Dynamical Regulation of Enzyme Cascade Amplification by a

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Dynamical Regulation of Enzyme Cascade Amplification by a Regenerated DNA Nanotweezer for Ultrasensitive Electrochemical DNA Detection Beibei Kou, Yaqin Chai, Yali Yuan, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00477 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Analytical Chemistry

Dynamical Regulation of Enzyme Cascade Amplification by a Regenerated DNA Nanotweezer for Ultrasensitive Electrochemical DNA Detection Beibei Kou, Yaqin Chai, Yali Yuan*, Ruo Yuan∗

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University）, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

∗

Corresponding authors at: Tel.: +86-23-68252277, fax: +86-23-68253172. E-mail: [email protected] (Y. L. Yuan); [email protected] (R. Yuan). 1

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ABSTRACT Traditional scaffolds such as metal nanoparticles, DNA origami remain a considerable challenge to regulate the enzyme cascade catalytic efficiency dynamically and reversibly on account of their irreversible conformation. To address these issues, a regenerated DNA tweezer was designed to dynamically regulate the interenzyme spacing for high-efficiency enzyme cascade amplification for homogeneous determination of target DNA related to cancer diseases. Initially, the enzyme-functionalized DNA tweezer was maintained at the opened state with relatively distant interenzyme distance (19 ~ 24 nm), leading to a low catalytic efficiency. Benefiting from target induced Mg2+-dependent DNAzyme cleavage recycling, the one input target could be transduced to multiple corresponding methylene blue (MB) labeled DNA (S5), which were served as not only signal probe to provide detectable electrochemical signal, but also fuel to switch DNA tweezer from opened to closed state, leading to cascaded enzymes close enough (5 ~ 10 nm) for enhancing the catalytic efficiency for sensitive target DNA analysis with a low detection limit down to 30 fM. In the presence of anti-fuels, the closed DNA tweezer easily switched back to opened state via one-step strand displacement, and the obtained DNA tweezer achieved regeneration for subsequently recycling target detection. With the dynamical regulation of interenzyme distance in an “open-close-open” way, the enzyme cascade catalytic efficiency became dynamically controllable and the DNA tweezer realized simply reutilization over five times, overcoming the drawbacks of inflexible, time-consuming operation and false positive 2


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signal induced by traditional scaffolds. More importantly, this method opened a new avenue for employing the arbitrary change of enzyme cascade catalytic efficiency for sensitive detection various biomolecules. KEYWORDS: regenerated DNA tweezer; dynamical regulation; enzyme cascade amplification; electrochemical DNA detection

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The accurate analysis of specific sequences of DNA has attracted great research interest in the fields of environmental monitoring, genetics therapy, early screening of cancers, etc.1,2 Owing to the DNA concentration with a very low level in biological samples, it is thus essential to explore a simple and sensitive method to detect nucleic acid. 3,4 The enzyme cascade amplification is defined as a chain of chemical reactions proceed in a concurrent fashion, having been gained much attention for application in DNA biosensor with high sensitivity.5-8 In a typical enzyme cascade reaction, the catalytic cascade needs effective channeling for substrates transfering from one enzymatic process to a subsequent catalytic process.9-11 Therefore, appropriate interenzyme distance to generate high local concentrations of the products and prohibit their diffusion is of great importance for high-efficiency enzyme cascade amplification.12-15 However, designing a reliable scaffold which is capable of efficiently regulating interenzyme distance in practical application is a great challenge. In previous work, we successfully demonstrated rigid and one-step synthesis scaffold PtNPs to regulate interenzyme distance for matrix metalloproteinases-2 (MMP-2) detection.16 Nevertheless, it was difficult to maintain the size and morphology of PtNPs due to its easy aggregation property.17 Moreover, the size-inherent feature as well as strong bonding capability with enzymes made PtNPs hard to regulate interenzyme distance dynamically and reversibly, thus resulting in poor regeneration ability of biosensor for target quantitative detection. It is thus imperative to develop a regenerated scaffold which interenzyme distance can be easily manipulated. 4


