Biosensors and Bioelectronics 74 (2015) 222–226
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A split G-quadruplex-based DNA nano-tweezers structure as a signaltransducing molecule for the homogeneous detection of specific nucleic acids Keisuke Nakatsuka a, Hajime Shigeto a,b, Akio Kuroda a, Hisakage Funabashi b,n a Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan b Institute for Sustainable Sciences and Development, Hiroshima University, 1-3-2 Kagamiyama, Higashihiroshima, Hiroshima 739-8527, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 28 April 2015 Received in revised form 12 June 2015 Accepted 23 June 2015 Available online 25 June 2015
A portable method of specific nucleic acid detection would be very useful for monitoring public health in a variety of settings for point-of-care and point-of-need testing. However, conventional methods for the detection of nucleic acids are not ideal for use in the field, as they require skilled operators and complex equipment. Here, we constructed a method for specific nucleic acid detection using a split G-quadruplex (Gq) structure that can recognize target nucleic acids without competitive reactions in a bimolecular reaction and directly produce a detectable signal based on peroxidase activity. We developed a single signal-transducing molecule with a split Gq-based DNA-nano tweezers (NT) structure that self-assembles from three single-stranded DNAs through simple mixing, and detects its target without requiring any washing steps. A model target, a partial norovirus mRNA (NV-RNA), was specifically recognized by the split Gq-based DNA-NT, causing it to undergo a structural change that restored its peroxidase activity. The peroxidase activity was measured by following the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the NV-RNA concentration. The lower detection limit was 4 nM. Our results demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT structure as a nucleic acid signal-transducing molecule in a homogenous assay format. Also the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device. & 2015 Elsevier B.V. All rights reserved.
Keywords: Homogeneous assay Norovirus Nucleic acid detection Point-of-care testing Signal-transducing molecule Split G-quadruplex
1. Introduction The detection of specific DNA and RNA is widely used not only for diagnostic purposes, but also for monitoring public health with regard to epidemics, food safety, and environmental pollution. The detection of specific nucleic acids enables a genetic marker analysis in addition to simple target detection. Therefore, the detection in a portable format has great potential for use in a wide variety of applications, such as point-of-care testing (POCT) and point-of-need testing (PONT) (Ahmad and Hashsham, 2012; Craw and Balachandran, 2012; Gubala et al., 2012; Hartman et al., 2013; Niemz et al., 2011). However, conventional methods such as PCRbased methods and DNA arrays require skilled operation, special equipment, and time-consuming complex procedures, which are n Correspondence to: Room 404, VBL Office, 2-313 Kagamiyama, Higashihiroshima, Hiroshima 739-8527, Japan. E-mail address: [email protected] (H. Funabashi).
http://dx.doi.org/10.1016/j.bios.2015.06.055 0956-5663/& 2015 Elsevier B.V. All rights reserved.
not ideal for a portable device. Thus, the development of a novel easy-to-use nucleic acid detection principle is highly desirable. We believe that the development of a signal-transducing molecule capable of recognizing target nucleic acids and directly producing a readily detectable signal is ideal for constructing a convenient nucleic acid detection method. Also, such a signaltransducing molecule should be able to produce a signal only when it recognizes its target, enabling its detection in a homogeneous assay that does not require washing steps and therefore is readily applicable to a portable format. To develop such a signaltransducing molecule, we focused on a G-quadruplex (Gq) structure (Sen and Gilbert, 1988) as a signal generator. A Gq is one of the well-characterized DNAzymes that has a guanine-rich sequence and exhibits peroxidase activity with the help of hemin as a co-factor (Travascio et al., 1998; Travascio et al., 2001). Because many detection methods for peroxidase activity are available, a Gq is often used in DNA-based sensor devices as a signal generator for specific nucleic acid detection (Lu et al., 2013; Roembke et al., 2013).
K. Nakatsuka et al. / Biosensors and Bioelectronics 74 (2015) 222–226
One approach to add target-recognition ability to a Gq is the use of a split Gq sequence. Each split Gq sequence is connected to a consecutive complementary sequence for a target nucleic acid as a target recognition site. The binding of the sites to the target nucleic acid brings the split Gqs into close proximity with one another. Therefore, the complemented split Gqs recover their peroxidase activity only when these split Gq-based probes bind to the consecutive target sequences (He et al., 2012; Jiang et al., 2014; Kolpashchikov et al., 2008; Nakayama and Sintim, 2009; Ren et al., 2014). Although the design for the probes with this type of target recognition mechanism seems rather simple, it could result in a slow response because this method needs three molecules: a target and two split Gq probes, to form a complex for the generation of a peroxidase activity-based signal. Furthermore, such a separated format is not ideal for the immobilization of sensing probes that is often necessary for the development of a portable device such as an electrochemical sensing system. The other common approach for addition of target-recognition ability to Gq is the use of a molecular beacon structure (MB). A MB, composed of single-stranded DNA (ssDNA), forms a stem-loop structure. In many MB-based split Gqs, the stem part is composed of a split Gq sequence and its complementary sequence. These sequences form a double-stranded DNA (dsDNA) structure as a stem, which inhibits the ability of the MB-based split Gq to form the quadruplex structure that is necessary for the molecule to have peroxidase activity. When a target nucleic acid hybridizes with its complementary sequence on the probe, forming a double-stranded structure, the stem portion is pulled apart and thus the Gq part becomes a free ssDNA that recovers its ability to form a quadruplex structure (Xiao et al., 2004). In this mechanism, the