Article pubs.acs.org/ac
Self-Phosphorylating Deoxyribozyme Initiated Cascade Enzymatic Amplification for Guanosine-5′-triphosphate Detection Lida Wang, Yang Liu, and Jinghong Li* Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China S Supporting Information *
ABSTRACT: The self-phosphorylating deoxyribozymes identified by in vitro selection can catalyze their own phosphorylation by utilizing phosphate donor guanosine-5′-triphosphate (GTP) which plays a critical role in a majority of cellular processes. On the basis of the unique properties of self-phosphorylating deoxyribozymes, we report a novel GTP sensor coupled with λ exonuclease cleavage reaction and nicking enzyme assisted fluorescence signal amplification process. The deoxyribozymes with special catalytic and structural characteristics display good stability compared to protein and RNA enzymes. We combined these properties with enzymatic recycling cleavage strategy to build a sensor which produced enhanced fluorescence signal. Sensitive and selective detection of GTP was successfully realized with the well-designed deoxyribozyme-based sensing platform by taking advantage of the selfphosphorylating ability of the kinase deoxyribozyme, efficient digestion capacity of λ exonuclease, and enzymatic recycling amplification of nicking enzyme. The method not only provides a platform for detecting GTP but also shows great potential in analyzing a variety of targets by combining deoxyribozymes with signal amplification strategy.
T
substrates and cofactors,10,11 these self-phosphorylating deoxyribozymes can discriminate clearly between a particular NTP and closely related analogues, showing good selectivity. However, only few papers about sensors based on selfphosphorylating deoxyribozymes have been reported in previous studies.15 It is still in early stage to turn these selfphosphorylating deoxyribozymes into practical sensors for monitoring targets. Detection of nucleosides and nucleotides is of paramount importance as they represent the fundamental building blocks that can be used to understand all life forms.16−18 Among the family of nucleosides and nucleotides, guanosine-5′-triphosphate (GTP) plays a key role in many important biological processes in the cell.19,20 As one of the four vital essential ribonucleoside triphosphates for the synthesis of RNA, it plays an important role in the transcription process. It acts as a source of energy for translation and other important cellular processes.21 Therefore, it is essential to develop a sensitive sensor for GTP due to its importance. Different designed methods have been used to realize the detection of GTP. For example, synthesized fluorescent dyes, such as water-soluble imidazolium anthracene derivative and benzimidazolium with unique specificity, have been applied for GTP detection in solutions.22 GTP detection making use of fluorescence has
o date, almost all natural enzymes are made of protein and RNA.1−3 While DNA is known for its double-stranded form to store genetic information, its chemical similarities to naturally occurring ribozymes suggest that DNA may have catalytic capabilities.4,5 The identification of the first artificial deoxyribozyme by in vitro selection in 19946 revealed that DNA is capable of catalyzing specific biochemical reactions beyond merely acting as the genetic material. Since then, many other catalytic DNA molecules have been developed to catalyze a wide variety of chemical reactions, including DNA phosphorylation,7 DNA-catalyzed RNA cleavage,6,8 DNA deglycosylation,9 etc. Among the family of deoxyribozymes, self-phosphorylating deoxyribozymes are single-stranded DNA molecules capable of self-phosphorylation at their 5′ ends.10 These self-phosphorylating deoxyribozymes have been isolated from a large pool of random sequences by multiple cycles of in vitro selection.11,12 In the presence of deoxynucleoside 5′triphosphates (dNTPs) or nucleoside 5′-triphosphates (NTPs), which serve as the source of activated phosphate, and cofactors, deoxyribozymes could undergo reversible conformation changes which transfer from inactivity to activity and catalyze the transfer of the terminal phosphate groups of substrates to the 5′-hydroxyl terminus of DNA itself. Compared to traditional protein enzymes and ribozymes, deoxyribozymes show great practical advantages, such as the simplicity of oligonucleotide synthesis, higher stability against chemical and nuclease degradation, and less cost.13,14 Therefore, deoxyribozymes have shown great potential as biosensors for different targets. In addition, because of the requirements of very specific © 2014 American Chemical Society
Received: May 17, 2014 Accepted: June 27, 2014 Published: June 27, 2014 7907
dx.doi.org/10.1021/ac501842t | Anal. Chem. 2014, 86, 7907−7912
Analytical Chemistry
Article
Scheme 1. Schematic Representation of the Fluorescent Assay for GTP Detection Based on GTP-Triggered SelfPhosphorylating Deoxyribozyme and Nicking Enzyme Assisted Fluorescence Signal Amplificationa
a
(1) DK3X used GTP as the source of activated phosphate to catalyze its self-phosphorylation. (2) DK3X was hybridized with another DNA. (3) Hybridized phosphorylated dsDNA was immediately cleaved by λ exonuclease. (4) The nicking enzyme catalyzed the multiple cleavage of MB, resulting in the enhancement of fluorescence signal.
