is more easily cleaved by S1 nuclease, PS2.M is thus used as the digestion substrate to construct the label-free assay of S1 nuclease activity. It should be pointed out that PPIX is involved after the cleavage process so as to avoid the interference from PPIX toward the activity of S1 nuclease. To validate that the observed fluorescence decrease is due to the degradation of G-quadruplex DNA caused by S1 nuclease, a number of control experiments are further conducted. Since S1 nuclease is a Zn2+-dependent glycoprotein and requires to be activated by low concentrations of Zn2+, cleavage of PS2.M in buffer conditions without Zn2+ is also performed. As shown in Supporting Information Figure S2, the fluorescence of PPIX still can be increased upon binding with G-quadruplexes as S1 nuclease does not work effectively under this condition (Supporting Information Figure S2, curve a). If S1 nuclease is first heated at 90 °C for 10 min to undergo an irreversible inactivation, we find that the activity of this heat-treated S1 nuclease is reduced greatly, accompanied by high fluorescence response of PPIX (Supporting Information Figure S2, curve b). These results confirm that the low fluorescent response of PPIX mentioned above really comes from the degradation of PS2.M caused by S1 nuclease. Figure 2A shows the change of fluorescence spectra upon adding different concentrations of S1 nuclease into the PS2.M

Figure 3. (A) Fluorescent spectra of solutions: (a) PS2.M + S1 nuclease + PPIX, (b) PW17 + S1 nuclease + PPIX, (c) PS2.M + K+ (10 mM) + S1 nuclease + PPIX, and (d) PW17 + K+ (10 mM) + S1 nuclease + PPIX. (B) Fluorescence spectra of the PW17/PPIX system in the presence of 20 U mL−1 S1 nuclease and various concentrations of K+. (C) The saturated fluorescence of PW17/PPIX system with various concentrations of K+ in the presence of 20 U mL−1 S1 nuclease, with a linear range of 0−0.2 mM (the inset). (D) Selectivity of PW17/PPIX system toward K+ in the presence of 20 U mL−1 S1 nuclease. The concentration of K+ was 5 mM; the concentration of Na+ was 150 mM; and the concentration of the other control metal ions was 10 mM. Error bars represent standard deviations from three repeated experiments.

Figure 2. (A) Fluorescence spectra of the PS2.M/PPIX system for analyzing different concentrations of S1 nuclease. (B) The saturated fluorescence of PS2.M/PPIX system with various concentrations of S1 nuclease, with a linear range of 0.04−0.4 U mL−1 (the inset). 2433

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can be observed (Supporting Information Figure S6B). These results indicate that the present strategy is easily amenable to a high-throughput format.

if the degradation reaction is conducted in the presence of 10 mM K+, the fluorescence of PS2.M/PPIX and PW17/PPIX systems with much stronger intensity is detected (Figure 3A, curves c and d). K+ can stabilize the G-quadruplex structure, and accordingly reduces the activity of S1 nuclease toward Gquadruplex DNA. The thermal stabilities (Tm) are measured as 54 and 59 °C for Na+-stabilized PS2.M and PW17. (Supporting Information Figure S3, curves a and b) Addition of K+ causes the Tm value of PS2.M increasing from 54 to 66 °C (Figure S3, curve c), and for PW17, its Tm value is even higher than 80 °C (Supporting Information Figure S3, curve d). Native PAGE in Supporting Information Figure S4 is used to verify K+-induced inhibition of S1 nuclease. In the presence of K+, the bands representing PW17 (Supporting Information Figure S4, lane 1) still exist for S1 nuclease-treated PW17 (Supporting Information Figure S4, lane 2), confirming the reduced activity of S1 nuclease toward PW17. However, in the absence of K+, no obvious band is observed after PW17 is treated with S1 nuclease (Supporting Information Figure S4, lane 4), indicating the hydrolysis of PW17.46 Because of the PW17 system showing more obvious response (Figure 3A, curve d), it is used to construct a sensitive sensor for K+. As shown in Figure 3B, the emission response of PPIX is low after the enzymatic digestion by 20 U mL−1 S1 nuclease. (Figure 3B, curve a) As the concentration of K+ increased from 5 × 10−3 to 15 mM, the activity of S1 nuclease is gradually reduced, and the fluorescent intensity is continually enhanced with a saturation at 10 mM K+ (Figure 3B, curves b−j). The inset in Figure 3C shows a good linear relationship (R2 = 0.985) in the concentration range from 0 to 200 μM. It is observed that 5 μM K+ can be distinctly detected. Here the presence of S1 nuclease provides a zero-background signal to improve overall sensitivity. For example, by means of S1 nuclease, 2.5 mM K+ can induce 11.6-fold fluorescent increase (Figure S5, curves a and b), but only 0.25-fold increase for the system without S1 nuclease (Figure S5, curves c and d). To test the specificity of this K+ sensor, the possible interference of some common metal ions (e.g., Na+, Ca2+, Mg2+, and heavy metals) is investigated. Figure 3D confirms a good selectivity of the proposed sensor for K+ over other tested ions. Especially, 310 mM Na+ is included in the enzymatic buffer solutions and further addition of 150 mM Na+ still show no interference effect (Figure 3D), which demonstrates another advantage of the present sensor. It is worth emphasizing that Na+-stabilized G-quadruplex structure would be cleaved by S1 nuclease, so S1 nuclease also plays important role in the aspect of selectivity. Compared with conventional K+-selective electrode, the present strategy with good selectivity is timesaving, sensitive and easily adaptable to high-throughput screening.47,48 Assay of S1 Nuclease and K+ in a High-Throughput Format. Currently, high-throughput microplate-based methods have gained much attention due to their unique advantages.18,49 For example, a large number of samples can be processed simultaneously on a microplate, which is absolutely efficient and labor-saving. In order to demonstrate the feasibility of highthroughput assay, simultaneous detection of various concentrations of S1 nuclease and K+ is performed in a 96-well microplate (Supporting Information Figure S6). As expected, the sample without S1 nuclease shows high fluorescence, and the fluorescence intensities decrease as the increased concentration of S1 nuclease (Supporting Information Figure S6A). While in the presence of K+, high fluorescence intensity