sensing probe works as an independent single molecule, recognizing a target in a bimolecular reaction, and thus it is possible to immobilize the probe onto a solid surface easily (Freeman et al., 2011; Meng et al., 2013; Pelossof et al., 2010). However, this principle relies on the competition between the force holding the stem portion together, and the force, driven by the target hybridization, pulling the stem portion apart, which presumably induces a slow response. For instance, in case of the design of MBs, it was reported that the increase of stem length with two to four nucleotides decreased the hybridization rate between one and two orders of magnitude (Tsourkas et al., 2003). Also the relation between the stability of stem part and the rate of hybridization affected to a signal to background ratio (Monroy-Contreras and Vaca, 2011). Therefore the delicate optimization is essential to the design of target recognition site and the stem part for the MB-based split Gq. To overcome these problems with the methods described above, we employed a DNA nano-tweezers structure (DNA-NT) (Surana et al., 2011). Recently, we successfully detected a specific messenger RNA by using a fluorescence resonance energy transfer (FRET)-based DNA-NT (Funabashi et al., 2015). Upon recognition of the target nucleic acid, the FRET-based DNA-NT altered its structure from an open state to a closed state, reducing the distance between the pre-modified FRET pair fluorescent dyes and bringing them close enough together to generate a FRET signal. Therefore, this probe exhibited a signal only when it recognized a target nucleic acid. We hypothesized that this target-induced structural change of DNA-NT could also be used for switching a split-enzymatic activity, such as the complementation of split Gqs. This would allow it to become a versatile platform for the design of a novel signal-transducing molecule that directly produces a catalytic signal only when it recognizes specific nucleic acids. In our design, the split Gqs were added to both ends of the tweezers. This split Gq-based DNA-NT alters its structure from an open to a closed state, inducing the complementation of the split Gq with restored peroxidase activity (Fig. 1). It is expected that this split
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Fig. 1. Schematic of target nucleic acid detection with the split G-quadruplex (Gq)based DNA-NT. The split Gq-based DNA-NT was created by the self-assembly of three synthesized ssDNA oligonucleotides. When the split Gq-based DNA-NT recognizes a target nucleic acid, it alters its structure from an open state to a closed state. The structural change induces complementation of the split Gq, restoring its peroxidase activity. The peroxidase activity is measured by following the oxidizing reaction of ABTS in a colorimetric assay. Therefore, the split Gq-based DNA-NT can act as a signal-transducing molecule that produces a signal only when it recognizes the target nucleic acid, enabling specific nucleic acid detection in a homogeneous assay format.
Gq-based DNA-NT works as an independent sensing probe that recognizes a target nucleic acid without competitive reactions in a bimolecular reaction and directly produces enzymatic activity as a sensing signal. In this communication, we have demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT as a nucleic acid signal-transducing molecule. The restored peroxidase activity was measured by following the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which gave a greenish colorimetric response, and a partial norovirus mRNA (NV-RNA) was selected as a model target. Norovirus is one of the major viruses that cause human infectious intestinal diseases (Phillips et al., 2010). In many cases, norovirus infects humans orally via contaminated food. Therefore, the detection of this virus before intake has great potential to prevent norovirus infection (Hoffmann et al., 2012; Yan et al., 2003). However, current methods for the detection of norovirus, such as ELISA and RT-PCR, are too inconvenient to be used to detect norovirus in a POCT format. Thus, we believe that a new signal transducer molecule that recognizes NV-RNA would be of great benefit.
2. Material and methods 2.1. Production of DNA-NT and NV-RNA All nucleotide sequences used in this study were listed in the Supplementary material (Table S1). DNA oligonucleotides (O1, O2, and O3) were purchased from Integrated DNA technologies (IDT, MBL, Nagoya, Japan). Each oligonucleotide (5 μM) mixed in phosphate-buffered saline (Sigma-Aldrich, Tokyo, Japan) was incubated at 95 °C for 5 min and then gradually cooled to 10 °C to form the DNA-NT structure by self-assembly. The prepared DNA-NT was stored at 4 °C until needed. The partial sequence of norovirus capsid (norovirus Hu/GII.4/Hiroshima/216/2010/JPN gene for capsid protein: GenBank: AB555594.1, 282 nt, Table S1) was artificially synthesized with the additional restriction enzyme cleavage sites EcoRI and SalI (Eurofins Genomics, Tokyo, Japan). The gene was
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cloned into the EcoRI and SalI restriction sites of a pTNT vector (Promega KK, Tokyo, Japan) after restriction enzyme digestions. The NV-RNA was then produced with the vector using a TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) according to the manufacturer's instructions. The synthesized RNA was purified using an RNeasy Mini Kit (Qiagen K.K. Tokyo, Japan) according to the manufacturer's instructions and was stored at 80 °C. 2.2. Gel electrophoresis analysis
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2.3. Enzymatic reaction 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hemin purchased from Wako (Osaka, Japan) were dissolved in dimethyl sulfoxide (DMSO, Wako) to form 200 mM and 100 μM solutions, respectively. The DNA-NT (50 nM final), an experimental concentration of NV-RNA, and hemin (1 μM final) were mixed in 89 μL of working buffer (50 mM Tris–HCl, 150 mM NH4Cl, 20 mM KCl, and 0.03% Triton X-100, pH 7.5) and incubated for 1 h at room temperature. One microliter of ABTS (2 mM final) was then added into the reaction mixture and transferred to a 96-well clear-bottomed plate. The absorbance of the solution at 420 nm was measured immediately at 10-s intervals by a Spectra Max M5 (Molecular Devices Japan, Tokyo, Japan) after the addition of 10 μL of H2O2 (2 mM final) to become a final volume of 100 μL.