isothermal amplified fluorescence signal. In this work, the deoxyribozyme utilizes GTP to trigger its phosphorylation while the phosphorylated double helixes are cleaved by λ exonuclease to generate mononucleotides and ssDNA. Because of the different interaction intensities of reporter molecule beacon (MB) with the substrate strand in the form of ssDNA and dsDNA, good detection sensitivity and high signal to background ratio were achieved. This method shows huge potential for further researches on combining target depended deoxyribozymes with signal amplifying reports to build sensing platforms.
been reported in previous studies.15,23 In spite of the development of these methods, further improvement of the analytical performances including sensitivity, selectivity, and linear range, etc. is still in urgent need. It remains in early stage to incorporate signal amplification methods into self-phosphorylating deoxyribozymes to develop highly accurate, suitable methods for the determination of GTP. In an attempt to improve the sensitivity of target DNA detection, DNA nicking enzyme, as a highly specific type of endonuclease which can recognize a particular sequence in double-stranded DNA and cleave only one strand at the specific site, has been used. In recent years, it has attracted considerable research interests due to its great capability in triggering signal amplification.24,25 Compared to other amplification techniques like polymerase chain reaction (PCR), the nicking enzyme possesses various advantages such as it can be performed under isothermal conditions without the requirement of specialized instruments, which makes it simpler and less cost. Some attempts have been made based on nicking enzyme to realize the detection of nucleic acids, proteins, and small molecules.25−27 Herein, we designed a novel sensor for detecting GTP on the basis of target-triggered self-phosphorylating deoxyribozymes. Sensitive detection of GTP is achieved due to three reasons: First, the GTP depended deoxyribozyme, which was artificially created by in vitro selection, has high self-phosphorylating ability. Second the efficient digestion of double-stranded DNA (dsDNA) with 5′-phosphoryl termini can be achieved by λ exonuclease, which is a highly processive enzyme catalyzing the removal of 5′ mononucleotides from duplex DNA to generate single-stranded DNA (ssDNA). Third, nicking enzyme based enzymatic recycling cleavage strategy has been employed for
■
EXPERIMENTAL SECTION
Reagents and Samples. λ exonuclease (5 units/μL) was obtained from New England Biolabs (Beijing, China); guanosine-5′-triphosphate (GTP, 100 mM), adenosine-5′triphosphate (ATP, 100 mM), cytidine-5′-triphosphate (CTP, 100 mM), uridine-5′-triphosphate (UTP, 100 mM), and Nb.Mva1269I (10 units/μL) were bought from Fermentas. NaCl, HEPES, KCl, and MnCl2 were bought from Beijing DingGuo Biotech. Co., Ltd. All solutions were prepared using ultrapure water. The DNA sequences were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd.; molecule beacon (MB) modified with 5′FAM and 3′-Dabcyl was purified through high-performance liquid chromatography (HPLC). Other DNA sequences without modification were purified through denaturing polyacrylamide gel electrophoresis (PAGE). Sequences of oligonucleotide probes used in this work are listed as follows: DK3X, 5′-GGAGAGGTGCATTCACCGAGGCCGGGGCCGAGTTCGAGGCCTTTCCCCGCGCGGCC-3′; DK-C7908
dx.doi.org/10.1021/ac501842t | Anal. Chem. 2014, 86, 7907−7912
Analytical Chemistry
Article
