Novel Amplex Red Oxidases Based on Noncanonical DNA Structures

Feb 24, 2014 - Yongxi Zhao , Feng Chen , Qian Li , Lihua Wang , and Chunhai Fan ... Wei Zhang , Hong Zhang , Tianlun Jiang , Dongli Fan , and Yang Luo...
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Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection Shaoru Wang,† Boshi Fu,† Jiaqi Wang,† Yuelin Long,† Xiaoe Zhang,† Shuang Peng,† Pu Guo,† Tian Tian,*,† and Xiang Zhou*,†,‡ †

College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, State Key Laboratory of Virology, Wuhan University, Wuhan, Hubei 430072, P. R. of China ‡ State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China S Supporting Information *

ABSTRACT: G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or Gquadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.

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stranded secondary DNA structures, have been intensely studied recently because of their unique structures and potential biological roles.5 So far, very limited functional study or application was reported.6 Inspired by its similarity to G-quadruplexes, we are interested in exploring their potential roles as novel DNAzymes. Amplex Red is a fluorogenic substrate and a stable hydrogen peroxide probe.7 It has been exploited to develop a wide variety of assay kits for different targets. However, the effects of Amplex Red oxidation in known assay systems always rely on hydrogen peroxide, which largely limits its application in sensor development.8 Here, we demonstrate novel direct Amplex Red

NA is an important biopolymer that has multifaceted functions and plays important roles in vitro and in vivo.1 DNAzymes, catalytic DNA sequences, have attracted intense interest because of their advantages over protein-based enzymes.2 DNAzymes’ catalytic ability usually relies on their unique topologies.3 G-quadruplex could potentially be used as a variety of DNAzymes.4 As examples, G-quadruplex DNA enhances H2O2-participated peroxidation, such as the peroxidation of NADH4a or ABTS.2,4c It could also catalyze the direct oxidation of NADH by O2.4a UV absorption is always used to detect these systems, which usually suffers from sensitivity issues. Thus, developing new DNAzyme systems with enhanced sensitivity still remains imperative and meaningful. Besides G-quadruplex, several other noncanonical structures potentially could be used as DNAzymes. G-triplexes, three© 2014 American Chemical Society

Received: August 10, 2013 Accepted: February 24, 2014 Published: February 24, 2014 2925

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hemin/G4 complex was observed, while a much weaker fluorescence enhancement was produced in the presence of only hemin or the oligomer. We considered that the specific secondary structure of G3 (probable G-triplex) or G4 (Gquadruplex) was required for the catalytic system. This demonstrated that G3- or G4-complexed hemin could effectively enhance the direct oxidation of Amplex Red by O2 and that the G3 or G4 oligomer most likely functioned as a cocatalyst in the oxidation reaction. To determine the G-triplex structure of the G3 oligomer in the oxidation system, we collected circular dichroism (CD) spectra and conducted a CD melting study.5a The CD spectra of G3 oligomer could be easily distinguished from the G4 one (results shown in the right half of Figure S1, Supporting Information), indicating different preformed secondary structures. To prove the molecularity of the preformed intramolecular G-triplex compared to the intermolecular Gquadruplex, melting experiments were performed using various concentrations of strand. The results demonstrated that the Tm values remained unchanged as the G3 concentration was varied from 5.0 to 20 μM (Figure S2, Supporting Information, and Figure 3), which indicated intramolecular secondary structure

oxidases based on different noncanonical DNA structures, including the relatively novel G-triplex (Figure 1a) and the common G-quadruplex (Figure 1b) structure.

Figure 1. Illustration of G-triplex or G-quadruplex DNA-based Amplex Red oxidase.

We first prepared a G3 oligomer with a sequence of 5′TGGGTAGGGCGGG-3′ and a G4 one with a sequence of 5′TGGGTAGGGCGGGTTGGG-3′, which is a widely used Gquadruplex-based DNAzyme sequence. This G3 oligomer only contains three groups of GGG, so there should be no formed intramolecular G-quadruplex of it. A HEPES buffer solution (20 mM, pH 8.0) containing 300 mM NH4OAc and 1.0 μM G3 or G4 oligomer was prepared and denatured at 95 °C for 5 min, followed by incubation at 25 °C for 30 min. Then, hemin was added at a final concentration of 125 nM and incubated for an additional 30 min. The fluorescence was determined immediately after Amplex Red (final concentration at 100 μM) was added. As shown in Figure 2, an evident time-dependent increase in the fluorescence generated by the hemin/G3 or