3. Results and discussion 3.1. Construction of a split Gq-based DNA-NT A split Gq-based DNA-NT was created by the self-assembly of three synthesized ssDNA oligonucleotides (O1, O2, and O3; Table S1 and Fig. 1). Two of the oligonucleotides (O1 and O2) each have a target recognition site that is a complementary sequence to the target. The third oligonucleotide (O3) has split Gq sequences on each 5ʹ and 3ʹ end. The successful construction of split Gq-based DNA-NT was confirmed by native PAGE analysis (Fig. 2A). In native PAGE analysis, the migration rate of ssDNA does not simply depend on its base length because its steric structure is maintained. Therefore, we compared the position of each band on the gel to confirm the successful construction of split Gq-based DNA-NT. First, the affinities among these ssDNAs were evaluated by comparing the band positions observed in mixtures containing combinations of each pair of oligonucleotides. In the mixture of O1 and O2 (lane 4), two bands were observed. These corresponded to the original band positions of O1 (lane 1) and O2 (lane 2), which suggests that these oligonucleotides do not have any affinity for one another. Bands above the original band position of O3 (lane 3 and lane 8) were observed in lane 5 and lane 9 (O1 and O3), and lane 6 and lane 10 (O2 and O3), while bands in the original positions of O1 alone (lane 1) or O2 alone (lane 2) were not observed.
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15% native PAGE The successful assembly of DNA-NT was confirmed by either a 15% or an 8% native polyacrylamide gel electrophoresis (PAGE) analysis. Each combination of oligonucleotides after the self-assembling process was loaded (12 μL, 500 nM) to the gel and the gels were stained with Gel Star (Lonza, Rockland, ME, USA). The target recognition ability of DNA-NT was evaluated as follows. The solutions of the NV-RNA (3 μL, 2.5 μM) and the DNA-NT (1.5 μL, 5 μM) were first mixed and incubated for 1 h at room temperature. Then, the target–DNA-NT complex was analyzed by 2% agarose gel electrophoresis. Prior to the experiment, the NVRNA was heated for 5 min at 65 °C and then immediately cooled for 1 min at 4 °C to destroy the steric structure.
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Fig. 2. Characterization of split Gq-based DNA-NT by electrophoresis analysis. (A) Gel Star-stained native PAGE gel image to confirm split Gq-based DNA-NT formation. Run with a 15% gel (Lane 1 to Lane 7) and an 8% gel (Lane 8 to Lane 11). M1, DNA step ladder (10–300 bp; Wako); Lane 1, O1; Lane 2, O2; Lane 3, O3; Lane 4, O1 þO2; Lane 5, O1 þ O3; Lane 6, O2 þ O3; Lane 7, O1 þ O2 þ O3; Lane 8, O3; Lane 9, O1 þO3; Lane 10, O2 þO3; Lane 11, O1 þO2 þO3; M2, PCR 20 bp Low Ladder (Sigma). (B) Agarose gel electrophoresis image for the evaluation of targeting ability. M, 1 kb DNA ladder (Mixell, Hiroshima, Japan); Lane 1, split Gq DNA-NT for NV-RNA; Lane 2, control split Gq DNA-NT; Lane 3, NV-RNA; Lane 4, split Gq DNANT for NV-RNA þ NV-RNA; Lane 5, control split Gq DNA-NT þNV-RNA.
Although there were some leftover of O3 observed which did not contribute to the complex formation in all lanes, these results confirmed an affinity between O1 and O3 and between O2 and O3, implying the formation of a partial dsDNA structure. The construction of split Gq-based DNA-NT was analyzed in lane 7 and lane 11, which showed a mixture of all three oligonucleotides. Bands at the sizes attributed to the original oligonucleotides (O1, O2) were rarely detected, while two weak bands corresponding to O3 and the partial dsDNA structure (O2þ O3) and a clear thick band in a position similar to the dsDNA structure (O1þ O3) were observed in lane 7. The 8% gel native PAGE clearly separated this thick band and revealed the new band seen in a higher position (lane 11) than the dsDNA structure (O1 þO3) (lane 10) in addition to O3 (lane 8) and the partial dsDNA structure (O2þ O3) (lane 9). This result implied that when the three oligonucleotides were combined, they formed a single molecule, which is a split Gqbased DNA-NT. Therefore, we concluded that the split Gq-based DNA-NT can be generated by the simple mixing of equal molar ratios of O1, O2, and O3. 3.2. Target recognition ability of split Gq-based DNA-NT Agarose gel electrophoresis was conducted to determine whether the split Gq-based DNA-NT for NV-RNA has the ability to recognize its target RNA (Fig. 2B). The single bands were observed in lanes 1 and 2 correspond to the split Gq-based DNA-NT for NVRNA and a control DNA-NT that does not contain NV-RNA recognition sites, respectively. Despite loading with NV-RNA alone, multiple bands were observed in lane 3. However, when we analyzed the RNA products with denatured gel electrophoresis, only a single band appeared (Supplementary material, Fig. S1). Therefore, we believe that all these bands observed in lane 3 were attributed to NV-RNA, assumably forming steric structures and/or self-oligomers. In lane 4 (split Gq-based DNA-NT for NV-RNA þNV-RNA), a band corresponding to the position of the split Gq-based DNA-
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3.3. Measurement of enzymatic activity induced by target recognition It is expected that the split Gq-based DNA-NT alters its structure from an open state to a closed state upon forming a complex with its target. This structural change shortens the distance between the separated split Gqs, bringing them into proximity with one another, reconstructing the Gq and restoring its peroxidase activity. To demonstrate this concept, the peroxidase activity was measured by following the oxidizing reaction of ABTS to ABTS þ . (Majcherczyk et al., 1999), which exhibits colorimetric absorbance at 420 nm. Generally, when split functional domains are used in the development of complementation assays, it is essential to adjust the distal relationship of the split domains. Accordingly, we first varied the linker distance between the DNA-NT and the split Gqs by inserting different numbers of thymines [resulting molecules termed split Gq-based DNA-NT(Tn)]. A typical time course of peroxidase activities measured with split Gq-based DNA-NTs is shown in Fig. 3A. Surprisingly, almost all of the tested split Gqbased DNA-NTs exhibited peroxidase activity in the presence of the NV-RNA, except for DNA-NT(T0). We speculate that because the target sites have gaps of four bases between their recognition