DK3X in order to stabilize the double-stranded form of phosphorylated DNA, which can be selectively cleaved by λ exonuclease to yield mononucleotides and ssDNA.28,29 The released ssDNA hybridizes with the signal output unit MB which carries a specific recognition site for nicking enzyme, Nb.Mva1269I.30 MB maintains the stem−loop structure due to the binding of the complementary sequences at the ends,31,32 while this nicking enzyme can recognize the specific sequence of the hybridized double-stranded DNA but hydrolyzes only MB. The cleaved MB probe is too short to maintain the double-stranded conformation, resulting in the complete disconnection of the fluorophore from the quencher, and then a fluorescence signal appears.33 Also, the released DK-C-3 becomes available to hybridize to the next uncleaved MB probe and can finally undergo many cycles to trigger the cleavage of more MB probes, providing an amplifying fluorescence signal.34,35 Thus, the concentration of GTP could be easily reflected by the fluorescence signals change. Detection Capability of the Strategy. The determination of GTP in this method is composed of GTP-induced selfphosphorylation, λ exonuclease cleavage, and a nicking endonuclease assisted fluorescence signal amplification. Initially, the ability of self-phosphorylation of DK3X was assayed. The phosphorylated DNA exhibits significant DNA ligation activity and is able to join two DNA substrates (DK3X, acceptor DNA as shown in the Supporting Information) to generate ligated products.12,36 The reaction products were precipitated with ethanol and analyzed by 10% denaturing PAGE. As shown in Figure 1A, when the 32P-labeled DK3X was incubated with 10 and 1 mM GTP, respectively, significant amounts of ligated products (which were bigger than the DK3X
3, 5′-CCTCGGTGAATGCACCTCTCC-3′; MB, 5′6-FAMcgacga CTCGAGGTGCATTCATAT tcgtcg-3′ Dabcyl. DK3X represents the self-phosphorylating deoxyribozyme. Lower-case letters represent the stem portion of MB. The underlined sequences of the DK-C-3 are the sequences that are the same as the underlined sequences of DK3X and complementary to the underlined sequences of MB. The bold italic letters are the recognition site of Nb.Mva1269I. DNA Self-Phosphorylation Assay. The self-phosphorylation of DK3X was carried out in 30 μL of reaction buffer as previous reported protocols.12 As Mn2+ plays an important role in the catalysis of the self-phosphorylating deoxyribozyme, DK3X, the buffer we choose in our method for DNA selfphosphorylation assay is the reaction buffer used for the in vitro selection of deoxyribozyme. First, DK3X was dissolved in reaction buffer (400 mM NaCl, 100 mM KCl, 10 mM MnCl2, 50 mM HEPES, pH 8.0) at a concentration of 100 nM. The solution was first incubated for 5 min at room temperature, and GTP was then added to initiate the phosphorylation reaction. After being incubated for a certain length of time, the reaction was heated to 90 °C for 20 min and cooled to room temperature. The ability of self-phosphorylation of DK3X was assayed by using the protocols in the Supporting Information. The self-phosphorylation reactions were precipitated with ethanol and analyzed by 10% PAGE. DNA Hybridization and λ Exonuclease Reaction Assay. The hybridization between DK3X (100 nM) and DKC-3 (100 nM) was carried out by adding DK-C-3 to the obtained DNA self-phosphorylation solution. The mixture was heated at 90 °C for 2 min and cooled to room temperature for further use. Then, a certain amount of λ exonuclease and 10× λ exonuclease reaction buffer (670 mM glycine−KOH, 25 mM MgCl2, 0.1% (v/v) Triton X-100) were mixed into the reaction buffer. After the incubation at 37 °C for 20 min, the reaction was stopped by heating to 90 °C for 10 min and cooled to room temperature. The optimizations of the concentration of λ exonuclease and reaction time were 0.1−10 units, 0−50 min, respectively. The reaction pH was optimized from 7 to 9. Fluorescence Assay Procedures. To conduct the fluorescence assay, 50 μL of 200 nM MB and a certain amount of Nb.Mva1269I were added to the prepared reaction mixture, respectively. After incubating at 37 °C for 45 min, the solution was transferred into a cuvette and the fluorescence spectra were measured on the Cary Eclipse (Varian). Excitation wavelength and emission wavelength were 495 and 520 nm, respectively. Instruments. The temperature was controlled by using a PCR System (Bio-Rad, U.S.A.). Fluorescence changes over time were monitored on the Cary Eclipse (Varian). The images of gels were obtained using a Typhoon 9200 variable mode imager (GE Healthcare) and analyzed using Image Quant software (Molecular Dynamics).