Figure 3. Plot of Tm values for G3 oligomer at various strand concentrations. All experiments were conducted in 300 mM NH4OAc and 20 mM HEPES (pH 8.0).

formation.9 Additionally, fluorescence-labeled G3 (G3-FAM) and G4 (G4-FAM) were both analyzed by gel electrophoresis using TBE buffer containing 10 mM NH4OAc, and the preformed structures were retained. It has been demonstrated that G3-FAM had faster mobility than G4-FAM with a known preformed G-quadruplex (Figure S3, Supporting Information). This observation further suggested a preformed intramolecular G-triplex structure because the intermolecular structure would have a much larger molecular weight and would move much slower than a single-stranded intramolecular structure.10 Next, we performed a truncation study to prove the importance of the specific secondary structure in the G-triplex and G-quadruplex-based catalytic system. Thus, G3-1 oligomer and the G4-1 one (sequences shown in Table S1, Supporting Information) were assayed together. One guanine deletion on the G3 oligomer significantly decreased the catalytic activity of the reaction (Figure 4), which suggested the presence of a specific secondary structure of the G3 oligomer. Next, the interactions between hemin and the different oligomers were studied using a UV absorption titration assay. A significant change in the Soret absorption in response to titration with DNA could indicate a tight bond between the DNA and

Figure 2. Time-dependent fluorescence changes as a result of the catalyzed oxidation of Amplex Red to resorufin with: hemin/G4 oligomer (pink line); hemin/G3 oligomer (black line); hemin only (green line); G3 oligomer only (blue line); G4 oligomer (red line). The experiments were performed in a buffer solution containing 20 mM HEPES (pH 8.0), 300 mM NH4OAc, and 100 μM Amplex Red, in the presence or absence of 1.0 μM of different oligomers and/or 125 nM hemin. 2926

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the fluorescence increase correlated well with the length of the ventilation time; only a small increase above the background signal was observed when DNA was absent (results in Figure 5). This result mirrored a corresponding increase of fluorescent product upon longer exposure to O2 in the oxidation system and demonstrated an O2-dependent manner.

Figure 4. Time-dependent fluorescence changes as a result of the catalyzed oxidation of Amplex Red to resorufin with: hemin/G4 oligomer (black line); hemin/G3 oligomer (blue line); hemin/G4-1 (red line); hemin/G3-1 (pink line); hemin only (green line). The experiments were performed in a buffer solution containing 20 mM HEPES (pH 8.0), 300 mM NH4OAc, and 100 μM Amplex Red, in the presence or absence of 1.0 μM different oligomer and/or 125 nM hemin.

Figure 5. The activity of the hemin/G3-based oxidase could be enhanced, when O2 was bubbled into the system for different time intervals (the flow rate was 0.04 L/min). The experiments were performed in a buffer solution containing 20 mM HEPES (pH 8.0), 300 mM NH4OAc, and 100 μM Amplex Red, in the presence of 1.0 μM G3 oligomer and 125 nM hemin.

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hemin. A much smaller change was observed with G3-1 or G4-1 (Figure S4, Supporting Information). The above experiments collectively indicated an intramolecular G-triplex structure rather than an intermolecular quadruplex one with G3 oligomer. To further study whether the catalytic effect was dependent on a specific sequence or a specific topology, we tested several other G-triplex sequences that have been demonstrated recently, such as TTA(GGGTTA)3 (Tel-3G)5b and 5′-GGTTGGTGTGG-3′ (T3).5a Neither of these two Gtriplexes could effectively catalyze Amplex Red oxidation like G3 (results shown in Figure S5, Supporting Information). We suggest that this effect could be due to the difference in Gtriplex topology. We then prepared increasing concentrations of the G4 or G3 oligomers and performed a dynamic study using the following assay. It was indicated that the oxidation of Amplex Red in a system containing G4 or G3 proceeded extremely fast, and the fluorescence enhancement was very large. As shown in Figures S6 and S7, Supporting Information, as concentrations of the G4 or G3 oligomers were increased, the fluorescence enhancing rates correspondingly increased. Therefore, the signal output of the G-triplex or G-quadruplex-based sequence was concentration dependent. This feature was considered promising for Amplex Red oxidase-based biosensor development. The above results indicated the generation of a fluorescent product, which was proposed to be resorufin. To further verify this theory, we performed a MS analysis of the product using negative mode ESI. On the basis of the result (Figure S8, Supporting Information), the peak at 211.9 Da should be responsible for the generated resorufin, while the one at 255.9 Da corresponded to Amplex Red. It is well-known that Amplex Red is easily oxidized by hydrogen peroxide and catalyzed by peroxidase.12 In the newly developed system, neither hydrogen peroxide nor hydrogen peroxide generated by reagents through other coupled reactions, such as NADH oxidation or glucose oxidation, was added. In addition, we attempted to elucidate the reaction mechanism.4a To confirm that O2 mediated the oxidation of Amplex Red, we bubbled O2 into the reaction solution for different time intervals. It was demonstrated that the extent of