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sites (Funabashi et al., 2015), the DNA-NT(T0) could not sufficiently reduce the distance between the split Gqs to restore peroxidase activity even in the closed state. However, DNA-NT (T 41) showed clear peroxidase activity. Originally, the intact Gq sequence has the ability to bind hemin and form the Gq structure, so we suspected that the split Gqs might form a complementation format without the addition of a target. In fact, O3 itself showed a clear peroxidase activity (Supplementary material, Fig. S2). In contrast, in case of split Gq-based DNA-NTs, almost no enhanced activities comparing to hemin (without DNA-NT) were observed in the absence of target as shown in Fig. 3B except DNA-NT (T5). These results suggested that there was some active force to separate the split Gqs away against the affinity to form Gq spontaneously. We believe that the tweezers must be in an open state to actively separate the split Gqs. In case of DNA-NT (T5), the lengths of five Ts from both edges of the tweezers were considered to be long enough to meet the split Gqs even in an open state. The main body of the tweezers, which is composed of dsDNA, is negatively charged because of its phosphate groups, which may repel each other, thereby keeping the DNA-NT in an open state. Further investigation such as an AFM imaging analysis is definitely needed to reveal the real structure of split Gq-based DNA-NT. Nevertheless, it was suggested that there was some active force that helps avoid the reconstitution of the split Gq in the absence of the target nucleic acids and keeps the background signals at a low level. We decided to use the split Gq-based DNA-NT(T3) for further experiments, because it exhibited the best signal/noise ratio among the linker distances tested. The peroxidase activity measured at 120 s after the initiation of the enzymatic reaction with varying concentrations of the target NV-RNA successfully reflected the concentration of target RNA (Fig. 3C). The detection limit of 3 standard deviations from the average response of 0 nM was 4 nM. One of the reasons to limit the sensitivity was the background signal attributed to a peroxidase activity from free hemin
Absorbance (420 nm)
NT for NV-RNA (lane 1) became dimmer. In contrast, a band corresponding to the control DNA-NT (lane 2) was clearly observed in lane 5 (control split Gq DNA-NT þNV-RNA). Additionally, the bands corresponding to NV-RNA appeared to be slightly shifted from their original positions in the presence of the split Gq-based DNA-NT for NV-RNA (lane 4), but not in the presence of the control split Gq DNA-NT (lane 5), which suggests that these band shifts resulted from the binding of the split Gq-based DNA-NT to NVRNA. These results supported the conclusion that the split Gqbased DNA-NT for NV-RNA specifically recognizes NV-RNA even the target formed a different steric structure.
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Final NV-RNA concentration (nM) Fig. 3. Characterization of split Gq-based DNA-NT by the measurement of enzymatic activity. (A) Typical time courses of peroxidase activities in the presence of the NV-RNA (50 nM final) measured with split Gq-based DNA-NTs (50 nM final) that each have a different linker length [termed as a split Gq-based DNA-NT(Tn)]. (B) Comparison of colorimetric responses (Abs420 at 90 s from the initiation of the reaction) of split Gq-based DNA-NT(Tn) without NV-RNA and with NV-RNA. Fifty nanomolar (final) split Gqbased DNA-NT(Tn) and NV-RNA were used. (n¼3) (C) Dose response of the split Gq-based DNA-NT(T3). Colorimetric responses (Abs420 at 120 s from the initiation of the reaction. The Abs420 measured in the absence of the split Gq-based DNA-NT was subtracted as a baseline) are plotted. Fifty nanomolar (final) split Gq-based DNA-NT(T3) was used for each experiment.
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in a solution as seen in Fig. 3A. Therefore it is necessary to eliminate the effect from the free hemin when we develop a concrete POCT device. For instance, electrochemical detection after the immobilization of split Gq-based DNA-NT onto an electrode may be a good candidate to reduce the effect of peroxidase activity from free hemin in a bulk solution. Also, in such case, the complex, the split Gq-based DNA-NT and the target, is expected to be rather stable, and thus the target concentration effect to the electrode surface that improves the sensitivity is also expected. Nevertheless, these results proved that the split Gq-based DNA-NT can function as a signal-transducing molecule, recognizing target nucleic acids without competitive reactions in a bimolecular reaction and producing a sensing signal via restored peroxidase activity. The target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Also, during the detection of the target nucleic acids with this signal transducer, we simply mixed all components required for the reaction and measured the absorbance. This is a homogeneous assay that does not require washing steps. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device.