■
RESULTS AND DISCUSSION Strategy for GTP Detection. The developed strategy for GTP detection is based on GTP-responsive self-phosphorylation and nicking enzyme assisted fluorescence signal amplification. As shown in Scheme 1, our design includes three components: GTP depended deoxyribozyme (DK3X), complementary DNA (DK-C-3), and signal reporter (MB). DK3X, as previously reported deoxyribozyme, utilizes GTP as the source of activated phosphate and Mn2+ as the divalent metal cofactor to catalyze its own phosphorylation. DK-C-3 was designed to be complementary to part of the sequence of
Figure 1. (A) Ligation assay for GTP-dependent deoxyribozyme. PAGE gel with ligated products: DK3X marker (lane a); DK3X incubated in the presence of 10 mM GTP (lane b), 1 mM GTP (lane c), and the buffer alone (lane d). (B) Fluorescence responses of the sensing system to 1 mM GTP in the presence (curve a) and absence (curve c) of nicking endonuclease with corresponding backgrounds (curves b and d), respectively. Reactions were performed with 3 units of λ exonuclease, 10 units of Nb.Mva1269I, and 200 nM MB. 7909
dx.doi.org/10.1021/ac501842t | Anal. Chem. 2014, 86, 7907−7912
Analytical Chemistry
Article
marker in lane a) were observed (lanes b and c). In contrast, no ligated product was produced when GTP was replaced with dH2O (lane d). The results here indicated that DK3X showed phosphorylation activity in the presence of GTP. As expected, the deoxyribozyme DK3X was able to utilize GTP as the source of activated phosphate to catalyze the transfer of the terminal phosphate groups to the 5′-hydroxyl terminus of DNA itself. In order to assess the amplification function of the proposed sensing strategy, GTP-induced fluorescence enhancements in the presence and absence of nicking enzyme Nb.Mva1269I were recorded, respectively. Upon the addition of Nb.Mva1269I, the fluorescence signal was amplified through continuous cleavage of MB. As shown in Figure 1B, without the assistance of Nb.Mva1269I, the fluorescence intensity in the presence of GTP (curve c) was a little higher than the one without GTP (curve d), but the fluorescence change was not remarkable for the detection of GTP. On the contrary, by using nicking enzyme, a dramatic signal enhancement in the fluorescence intensity upon the addition of GTP was observed (curves a and b). By introducing nicking enzyme, we observed a 1021 ± 29 signal increase, while in the absence of nicking enzyme, only a 125 ± 5 increase in the signal was observed. The fluorescence intensity increment of the sensing system catalyzed by coupled MB and nicking enzyme was much more obvious than that by MB only. The main reason for this fact is that nicking enzyme could catalyze the cleavage of MB, converting the stable hybridized double-stranded DNA (DK-C3 and MB) into much less stable forms (DK-C-3 and two cleaved partial MB probes).37 Also, the released DK-C-3 became available to hybridize to the next uncleaved MB probe and could finally undergo many cycles to trigger the cleavage of more MB probes, providing an amplifying fluorescence signal for the sensitive detection of GTP. At the same time, in the absence of GTP, reporter MB could not compete with DK3X to hybridize with the DK-C-3 to open its hairpin structure. Since Nb.Mva1269I was disabled to cleave the hairpin-shaped MB,38 no remarkable fluorescent signal generated (curve b), thus lowering the background and showing excellent discrimination between presence and absence of the target GTP. Therefore, it was demonstrated that the established nicking enzyme assisted signal amplification strategy is effective for the detection of GTP. Optimization of the Detection Conditions. In order to achieve the best sensing performance, different conditions were optimized. First, the concentration and reaction time of λ exonuclease are crucial parameters as the ssDNA thus generated by λ exonuclease reaction serves as the trigger for the next enzymatic recycling cleavage of MB. λ exonuclease exhibits much higher activity on phosphorylated dsDNA than single-stranded DNA and nonphosphorylated DNA.39,40 An excess of λ exonuclease and cleavage time would make the fluorescence signals decrease slowly. As can be seen from Figure 2A, the fluorescence intensities increased with the increase of λ exonuclease concentration, and then reached highest at the concentration of 3 units indicting the complete degradation of the phosphorylated dsDNA. In Figure 2B, with the increase of incubation time, the fluorescence intensity increased at first, and then reached maximum in 20 min. When the reaction time was longer than 20 min, the fluorescence signal slowly decreased. Therefore, 3 units and 20 min for λ exonuclease cleavage process were chosen for further investigation. In addition, other significant factors such as incubation time of self-phosphorylation with GTP and pH of
Figure 2. (A) Optimization of λ exonuclease concentration. (B) Optimization of λ exonuclease reaction time. (C) Optimization of Nb.Mva1269I concentration. The assays were carried out in the reaction buffer, containing 1 mM GTP, 10 units of Nb.Mva1269I, and 200 nM MB.