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DNA nanostructure has currently emerged as a promising scaffold to organize biomolecules with nanoscale precision.18-22 In comparison with metal nanoparticles, two-dimensional DNA nanostructure showed more precise distance regulation and flexible operation based on their structural programmability and accurate addressability.23,24 For instance, many studies utilized DNA origami to tether sequential enzymes at predesigned positions, and systematically varied the interenzyme distance for optimal enzyme activity.25-27 Despite these successes, such strategies could only achieve static regulation of interenzyme distance since irreversible conformation of DNA origami. Furthermore, the folding of a long DNA scaffold by hundreds of short staple strands into nanostructures made the preparation of DNA origami complicated and time-consuming. In addition, three-dimensional DNA nanostructure such as DNA tetrahedron also existed above-mentioned problems in terms of distance regulation. It was worth mentioning that the switchable DNA machine provides ideas for dynamic regulation. For example, DNA tweezers could be acheived autonomous switchable motion with a addressable conformation change in response to external triggers, such as light28 or metal ions29, paving the way for the design of regenerated scaffold with high mechanical rigidity, dynamic tunability and continuous controllability.30-34 Unfortunately, these methods not only had the strict reaction conditions, but also needed to introduce toxic azobenzene moieties, which may lead to a reduction of enzyme activity. To solve above issues, a regenerated DNA tweezer which can be reversibly switched between “opened” and “closed” state with nucleic acid strand as external 5



triggers was designed and served as scaffold to regulate the interenzyme spacing for high-efficiency enzyme cascade amplification for homogeneous target DNA determination. As shown in Scheme 1, glucose oxidase (GOx) and horseradish peroxidase (HRP) as model enzymes were respectively modified with the arms of opened DNA tweezer, and at this time two enzymes is spatially separated with low catalytic efficiency. With the aid of target induced Mg2+-dependent DNAzyme cleavage recycling amplication, the output methylene blue (MB) labeled DNA (S5) was used as fuel to trigger switching of the DNA tweezer from opened to closed state, which also provided detectable electrochemical signal for sensitive analysis of target DNA. Here, it was worth mentioning that the proximity of cascaded enzymes facilitated the transport of substrates, bringing in efficient enzyme cascade amplification. More importantly, it could be switched back to opened state in the presence of anti-fuel, leading to the regeneration of DNA tweezer for consecutive target detection. In addition, the homogeneous detection improved the specificity and maneuverability of proposed biosensor. In a word, this work would open up an attractive method for employing reconfigurational switching of nanomechine to dynamically regulate the enzyme cascade catalytic efficiency for detecting various targets in the further. For instance, with the introduction of a series of DNA structures (e.g., anti-ATP aptamer, split G-quadruplex structures and i-motif) to DNA tweezers,

the configuration of DNA tweezer can reversibly switch in response to specific targets (ATP, metal ions and protons).

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EXPERIMENTAL SECTION Target

DNA

Induced

Mg2+-Dependent

DNAzyme

Cleavage

Recycling

Amplification Mg2+-dependent DNAzyme cleavage recycling system included hairpin DNA 1 (S1), subunit 2 (S2) and 3′-MB labeled hairpin DNA 3 (S3). Upon introduction of target DNA, hairpin-structured S1 was first opened, thus leading to the assembly of S1 and S2 to form active DNAzyme structure, and then combined with S3 to catalyze its cleavage in presence of Mg2+, resulting in the release of two fragments S4 and MB labeled S5 (cycle I). Interestingly, the released S4 could be act as secondary target DNA to open S1 for next cycle to produce amount of S5 (cycle II), which not only provided detectable electrochemical signal for target DNA analysis, but also was served as fuel to trigger switching of DNA tweezer from opened to closed state. Additionally, the remaining active DNAzyme could enter next target recycling. Before use, hairpin-structured S1, S3 were heated to 95 ºC for 5 min and then cooled to room temperature for 1 h. Then, 15 µL S1, 15 µL S2 and 15 µL S3 at the same concentration of 4 µM were incubated into 35 µL Tris-HCl buffer (20 mM KCl, 10 mM MgCl2 and 100 mM NaCl, pH 7.4) containing different concentrations of target DNA for 2 h at 37 °C. Assembly of Enzyme-functionalized DNA Tweezers To assemble DNA tweezers, equimolar A-E sequences were mixed together in TAE/Mg2+ buffer (pH 8.0)35 to reach a final concentration of 0.5 µM, followed by the annealing process in a ETC811 Thermal Cycler (Bio-Rad, U.S.A.).36 7



The enzyme-functionalized DNA tweezers were prepared by the following steps. First, 60 µL GOx (1 µM) and 120 µL HRP (1 µM) were mixed with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP, 20 mM) solution for 2 h to active amino groups of enzymes, respectively. In parallel, 80 µL DNA tweezers was pretreated with dithiothreitol (DTT, 50 mM) for 2 h to avoid the formation of S−S bond. Then, the actived enzymes-SPDP and pretreated DNA tweezers were mixed and reacted for 2 h. At last, the obtained enzyme-functionalized DNA tweezers were stored at 4 °C for further use. The cyclic addition of 0.75 µM fuel and anti-fuel strands for 40 min could achieve the reversible switches of DNA tweezers between opened and closed states.