4. Conclusions In this communication, we have designed a split Gq-based DNA-NT for the development of a signal-transducing molecule that recognizes target nucleic acids without competitive reactions in a bimolecular reaction and directly produces a readily detectable signal. The split Gq-based DNA-NT specifically recognized the NV-RNA as a model target and exhibited peroxidase activity in response to the NV-RNA. Notably, the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure, and the detection of target NVRNA by the split Gq-based DNA-NT does not require a washing operation, only a simple mixing process. From these results and discussions, we conclude that a split Gq-based DNA-NT acts as a signal-transducing molecule for specific target nucleic acid detection that is readily applicable to a basic platform for the development of a portable device.
Acknowledgments This work was partially supported by JSPS KAKENHI Grant numbers 26289314 and 25249027.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2015.06.055.
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A split G-quadruplex-based DNA nano-tweezers structure as a signaltransducing molecule for the homogeneous detection of specific nucleic acids Keisuke Nakatsuka a, Hajime Shigeto a,b, Akio Kuroda a, Hisakage Funabashi b,n a Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan b Institute for Sustainable Sciences and Development, Hiroshima University, 1-3-2 Kagamiyama, Higashihiroshima, Hiroshima 739-8527, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 28 April 2015 Received in revised form 12 June 2015 Accepted 23 June 2015 Available online 25 June 2015
A portable method of specific nucleic acid detection would be very useful for monitoring public health in a variety of settings for point-of-care and point-of-need testing. However, conventional methods for the detection of nucleic acids are not ideal for use in the field, as they require skilled operators and complex equipment. Here, we constructed a method for specific nucleic acid detection using a split G-quadruplex (Gq) structure that can recognize target nucleic acids without competitive reactions in a bimolecular reaction and directly produce a detectable signal based on peroxidase activity. We developed a single signal-transducing molecule with a split Gq-based DNA-nano tweezers (NT) structure that self-assembles from three single-stranded DNAs through simple mixing, and detects its target without requiring any washing steps. A model target, a partial norovirus mRNA (NV-RNA), was specifically recognized by the split Gq-based DNA-NT, causing it to undergo a structural change that restored its peroxidase activity. The peroxidase activity was measured by following the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the NV-RNA concentration. The lower detection limit was 4 nM. Our results demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT structure as a nucleic acid signal-transducing molecule in a homogenous assay format. Also the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device. & 2015 Elsevier B.V. All rights reserved.
Keywords: Homogeneous assay Norovirus Nucleic acid detection Point-of-care testing Signal-transducing molecule Split G-quadruplex
1. Introduction The detection of specific DNA and RNA is widely used not only for diagnostic purposes, but also for monitoring public health with regard to epidemics, food safety, and environmental pollution. The detection of specific nucleic acids enables a genetic marker analysis in addition to simple target detection. Therefore, the detection in a portable format has great potential for use in a wide variety of applications, such as point-of-care testing (POCT) and point-of-need testing (PONT) (Ahmad and Hashsham, 2012; Craw and Balachandran, 2012; Gubala et al., 2012; Hartman et al., 2013; Niemz et al., 2011). However, conventional methods such as PCRbased methods and DNA arrays require skilled operation, special equipment, and time-consuming complex procedures, which are n Correspondence to: Room 404, VBL Office, 2-313 Kagamiyama, Higashihiroshima, Hiroshima 739-8527, Japan. E-mail address: [email protected] (H. Funabashi).
http://dx.doi.org/10.1016/j.bios.2015.06.055 0956-5663/& 2015 Elsevier B.V. All rights reserved.
not ideal for a portable device. Thus, the development of a novel easy-to-use nucleic acid detection principle is highly desirable. We believe that the development of a signal-transducing molecule capable of recognizing target nucleic acids and directly producing a readily detectable signal is ideal for constructing a convenient nucleic acid detection method. Also, such a signaltransducing molecule should be able to produce a signal only when it recognizes its target, enabling its detection in a homogeneous assay that does not require washing steps and therefore is readily applicable to a portable format. To develop such a signaltransducing molecule, we focused on a G-quadruplex (Gq) structure (Sen and Gilbert, 1988) as a signal generator. A Gq is one of the well-characterized DNAzymes that has a guanine-rich sequence and exhibits peroxidase activity with the help of hemin as a co-factor (Travascio et al., 1998; Travascio et al., 2001). Because many detection methods for peroxidase activity are available, a Gq is often used in DNA-based sensor devices as a signal generator for specific nucleic acid detection (Lu et al., 2013; Roembke et al., 2013).