To further study the mechanism, we added 5.0 mM sodium pyruvate or sodium azide into the reaction system. We demonstrated that sodium pyruvate completely inhibited the generated fluorescence because it is a commonly used scavenger for H2O2.13 The generated fluorescence also did not change in the presence of 5 mM sodium azide, which is a widely used singlet oxygen (1O2) scavenger (Figure 6).14

Figure 6. Time-dependent fluorescence changes as a result of the catalyzed oxidation under the effect by added sodium azide or sodium pyruvate. The experiments were performed in a buffer solution containing 20 mM HEPES (pH 8.0), 300 mM NH4OAc, 100 μM Amplex Red, 125 nM hemin, and 500 nM G3 oligomer, in the presence or absence of 5 mM scavenger salts.

Therefore, the oxidation is likely to be the result of H2O2 generation because sodium pyruvate itself did not quench the fluorescence of resorufin (results shown in Figure S9, Supporting Information). This suggests an in situ generation of H2O2. Herein, we propose that, under the catalytic effect of hemin/G3, reaction occurs between water and O2 to generate 2927

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Figure 7. Schematic illustration of microRNA detection based on G-triplex Amplex Red oxidase.

H2O2, and simultaneously, the generated H2O2 oxidizes the Amplex Red to produce resorufin, causing a fluorescence enhancement. After demonstrating the high efficiency of the direct Amplex Red oxidase, we were interested in applying it to biomarker detection. MicroRNA (miRNA) detection is of considerable significance in both disease diagnosis and studying miRNA functions.15 To develop a sensitive detection method, signal amplification is an important factor.16 Different techniques have been widely used for signal amplification.17 Herein, from a distinct perspective, we designed a cascade amplification system for a sensitive detection. Double strand specific nuclease (DSN) is known to be capable of cleaving DNA strands other than RNA ones in DNA−RNA hybrids,18 and our newly developed system could be used as a signal output unit and a second signal amplifier. To separate the reacted probes from the unreacted ones, we introduced a novel solid-phase-based strategy for isolation, taking advantage of the streptavidin−biotin system. Only cleaved fragments corresponding to target miRNAs could remain in solution and function as a signaling flare, while the intact probes could be trapped and be removed by strepavidincoated beads. Then, we designed two models of our detection system for microRNA. A primary component of the probe model was the target-binding element (green fragment in Figure 7 and Figure S10, Supporting Information), which was complementary with the target miRNA. Duplex DNA−RNA hybrids would be generated in the presence of target miRNAs, and DNA could be cleaved by DSN enzyme, making detachment of biotin tag and the signaling segment. Another primary element next to the target-binding one was the signaloutput unit (blue fragment in Figure 7 and Figure S10, Supporting Information), which was designed as DNA Amplex Red oxidase sequences, including G-triplex ones or Gquadruplex ones. Human microRNA21 (miR21) was known to be involved in a wide range of human cancers.19 We first designed probe21G3 using a G-triplex signaling system and probe21-G4 using a G-quadruplex one toward an analyte of miR21 in our experiment (sequences shown in Table S1, Supporting Information). After the DSN digestion step, the established process for the oxidase reaction was applied. The experimental results showed that our newly developed systems were effective for miRNA detection, which had modest background and obvious signal enhancement (Figures 8 and S11, Supporting Information). Compared with some known probes for miRNAs,20 the design was pretty simple, using only one biotin-labeled probe and one small-molecule substrate. For our

Figure 8. Concentration-dependent response of probe21-G3 for miRNA detection. The concentration for probe21-G3 was at 1.0 μM, and 0.02 U of DSN was used in a 50 μL system of the first step. The buffer of the second step contained 20 mM HEPES (pH 8.0), 300 mM NH4OAc, 100 μM Amplex Red, and 125 nM hemin.

microRNA detection systems, a detection limit of 2.0 pM could be achieved, which was evidently better than the known method established by our group.18b Next, we further assessed the anti-interference properties of our newly developed strategies, which were pretty important for their further applications in diagnostic study.21 We used miR155, which was another miRNA playing important roles in a variety of pathological and physiological processes.22 In Figures 9 and S12, Supporting Information, dramatic decreases of absorbance by probe21-G3 or probe21-G4 could be observed when miR21 was replaced by miR155. The results demonstrated that our systems had a good specificity toward a detection of a specific target.