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Scheme 1 Illustration of the electrochemical DNA biosensor based on: (I) Mg2+-dependent DNAzyme cleavage recycling amplification and (II) the dynamical regulation of enzyme cascade reaction by regenerated DNA tweezer.

RESULTS AND DISCUSSIONS Characterization of enzymes-functionalized DNA tweezer To verify that we have obtained enzymes-functionalized DNA tweezer successfully, 12% native PAGE was implemented. As shown in Figure 1, the single strand DNA E with the lowest molecular weight was run fastest (lane 1). After the hybridization of the five DNA strands (A-E) by the annealing process, the band with reduced mobility in lane 2 was observed, illustrating the successful self-assembly of the opened DNA tweezer. In addition, HRP-functionalized opened DNA tweezer exhibited a lower electrophoretic mobility (lane 3) than that of opened DNA tweezer without enzymes (lane 2), but possessed a faster mobility compared to enzymes-functionalized opened DNA tweezer (lane 4), indicating that GOx and HRP effectively attached to the opened DNA tweezer, respectively. Atomic force microscopy (AFM) was further confirmed the structural conformation of opened and closed tweezers (Figure S1, see Supporting Information). We could see that inter-arm distance for opened DNA tweezers was 19 ~ 24 nm and closed DNA tweezers was 5 ~ 10 nm.

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Figure 1. PAGE characterization of the enzymes-functionalized DNA tweezer. Lane 1: E (5 µM); Lane 2: opened DNA tweezer; Lane 3: HRP-functionalized opened DNA tweezer; Lane 4: enzymes-functionalized opened DNA tweezer.

The Validation of Enzyme Cascade Amplification Strategy To verify the successful construction of the regenerated biosensor, differential pulse voltammetry (DPV) was measured in phosphate buffered solution (PBS, pH 7.4). As displayed in Figure 2A, the DNA tweezer was kept in the opened state at the beginning, no DPV peak was observed (curve a) owing to the lack of electroactive species in this case. When DNA tweezer hybridized with the fuel, the DPV signal significantly increased (curve b) because fuel triggered the switching of the opened DNA tweezer to closed state. During this process, the spatial distance between cascaded enzymes were in close proximity, thereby resulting in efficient enzyme cascade amplification. After the successive strand displacement of anti-fuel, the DPV peak currents exhibited rapidly decreased (curve c) on account of DNA tweezer switched back to the opened state

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again. These results demonstrated the successful regeneration of constructed biosensor. We also studied the contribution of glucose in detection solution to the efficiency of enzyme cascade amplification. As depicted in Figure 2B, the addition of 4.0 mM glucose in detection solution led to a significant enhancement in current response compared with the absence of glucose, which was ascribed the fact that H2O2 generated by GOx accumulated on surface of HRP with high local concentrations and subsequently would be decomposed to facilitate the oxidation of MB, leading to prominent enhancement of biosensor sensitivity.

Figure 2 (A) DPV responses of the proposed biosensor with the original opened state of DNA tweezer (curve a); closed state of DNA tweezer (curve b); and regenerated DNA tweezer with opened state (curve c); (B) Signal changes of the DPV peak without and with 4.0 mM glucose in the detection solution containing 0.1 M PBS (pH 7.4).

The Efficiency of Distance-Dependent Enzyme Cascade Amplification To confirm the enzyme cascade catalytic efficiency closely related to interenzyme distance between GOx and HRP, we explored the performance of proposed biosensor in three different states of DNA tweezer. As depicted in Figure 3, the biosensor with closed 11



DNA tweezer (Figure 3C) showed much greater DPV peak current in comparison with that obtained by the biosensor with free DNA tweezer, opened DNA tweezer (Figure 3A-B), respectively. Such a phenomenon can be ascribed to the fact that close proximity of enzymes would efficiently facilitate transport of reaction products, resulting in efficient enzyme cascade amplification for target DNA detection.

Figure 3 DPV curves of the different format biosensor in 1 mL PBS (pH 7.4) with three states of DNA tweezer: (A) free DNA tweezer, (B) opened DNA tweezer, (C) closed DNA tweezer.