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One approach to add target-recognition ability to a Gq is the use of a split Gq sequence. Each split Gq sequence is connected to a consecutive complementary sequence for a target nucleic acid as a target recognition site. The binding of the sites to the target nucleic acid brings the split Gqs into close proximity with one another. Therefore, the complemented split Gqs recover their peroxidase activity only when these split Gq-based probes bind to the consecutive target sequences (He et al., 2012; Jiang et al., 2014; Kolpashchikov et al., 2008; Nakayama and Sintim, 2009; Ren et al., 2014). Although the design for the probes with this type of target recognition mechanism seems rather simple, it could result in a slow response because this method needs three molecules: a target and two split Gq probes, to form a complex for the generation of a peroxidase activity-based signal. Furthermore, such a separated format is not ideal for the immobilization of sensing probes that is often necessary for the development of a portable device such as an electrochemical sensing system. The other common approach for addition of target-recognition ability to Gq is the use of a molecular beacon structure (MB). A MB, composed of single-stranded DNA (ssDNA), forms a stem-loop structure. In many MB-based split Gqs, the stem part is composed of a split Gq sequence and its complementary sequence. These sequences form a double-stranded DNA (dsDNA) structure as a stem, which inhibits the ability of the MB-based split Gq to form the quadruplex structure that is necessary for the molecule to have peroxidase activity. When a target nucleic acid hybridizes with its complementary sequence on the probe, forming a double-stranded structure, the stem portion is pulled apart and thus the Gq part becomes a free ssDNA that recovers its ability to form a quadruplex structure (Xiao et al., 2004). In this mechanism, the sensing probe works as an independent single molecule, recognizing a target in a bimolecular reaction, and thus it is possible to immobilize the probe onto a solid surface easily (Freeman et al., 2011; Meng et al., 2013; Pelossof et al., 2010). However, this principle relies on the competition between the force holding the stem portion together, and the force, driven by the target hybridization, pulling the stem portion apart, which presumably induces a slow response. For instance, in case of the design of MBs, it was reported that the increase of stem length with two to four nucleotides decreased the hybridization rate between one and two orders of magnitude (Tsourkas et al., 2003). Also the relation between the stability of stem part and the rate of hybridization affected to a signal to background ratio (Monroy-Contreras and Vaca, 2011). Therefore the delicate optimization is essential to the design of target recognition site and the stem part for the MB-based split Gq. To overcome these problems with the methods described above, we employed a DNA nano-tweezers structure (DNA-NT) (Surana et al., 2011). Recently, we successfully detected a specific messenger RNA by using a fluorescence resonance energy transfer (FRET)-based DNA-NT (Funabashi et al., 2015). Upon recognition of the target nucleic acid, the FRET-based DNA-NT altered its structure from an open state to a closed state, reducing the distance between the pre-modified FRET pair fluorescent dyes and bringing them close enough together to generate a FRET signal. Therefore, this probe exhibited a signal only when it recognized a target nucleic acid. We hypothesized that this target-induced structural change of DNA-NT could also be used for switching a split-enzymatic activity, such as the complementation of split Gqs. This would allow it to become a versatile platform for the design of a novel signal-transducing molecule that directly produces a catalytic signal only when it recognizes specific nucleic acids. In our design, the split Gqs were added to both ends of the tweezers. This split Gq-based DNA-NT alters its structure from an open to a closed state, inducing the complementation of the split Gq with restored peroxidase activity (Fig. 1). It is expected that this split
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Fig. 1. Schematic of target nucleic acid detection with the split G-quadruplex (Gq)based DNA-NT. The split Gq-based DNA-NT was created by the self-assembly of three synthesized ssDNA oligonucleotides. When the split Gq-based DNA-NT recognizes a target nucleic acid, it alters its structure from an open state to a closed state. The structural change induces complementation of the split Gq, restoring its peroxidase activity. The peroxidase activity is measured by following the oxidizing reaction of ABTS in a colorimetric assay. Therefore, the split Gq-based DNA-NT can act as a signal-transducing molecule that produces a signal only when it recognizes the target nucleic acid, enabling specific nucleic acid detection in a homogeneous assay format.
Gq-based DNA-NT works as an independent sensing probe that recognizes a target nucleic acid without competitive reactions in a bimolecular reaction and directly produces enzymatic activity as a sensing signal. In this communication, we have demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT as a nucleic acid signal-transducing molecule. The restored peroxidase activity was measured by following the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which gave a greenish colorimetric response, and a partial norovirus mRNA (NV-RNA) was selected as a model target. Norovirus is one of the major viruses that cause human infectious intestinal diseases (Phillips et al., 2010). In many cases, norovirus infects humans orally via contaminated food. Therefore, the detection of this virus before intake has great potential to prevent norovirus infection (Hoffmann et al., 2012; Yan et al., 2003). However, current methods for the detection of norovirus, such as ELISA and RT-PCR, are too inconvenient to be used to detect norovirus in a POCT format. Thus, we believe that a new signal transducer molecule that recognizes NV-RNA would be of great benefit.
2. Material and methods 2.1. Production of DNA-NT and NV-RNA All nucleotide sequences used in this study were listed in the Supplementary material (Table S1). DNA oligonucleotides (O1, O2, and O3) were purchased from Integrated DNA technologies (IDT, MBL, Nagoya, Japan). Each oligonucleotide (5 μM) mixed in phosphate-buffered saline (Sigma-Aldrich, Tokyo, Japan) was incubated at 95 °C for 5 min and then gradually cooled to 10 °C to form the DNA-NT structure by self-assembly. The prepared DNA-NT was stored at 4 °C until needed. The partial sequence of norovirus capsid (norovirus Hu/GII.4/Hiroshima/216/2010/JPN gene for capsid protein: GenBank: AB555594.1, 282 nt, Table S1) was artificially synthesized with the additional restriction enzyme cleavage sites EcoRI and SalI (Eurofins Genomics, Tokyo, Japan). The gene was
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cloned into the EcoRI and SalI restriction sites of a pTNT vector (Promega KK, Tokyo, Japan) after restriction enzyme digestions. The NV-RNA was then produced with the vector using a TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) according to the manufacturer's instructions. The synthesized RNA was purified using an RNeasy Mini Kit (Qiagen K.K. Tokyo, Japan) according to the manufacturer's instructions and was stored at 80 °C. 2.2. Gel electrophoresis analysis
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2.3. Enzymatic reaction 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hemin purchased from Wako (Osaka, Japan) were dissolved in dimethyl sulfoxide (DMSO, Wako) to form 200 mM and 100 μM solutions, respectively. The DNA-NT (50 nM final), an experimental concentration of NV-RNA, and hemin (1 μM final) were mixed in 89 μL of working buffer (50 mM Tris–HCl, 150 mM NH4Cl, 20 mM KCl, and 0.03% Triton X-100, pH 7.5) and incubated for 1 h at room temperature. One microliter of ABTS (2 mM final) was then added into the reaction mixture and transferred to a 96-well clear-bottomed plate. The absorbance of the solution at 420 nm was measured immediately at 10-s intervals by a Spectra Max M5 (Molecular Devices Japan, Tokyo, Japan) after the addition of 10 μL of H2O2 (2 mM final) to become a final volume of 100 μL.