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CONCLUSION In conclusion, we have demonstrated a unique G-quadruplexbased platform for sensitive, facile and label-free assay of endonuclease activity. Compared with previous studies where labeled substrate is needed, the assay presented here is not only sensitive and reliable, but also simple in its operation and easily adaptable to high-throughput screening. In addition, this assay only involves G-quadruplex DNA as substrate and signal responser, which offers advantages of simplicity and cost efficiency. Furthermore, this system is used for the selective detection of K+ since K+ can reduce the activity of S1 nuclese toward G-quadruplex DNA. These findings may contribute to the future application of G-quadruplex-based technologies to a wide range of fields, such as biology, biomedicine, and bioanalysis.

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ASSOCIATED CONTENT

* Supporting Information S

Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: (+86) 431-85689711. Tel: (+86) 431-85262101. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 20935003, 21075116), and 973 project (Nos. 2011CB911002, 2010CB933603)

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REFERENCES

(1) Grindley, N. D. F.; Whiteson, K. L.; Rice, P. A. Annu. Rev. Biochem. 2006, 75, 567−605. (2) Lieber, M. R. Bioessays 1997, 19, 233−240. (3) Marti, T. M.; Fleck, O. Cell. Mol. Life Sci. 2004, 61, 336−354. (4) Norberg, P.; Bergstrom, T.; Liljeqvist, J.-A. J. Clin. Microbiol. 2006, 44, 4511−4514. (5) West, S. C. Nat. Rev. Mol. Cell Biol. 2003, 4, 435−445. (6) Xu, X.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 3468−3470. (7) Antonios, G. K.; Wang, Z. X.; Andrew, D. B.; Richard, C.; Mathias, B. Angew. Chem., Int. Ed. 2003, 115, 201−204. (8) Ghadiali, J. E.; Stevens, M. M. Adv. Mater. 2008, 20, 4359−4363. (9) Kong, R.-M.; Zhang, X.-B.; Zhang, L. L.; Huang, Y.; Lu, D.-Q.; Tan, W. H.; Shen, G.-L.; Yu, R. Q. Anal. Chem. 2011, 83, 14−17. (10) Connolly, A. R.; Trau, M. Angew. Chem., Int. Ed. 2010, 49, 2720−2723. (11) Cui, L.; Lin, X.; Lin, N.; Song, Y.; Zhu, Z.; Chen, X.; Yang, C. J. Chem. Commun. 2012, 48, 194−196. (12) Hiramatsu, K.; Miura, H.; Kamei, S.; Iwasaki, K.; Kawakita, M. J. Biochem. 1998, 124, 231−236. (13) Jeltsch, A.; Fritz, A.; Alves, J.; Wolfes, H.; Pingoud, A. Anal. Biochem. 1993, 213, 234−240. (14) McLaughlin, L. W.; Benseler, F.; Graeser, E.; Piel, N.; Scholtissek, S. Biochemistry 1987, 26, 7238−7245. (15) Spitzer, S.; Eckstein, F. Nucleic Acids Res. 1988, 16, 11691− 11704. 2434