the λ exonuclease reaction for this sensing system were also studied. Because the phosphorylated DNA is able to join two DNA substrates (DK3X, acceptor DNA as shown in the Supporting Information) to generate ligated products, the percentage of the successful self-phosphorylated DK3X could be easily reflected by the percent ligation values. As seen in Figure S1 (Supporting Information), the percent ligation values increased with the GTP incubation time and then reached a plateau at 30 min; longer incubation time did not enhance it further. It indicated that the self-phosphorylation of DK3X achieved saturation. Also, pH of the reaction buffer has a strong influence on the efficiency of λ exonuclease, and experimental results in Supporting Information Figure S3 showed that pH 8.8 could provide the maximum fluorescence intensity for the sensing system. 7910
dx.doi.org/10.1021/ac501842t | Anal. Chem. 2014, 86, 7907−7912
Analytical Chemistry
Article
The sensing performance of the system is also closely related to the effect of nicking enzyme, Nb.Mva1269I. Thus, we further optimized the concentration and the incubation time in order to get the best performance, minimize the use of reagents, and reduce the assay time as well. As can be seen from Figure 2C, the fluorescence intensity of the sensing system was found to increase with the amount of Nb.Mva1269I and trend to a constant value at an amount of 10 units of Nb.Mva1269I. In Supporting Information Figure S2, fluorescence intensity exhibited a rapid increase with a further increase in the reaction time after the addition of Nb.Mva1269I at 5 min and then reached equilibrium at 45 min. The results here suggested the complete enzymatic cleavage due to the exhaustion of MB or inactivation of Nb.Mva1269I.41,42 Thus, the optimized Nb.Mva1269I concentration and cleavage time were chosen to be 10 units and 45 min. Sensitivity and Specificity of the Detection Conditions. The proposed amplified sensing system is sensitive and specific to GTP. To demonstrate the ability of the described strategy to sensitively quantify GTP, a series of concentrations of GTP (ranging from 0.5 μM to 1 mM) were measured based on the optimal assay conditions. Figure 3A shows the fluorescence emission spectra of the sensing system upon the addition of GTP at different concentrations. A gradual increase in fluorescence intensity was observed as GTP concentration increased, thus indicating that the concentration of GTP could be easily reflected by the fluorescence signals change. Figure 3B displays the relationship between the fluorescence intensities and GTP concentrations. The fluorescence intensities and GTP concentrations exhibited a good linear relationship from 0.5 to 10 μM as shown in Figure 3C. This new system is very sensitive to GTP, with the detection limit of 1 μM determined by 3 times the standard deviation corresponding to the blank sample detection. Fluorescence sensors reported in previous studies can detect GTP in the micromolar to millimolar range.21−23 Therefore, the detection sensitivity of our method reveals that it is comparable to many existing homogeneous assay techniques for GTP detection.15,22 Besides sensitivity, selectivity is another important issue to assess the performance of a sensor. To validate the selectivity of the described strategy, the three biologically relevant ribonucleoside triphosphates, ATP, CTP, and UTP, were tested by using the same reaction protocol described above. As shown in Figure 4, these control groups exhibited minimal signal and high signal was only obtained when the specific GTP was induced as the source of activated phosphate for the reason that DK3X can only use GTP to catalyze its selfphosphorylation. Traditionally, T4 polynucleotide kinase is used to catalyze phosphorylation of DNA, but it has no significant NTPs or dNTPs restrictions.10 Here, the results demonstrate that the fluorescence signal of our system was specifically triggered by GTP, which indicated that the proposed sensing system could offer a high specificity. Overall, these experimental results indicate that the proposed fluorescence sensing system on the recognition of GTP is successfully realized.