3. Results and discussion 3.1. Construction of a split Gq-based DNA-NT A split Gq-based DNA-NT was created by the self-assembly of three synthesized ssDNA oligonucleotides (O1, O2, and O3; Table S1 and Fig. 1). Two of the oligonucleotides (O1 and O2) each have a target recognition site that is a complementary sequence to the target. The third oligonucleotide (O3) has split Gq sequences on each 5ʹ and 3ʹ end. The successful construction of split Gq-based DNA-NT was confirmed by native PAGE analysis (Fig. 2A). In native PAGE analysis, the migration rate of ssDNA does not simply depend on its base length because its steric structure is maintained. Therefore, we compared the position of each band on the gel to confirm the successful construction of split Gq-based DNA-NT. First, the affinities among these ssDNAs were evaluated by comparing the band positions observed in mixtures containing combinations of each pair of oligonucleotides. In the mixture of O1 and O2 (lane 4), two bands were observed. These corresponded to the original band positions of O1 (lane 1) and O2 (lane 2), which suggests that these oligonucleotides do not have any affinity for one another. Bands above the original band position of O3 (lane 3 and lane 8) were observed in lane 5 and lane 9 (O1 and O3), and lane 6 and lane 10 (O2 and O3), while bands in the original positions of O1 alone (lane 1) or O2 alone (lane 2) were not observed.
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Fig. 2. Characterization of split Gq-based DNA-NT by electrophoresis analysis. (A) Gel Star-stained native PAGE gel image to confirm split Gq-based DNA-NT formation. Run with a 15% gel (Lane 1 to Lane 7) and an 8% gel (Lane 8 to Lane 11). M1, DNA step ladder (10–300 bp; Wako); Lane 1, O1; Lane 2, O2; Lane 3, O3; Lane 4, O1 þO2; Lane 5, O1 þ O3; Lane 6, O2 þ O3; Lane 7, O1 þ O2 þ O3; Lane 8, O3; Lane 9, O1 þO3; Lane 10, O2 þO3; Lane 11, O1 þO2 þO3; M2, PCR 20 bp Low Ladder (Sigma). (B) Agarose gel electrophoresis image for the evaluation of targeting ability. M, 1 kb DNA ladder (Mixell, Hiroshima, Japan); Lane 1, split Gq DNA-NT for NV-RNA; Lane 2, control split Gq DNA-NT; Lane 3, NV-RNA; Lane 4, split Gq DNANT for NV-RNA þ NV-RNA; Lane 5, control split Gq DNA-NT þNV-RNA.
Although there were some leftover of O3 observed which did not contribute to the complex formation in all lanes, these results confirmed an affinity between O1 and O3 and between O2 and O3, implying the formation of a partial dsDNA structure. The construction of split Gq-based DNA-NT was analyzed in lane 7 and lane 11, which showed a mixture of all three oligonucleotides. Bands at the sizes attributed to the original oligonucleotides (O1, O2) were rarely detected, while two weak bands corresponding to O3 and the partial dsDNA structure (O2þ O3) and a clear thick band in a position similar to the dsDNA structure (O1þ O3) were observed in lane 7. The 8% gel native PAGE clearly separated this thick band and revealed the new band seen in a higher position (lane 11) than the dsDNA structure (O1 þO3) (lane 10) in addition to O3 (lane 8) and the partial dsDNA structure (O2þ O3) (lane 9). This result implied that when the three oligonucleotides were combined, they formed a single molecule, which is a split Gqbased DNA-NT. Therefore, we concluded that the split Gq-based DNA-NT can be generated by the simple mixing of equal molar ratios of O1, O2, and O3. 3.2. Target recognition ability of split Gq-based DNA-NT Agarose gel electrophoresis was conducted to determine whether the split Gq-based DNA-NT for NV-RNA has the ability to recognize its target RNA (Fig. 2B). The single bands were observed in lanes 1 and 2 correspond to the split Gq-based DNA-NT for NVRNA and a control DNA-NT that does not contain NV-RNA recognition sites, respectively. Despite loading with NV-RNA alone, multiple bands were observed in lane 3. However, when we analyzed the RNA products with denatured gel electrophoresis, only a single band appeared (Supplementary material, Fig. S1). Therefore, we believe that all these bands observed in lane 3 were attributed to NV-RNA, assumably forming steric structures and/or self-oligomers. In lane 4 (split Gq-based DNA-NT for NV-RNA þNV-RNA), a band corresponding to the position of the split Gq-based DNA-
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3.3. Measurement of enzymatic activity induced by target recognition It is expected that the split Gq-based DNA-NT alters its structure from an open state to a closed state upon forming a complex with its target. This structural change shortens the distance between the separated split Gqs, bringing them into proximity with one another, reconstructing the Gq and restoring its peroxidase activity. To demonstrate this concept, the peroxidase activity was measured by following the oxidizing reaction of ABTS to ABTS þ . (Majcherczyk et al., 1999), which exhibits colorimetric absorbance at 420 nm. Generally, when split functional domains are used in the development of complementation assays, it is essential to adjust the distal relationship of the split domains. Accordingly, we first varied the linker distance between the DNA-NT and the split Gqs by inserting different numbers of thymines [resulting molecules termed split Gq-based DNA-NT(Tn)]. A typical time course of peroxidase activities measured with split Gq-based DNA-NTs is shown in Fig. 3A. Surprisingly, almost all of the tested split Gqbased DNA-NTs exhibited peroxidase activity in the presence of the NV-RNA, except for DNA-NT(T0). We speculate that because the target sites have gaps of four bases between their recognition