Optimization of Experimental Conditions The S5 from target induced Mg2+-dependent DNAzyme cleavage recycling amplification could switch opened DNA tweezer to closed state, so Mg2+-dependent DNAzyme cleavage time had great effect on the efficiency of enzyme cascade ampliﬁcation, and the optimum cleavage time of Mg2+-dependent DNAzyme was investigated under 10 nM target DNA. As displayed in Figure 4A, the DPV peak current increased with the increment of Mg2+-dependent DNAzyme cleavage time and almost remained constant after 60 min. Consequently, 60 min was chosen as the optimal cleavage time. Furthermore, the glucose concentration in testing buffer solution had a vital impact on electrochemical 12


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performance because glucose was oxidized by GOx to yield H2O2 for accelerating the oxidation of MB with the help of HRP. To investigate the effect of glucose concentration, the electrochemical biosensor was measured in PBS solution (pH 7.4) with different concentration of glucose. From Figure 4B, it was found that the DPV peak current increased continually with glucose concentration ranging from 0 to 4.0 mM, and tended to level off after 4.0 mM. Hence, the optimal concentration of glucose was 4.0 mM in the following experiments.

Figure 4 Optimization of (A) Mg2+-dependent DNAzyme cleavage time, and (B) glucose concentration in the detection solution.

Performance of Proposed Biosensor The dependence of the DPV signal upon target DNA concentrations was investigated under the optimal conditions. As presented in Figure 5A, the incremental target concentrations ranging from 0.1 pM to 100 nM led to a gradual DPV signal enhancement. Figure 5B indicated a prominent liner relationship between DPV signal and the logarithm of target concentration with a linear regression equation of I = - 0.192 lgctarget -1.746 (r = -0.990) and the estimated detection limit was 30 fM. Impressively, from Table 1 and 2, we could see that the proposed biosensor exhibited higher sensitivity in comparison with reported 13



literatures for target DNA detection on account of target induced Mg2+-dependent DNAzyme cleavage recycling amplification and efficient enzyme cascade amplification dynamically regulated by DNA nanotweezer.

Figure 5 (A) The DPVs of proposed biosensor towards various target DNA concentrations: 0.1 pM, 1 pM, 10 pM, 0.1 nM, 1 nM, 10 nM and 100 nM. (B) The corresponding calibration curve

(error bars: SD; n = 3).

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Regenerability of the Biosensor The difficult states of DNA tweezer could be achieved reversible conversion by cyclic addition fuel and anti-fuel, resulting in the regeneration of the proposed biosensor. As exhibited in Figure 6, the proposed biosensor could be regenerated over five times for target DNA assay, which demonstrated the ideal regeneration of the constructed biosensor.

Figure 6 The regeneration of the proposed biosensor with 10 nM target DNA in five times.

Clinical Serum Samples Analysis To demonstrate the potential application of proposed biosensor for real samples, 50-fold diluted healthy serum samples with different concentrations of target (0.01, 0.1, 1, and 10 nM) were monitored. It was found from Table 3 that recovery ranged from 96.0% to 104% and RSD was between 2.4% and 4.1%, indicating our proposed method could be applied to real samples.

CONCLUSION 15



In short, a universal electrochemical assay was established for highly sensitive target DNA detection on basis of regenerated and distance-controllable DNA tweezer regulated interenzyme spacing for highly efficient enzyme cascade amplification. This study included the following three major innovations: (i) The switching of DNA tweezer from opened to closed state bought in cascaded enzymes close enough, thus leading to efficient enzyme cascade amplification for enhancing the sensitivity of biosensor. (ii) The “open-close-open” strategy based on dynamical regulation of interenzyme distance realized the regeneration of DNA tweezer for consecutive target detection, which had the advantages of flexible operation, saving time in comparison with the conventional scaffolds. (iii) The homogeneous target detection endowed proposed biosensor with powerful reliability and reproducibility. In addition, the present method paves a new pattern to dynamically regulate enzyme cascade reaction though configuration switching of nanomechine for various target analysis. ASSOCIATED CONTENT Supporting Information Chemicals and materials, apparatus, native polyacrylamide gel electrophoresis, AFM characterization of enzymes-functionalized DNA tweezer, and specificity and reproducibility of the biosensor were supplied in Supporting Information. ACKNOWLEDGEMENT This work was supported by the NNSF of China (21775124, 21505107, 21575116, 21675129), and the Natural Science Foundation Project of Chongqing City (cstc2018jcyjAX0085). 16


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Dynamical Regulation of Enzyme Cascade Amplification by a

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