Figure 3. (A) Fluorescence intensity−wavelength curves with different concentrations of GTP (top to bottom, 1, 0.5, 0.3, and 0.1 mM, 50, 10, 5, 3, 1, 0.5 μM in reaction buffer). (B) Dependence of fluorescence intensity at 520 nm on GTP concentration. The curve shows a linear range from 0.5 to 10 μM (C). The concentrations of λ exonuclease, Nb.Mva1269I, and MB were 3 units, 10 units, and 200 nM, respectively.
use dNTPs and NTPs as the phosphorylation substrates have been discovered. In this work, a sensitive biosensor on the basis of self-phosphorylation deoxyribozyme and enzymatic recycling cleavage strategy was developed for the detection of GTP. Due to the efficient cleavage capacity of λ exonuclease and nicking enzyme based catalytic turnover for signal amplification, this approach exhibits a very high sensitivity for GTP detection, with a low detection limit down to 1 μM. Additionally, as this deoxyribozyme only uses GTP as the source of activated phosphate to catalyze its own phosphorylation, it indicates that the selectivity may be suitable to cellular applications by sensing GTP in the presence of other similar molecules, such as ATP,
■
CONCLUSION In summary, we have successfully utilized self-phosphorylating deoxyribozyme to build a novel amplified fluorescence sensing system for sensitive detection of GTP which plays a key role in many important biological processes. Through in vitro selection, different self-phosphorylating deoxyribozymes that 7911
dx.doi.org/10.1021/ac501842t | Anal. Chem. 2014, 86, 7907−7912
Analytical Chemistry
Article
(12) McManus, S. A.; Li, Y. Biochemistry 2007, 46, 2198−2204. (13) Silverman, S. K. Acc. Chem. Res. 2009, 42, 1521−1531. (14) Liu, J.; Lu, Y. Angew. Chem. 2007, 119, 7731−7734. (15) McManus, S. A.; Li, Y. J. Am. Chem. Soc. 2013, 135, 7181−7186. (16) Nakano, S.; Fukuda, M.; Tamura, T.; Sakaguchi, R.; Nakata, E.; Morii, T. J. Am. Chem. Soc. 2013, 135, 3465−3473. (17) Nyborg, J.; Kjeldgaard, M. Curr. Opin. Biotechnol. 1996, 7, 369− 375. (18) McCleskey, S. C.; Griffin, M. J.; Schneider, S. E.; McDevitt, J. T.; Anslyn, E. V. J. Am. Chem. Soc. 2003, 125, 1114−1115. (19) Bourne, H. R.; Sanders, D. A.; McCormick, F. Nature 1990, 348, 125−132. (20) Sepuri, N. B. V.; Schülke, N.; Pain, D. J. Biol. Chem. 1998, 273, 1420−1424. (21) Janiak, F.; Dell, V. A.; Abrahamson, J. K.; Watson, B. S.; Miller, D. L.; Johnson, A. E. Biochemistry 1990, 29, 4268−4277. (22) Kwon, J. Y.; Singh, N. J.; Kim, H. N.; Kim, S. K.; Kim, K. S.; Yoon, J. J. Am. Chem. Soc. 2004, 126, 8892−8893. (23) Wang, Y.; Li, Z.; Weber, T. J.; Hu, D.; Lin, C.-T.; Li, J.; Lin, Y. Anal. Chem. 2013, 85, 6775−6782. (24) Walker, G. T.; Little, M. C.; Nadeau, J. G.; Shank, D. D. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 392−396. (25) Duan, R.; Zuo, X.; Wang, S.; Quan, X.; Chen, D.; Chen, Z.; Jiang, L.; Fan, C.; Xia, F. Nat. Protoc. 