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sites (Funabashi et al., 2015), the DNA-NT(T0) could not sufficiently reduce the distance between the split Gqs to restore peroxidase activity even in the closed state. However, DNA-NT (T 41) showed clear peroxidase activity. Originally, the intact Gq sequence has the ability to bind hemin and form the Gq structure, so we suspected that the split Gqs might form a complementation format without the addition of a target. In fact, O3 itself showed a clear peroxidase activity (Supplementary material, Fig. S2). In contrast, in case of split Gq-based DNA-NTs, almost no enhanced activities comparing to hemin (without DNA-NT) were observed in the absence of target as shown in Fig. 3B except DNA-NT (T5). These results suggested that there was some active force to separate the split Gqs away against the affinity to form Gq spontaneously. We believe that the tweezers must be in an open state to actively separate the split Gqs. In case of DNA-NT (T5), the lengths of five Ts from both edges of the tweezers were considered to be long enough to meet the split Gqs even in an open state. The main body of the tweezers, which is composed of dsDNA, is negatively charged because of its phosphate groups, which may repel each other, thereby keeping the DNA-NT in an open state. Further investigation such as an AFM imaging analysis is definitely needed to reveal the real structure of split Gq-based DNA-NT. Nevertheless, it was suggested that there was some active force that helps avoid the reconstitution of the split Gq in the absence of the target nucleic acids and keeps the background signals at a low level. We decided to use the split Gq-based DNA-NT(T3) for further experiments, because it exhibited the best signal/noise ratio among the linker distances tested. The peroxidase activity measured at 120 s after the initiation of the enzymatic reaction with varying concentrations of the target NV-RNA successfully reflected the concentration of target RNA (Fig. 3C). The detection limit of 3 standard deviations from the average response of 0 nM was 4 nM. One of the reasons to limit the sensitivity was the background signal attributed to a peroxidase activity from free hemin
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NT for NV-RNA (lane 1) became dimmer. In contrast, a band corresponding to the control DNA-NT (lane 2) was clearly observed in lane 5 (control split Gq DNA-NT þNV-RNA). Additionally, the bands corresponding to NV-RNA appeared to be slightly shifted from their original positions in the presence of the split Gq-based DNA-NT for NV-RNA (lane 4), but not in the presence of the control split Gq DNA-NT (lane 5), which suggests that these band shifts resulted from the binding of the split Gq-based DNA-NT to NVRNA. These results supported the conclusion that the split Gqbased DNA-NT for NV-RNA specifically recognizes NV-RNA even the target formed a different steric structure.
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Final NV-RNA concentration (nM) Fig. 3. Characterization of split Gq-based DNA-NT by the measurement of enzymatic activity. (A) Typical time courses of peroxidase activities in the presence of the NV-RNA (50 nM final) measured with split Gq-based DNA-NTs (50 nM final) that each have a different linker length [termed as a split Gq-based DNA-NT(Tn)]. (B) Comparison of colorimetric responses (Abs420 at 90 s from the initiation of the reaction) of split Gq-based DNA-NT(Tn) without NV-RNA and with NV-RNA. Fifty nanomolar (final) split Gqbased DNA-NT(Tn) and NV-RNA were used. (n¼3) (C) Dose response of the split Gq-based DNA-NT(T3). Colorimetric responses (Abs420 at 120 s from the initiation of the reaction. The Abs420 measured in the absence of the split Gq-based DNA-NT was subtracted as a baseline) are plotted. Fifty nanomolar (final) split Gq-based DNA-NT(T3) was used for each experiment.
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in a solution as seen in Fig. 3A. Therefore it is necessary to eliminate the effect from the free hemin when we develop a concrete POCT device. For instance, electrochemical detection after the immobilization of split Gq-based DNA-NT onto an electrode may be a good candidate to reduce the effect of peroxidase activity from free hemin in a bulk solution. Also, in such case, the complex, the split Gq-based DNA-NT and the target, is expected to be rather stable, and thus the target concentration effect to the electrode surface that improves the sensitivity is also expected. Nevertheless, these results proved that the split Gq-based DNA-NT can function as a signal-transducing molecule, recognizing target nucleic acids without competitive reactions in a bimolecular reaction and producing a sensing signal via restored peroxidase activity. The target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Also, during the detection of the target nucleic acids with this signal transducer, we simply mixed all components required for the reaction and measured the absorbance. This is a homogeneous assay that does not require washing steps. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device.
4. Conclusions In this communication, we have designed a split Gq-based DNA-NT for the development of a signal-transducing molecule that recognizes target nucleic acids without competitive reactions in a bimolecular reaction and directly produces a readily detectable signal. The split Gq-based DNA-NT specifically recognized the NV-RNA as a model target and exhibited peroxidase activity in response to the NV-RNA. Notably, the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure, and the detection of target NVRNA by the split Gq-based DNA-NT does not require a washing operation, only a simple mixing process. From these results and discussions, we conclude that a split Gq-based DNA-NT acts as a signal-transducing molecule for specific target nucleic acid detection that is readily applicable to a basic platform for the development of a portable device.
Acknowledgments This work was partially supported by JSPS KAKENHI Grant numbers 26289314 and 25249027.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2015.06.055.
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