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CONCLUSIONS Although G-quadruplex DNAzyme-catalyzed oxidation of Amplex Red by H2O2 has been reported in other works,23 we first demonstrated that the complexes formed by a relatively new G-triplex structure (or a common G-quadruplex one) and hemin could directly catalyze the oxidation of Amplex Red by O2 (air). A circular dichroism study and UV titration study were carried out by us to determine and confirm the specific Gtriplex structure of the G3 oligomer. In the presence of hemin and G-triplex or G-quadruplex DNA, the fluorescent signal was remarkably enhanced due to an oxidation reaction. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on 2928

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for 5 min in a buffer containing 20 mM HEPES (pH 8.0) and 300 mM NH4OAc; samples were then incubated at room temperature for 1 h, and then, hemin was added, making the final concentration 125 nM. Finally, Amplex Red was added at a final concentration of 100 μM. The fluorescence detection was immediately conducted in the Kinetics mode of a LS55 PerkinElmer. The excitation and emission wavelengths were set at 563 and 587 nm, respectively. Specificity Assay of DNA Oxidase System. To verify the specificity, we used miR155 and miR21 for the test of probe21G3 or probe21-G4, respectively. DSN reaction and DNA oxidase reaction were silimarly performed, and fluorescence spectra were recorded by a LS55 Perkin-Elmer. The concentration of probe21-G3 or probe21-G4 was at 1.0 μM, 0.02 U of DSN, and the same amount of different microRNAs was used in a 50 μL system of the first step. The buffer of the fluorescence recording step contained 20 mM HEPES (pH 8.0), 300 mM NH4OAc, 100 μM Amplex Red, and 125 nM hemin. Signal enhancement between target miR21 and the other one (miR155) was compared.

Figure 9. Specificity test of probe21-G3 by miR155 with varied sequence of miR21 in Amplex Red oxidase system. The concentration of probe21-G3 was at 1.0 μM, 0.02 U of DSN, and the indicated amount of microRNA was used in a 50 μL system of the first step. The buffer of the second step contained 20 mM HEPES (pH 8.0), 300 mM NH4OAc, 100 μM Amplex Red, and 125 nM hemin.

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DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, we successfully investigated the applications of the novel enzymes in microRNA detection. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. The advantages of using newly developed methods over the conventional one included a lower detection limit, a decreased background fluorescence, and a wider range of detection.18b Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.

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.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: (+)86-27-68756663. *E-mail: [email protected]. Fax: (+)86-27-68756663. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Basic Research Program of China (973 Program) (2012CB720600, 2012CB720603, 2012CB720605), the National Science of Foundation of China (No. 91213302, 81373256, 21372182), and the National Grand Program on Key Infectious Disease (2012ZX10003002014). This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT1030).

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EXPERIMENTAL SECTION Circular Dichroic Studies. CD experiments utilizing a Jasco-810 spectropolarimeter (Jasco, Easton, MD, USA) were measured at room temperature using a quartz cell with a 1 cm path length; CD spectra were collected from 220 to 330 nm at scanning speed of 200 nm/min. The bandwidth was 5 nm, and the response time was 2 s. All CD spectra were baselinecorrected for signal contributions due to the buffer and were the average of at least two runs. UV Titration Study of Hemin by DNAs. Different concentrations of DNA were prepared and heated at 95 °C for 5 min in buffer containing 20 mM HEPES (pH 8.0) and 300 mM NH4OAc. The samples were incubated at room temperature for 30 min, and then, hemin was added to a final concentration of 0.125 μM. A Shimadzu UV-2550 UV−vis spectrophotometer in Spectrum mode was used for detection. Procedure of MicroRNA Detection. The DSN cleavage reaction was performed in 1× DSN master buffer, which contained 50 mM Tris−HCl, 5 mM MgCl2, and 1 mM DTT at pH 8.0. A 50 μL sample was incubated at 55 °C for 20 min and then cooled to room temperature in 25 min. The optimized recipe is as follows: 1 μM of DNA probe, 0.02 U DSN enzyme, and different amounts of microRNAs. DSN digestion samples (each 50 μL) were incubated with 15 μL of strepavidin coated beads at 37 °C for 30 min, followed by a separation by ferromagnet. The resulting supernatants were heated at 95 °C

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