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Analytical Chemistry

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(16) Huang, Y.; Zhao, S.; Liang, H.; Chen, Z.-F.; Liu, Y.-M. Chem. Eur. J. 2011, 20, 7313−7319. (17) Li, W.; Liu, Z.; Lin, H.; Nie, Z.; Chen, J.; Xu, X.; Yao, S. Anal. Chem. 2010, 82, 1935−1941. (18) Lee, J.; Kim, Y.-K.; Min, D.-H. Anal. Chem. 2011, 83, 8906− 8912. (19) Xu, X.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 3468−3470. (20) Zhou, Z.; Zhu, C.; Ren, J.; Dong, S. Anal. Chim. Acta 2012, 740, 88−92. (21) Li, T.; Dong, S.; Wang, E. Anal. Chem. 2009, 81, 2144−2149. (22) Li, B.; Wei, H.; Dong, S. Chem. Commun. 2007, 73, 73−75. (23) Wang, K.; You, M.; Chen, Y.; Han, D.; Zhu, Z.; Huang, J.; Williams, K.; Yang, C. J.; Tan, W. Angew. Chem., Int. Ed. 2011, 50, 6098−6101. (24) Nakayama, S.; Sintim, H. O. Mol. BioSyst. 2010, 6, 95−97. (25) Fu, R.; Jeon, K.; Jung, C.; Park, H. G. Chem. Commun. 2011, 47, 9876−9878. (26) Shi, Y.; Huang, W. T.; Luo, H. Q.; Li, N. B. Chem. Commun. 2011, 47, 4676−4678. (27) Zhou, Z.; Du, Y.; Zhang, L.; Dong, S. Biosens. Bioelectron. 2012, 34, 100−105. (28) Feng, F.; Tang, Y.; He, F.; Yu, M.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2007, 19, 3490−3495. (29) Butour, J. L.; Mazard, A. M.; Vieussens, C.; Johnson, N. P. Chemi. Biol. Interact. 1990, 73, 195−205. (30) Harada, F.; Dahlberg, J. E. Nucleic Acids Res. 1975, 2, 865−871. (31) Gite, S.; Shankar, V. Eur. J. Biochem. 1992, 210, 437−441. (32) Rushizky, G. W. Gene. Amplif. Anal. 1981, 2, 205−215. (33) Wiegand, R. C.; Godson, G. N.; Radding, C. M. J. Biol. Chem. 1975, 250, 8848−8855. (34) Drew, H. R. J. Mol. Biol. 1984, 176, 535−557. (35) Sun, D.; Hurley, L. H. J. Med. Chem. 2009, 52, 2863−2874. (36) Sen, D.; Gilbert, W. Nature 1990, 344, 410−414. (37) Yang, X.; Pu, F.; Ren, J.; Qu, X. Chem. Commun. 2011, 47, 8133−8135. (38) Teresa, M.; Gomes, S. R.; Tavares, K. S.; Oliveira, J. Analyst 2000, 125, 1983−1986. (39) Huang, C. C.; Chang, H. T. Chem. Commun. 2008, 12, 1461− 1463. (40) Kong, D. M.; Guo, J. H.; Yang, W.; Ma, Y. E.; Shen, H. X. Biosens. Bioelectron. 2009, 25, 88−93. (41) Shi, C.; Gu, H. X.; Ma, C. P. Anal. Biochem. 2010, 400, 99−102. (42) Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S. Angew. Chem., Int. Ed. 2005, 44, 5067−5070. (43) Nagatoishi, S.; Nojima, T.; Galezowska, E.; Gluszynska, A.; Juskowiak, B.; Takenaka, S. Anal. Chim. Acta 2007, 581, 125−131. (44) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286−14287. (45) Radi, A. E.; O’Sullivan, C. K. Chem. Commun. 2006, 32, 3432− 3434. (46) Zhao, C.; Qu, K.; Song, Y.; Ren, J.; Qu, X. Adv. Funct. Mater. 2011, 21, 583−590. (47) Mousavi, Z.; Teter, A.; Lewenstam, A.; Maj-Zurawska, M.; Ivaska, A.; Bobacka, J. Electroanalysis 2011, 23, 1352−1358. (48) Ping, J.; Wang, Y.; Wu, J.; Ying, Y. Electrochem. Commun. 2011, 13, 1529−1532. (49) Leung, C.-H.; Chan, D. S.-H.; Man, B. Y.-W.; Wang, C.-j.; Lam, W.; Cheng, Y.-C.; Fong, W.-F.; Hsiao, W.-L. W.; Ma, D.-L. Anal. Chem. 2011, 83, 463−466.

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