2014, 9, 597−607. (26) Xue, L.; Zhou, X.; Xing, D. Anal. Chem. 2012, 84, 3507−3513. (27) Kong, R.; Zhang, X.; Chen, Z.; Meng, H.; Song, Z.; Tan, W.; Shen, G.; Yu, R. Anal. Chem. 2011, 83, 7603−7607. (28) Subramanian, K.; Rutvisuttinunt, W.; Scott, W.; Myers, R. S. Nucleic Acids Res. 2003, 31, 1585−1596. (29) Song, C.; Zhao, M. Anal. Chem. 2009, 81, 1383−1388. (30) Guo, Y.; Sun, X.; Fu, Y.; Mao, Y. Sens. Actuators, B 2014, 193, 107−112. (31) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303−308. (32) Tan, W.; Fang, X.; Li, J.; Liu, X. Chem.Eur. J. 2000, 6, 1107− 1111. (33) Yan, L.; Zhou, J.; Zheng, Y.; Gamson, A. S.; Roembke, B. T.; Nakayama, S.; Sintim, H. O. Mol. BioSyst. 2014, 10, 970−1003. (34) Lu, L.-M.; Zhang, X.-B.; Kong, R.-M.; Yang, B.; Tan, W. J. Am. Chem. Soc. 2011, 133, 11686−11691. (35) Gerasimova, Y. V.; Peck, S.; Kolpashchikov, D. M. Chem. Commun. 2010, 46, 8761−8763. (36) Levy, M.; Ellington, A. D. Chem. Biol. 2002, 9, 417−426. (37) Yin, B.; Liu, Y.; Ye, B. J. Am. Chem. Soc. 2012, 134, 5064−5067. (38) Chen, F.; Zhao, Y. Analyst 2013, 138, 284−289. (39) Little, J. W. J. Biol. Chem. 1967, 242, 679−686. (40) Lin, L.; Liu, Y.; Zhao, X.; Li, J. Anal. Chem. 2011, 83, 8396− 8402. (41) Zou, B.; Ma, Y.; Wu, H.; Zhou, G. Angew. Chem., Int. Ed. 2011, 50, 7395−7398. (42) Li, J. J.; Chu, Y.; Lee, B. Y.-H.; Xie, X. S. Nucleic Acids Res. 2008, 36, e36.
Figure 4. Fluorescence intensity of the sensor incubated with 100 μM GTP, ATP, CTP, and UTP, respectively. The concentrations of λ exonuclease, Nb.Mva1269I, and MB were 3 units, 10 units, and 200 nM, respectively.
CTP, and UTP. The positive results achieved here might become an initial guide for the potential applications of the deoxyribozymes in practical investigation. As deoxyribozymes are selected to react with a wide range of targets, this system shows that deoxyribozymes have great potential in providing platforms for analyzing a variety of targets by combining with signal amplification strategy.
■
ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 86-10-62795290. Fax: 86-10-62771149. E-mail: jhli@ mail.tsinghua.edu.cn. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (No. 2011CB935704), the National Natural Science Foundation of China (No. 21235004, No. 21327806), and Tsinghua University Initiative Scientific Research Program.
■
REFERENCES
(1) Doudna, J. A.; Cech, T. R. Nature 2002, 418, 222−228. (2) Altman, S.; Baer, M.; Bartkiewicz, M.; Gold, H.; Guerrier-Takada, C.; Kirsebom, L. A.; Lumelsky, N.; Peck, K. Gene 1989, 82, 63−64. (3) Kruger, K.; Grabowski, P. J.; Zaug, A. J.; Sands, J.; Gottschling, D. E.; Cech, T. R. Cell 1982, 31, 147−157. (4) Breaker, R. R. Nat. Biotechnol. 1997, 15, 427−431. (5) Silverman, S. K. Org. Biomol. Chem. 2004, 2, 2701−2706. (6) Breaker, R. R.; Joyce, G. F. Chem. Biol. 1994, 1, 223−229. (7) Wang, W.; Billen, L. P.; Li, Y. Chem. Biol. 2002, 9, 507−517. (8) Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 4262−4266. (9) Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 7802−7807. (10) Li, Y.; Breaker, R. R. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2746−2751. (11) Achenbach, J. C.; Jeffries, G. A.; McManus, S. A.; Billen, L. P.; Li, Y. Biochemistry 2005, 44, 3765−3774. 7912
dx.doi.org/10.1021/ac501842t | Anal. Chem. 2014, 86, 7907−7912
