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Engineering DNA Three-way Junction with Multifunctional Moieties: Sensing Platform for Bioanalysis Libing Zhang, Shaojun Guo, Jinbo Zhu, Zhixue Zhou, Tao Li, Jing Li, Shaojun Dong, and Erkang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02468 • Publication Date (Web): 28 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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Analytical Chemistry

Engineering DNA Three-way Junction with Multifunctional Moieties: Sensing Platform for Bioanalysis Libing Zhang,† Shaojun Guo,‡ Jinbo Zhu,† Zhixue Zhou,† Tao Li,§ Jing Li,† Shaojun Dong*† and Erkang Wang*†

†

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China ‡

Materials Science & Engineering, College of Engineering, Peking University, Beijing

100871, China §Department

of Chemistry, University of Science and Technology of China, Hefei, Anhui

230026, China *Corresponding authors: E-mail: [email protected]; [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

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ABSTRACT Functionalization of DNA nanostructures is critical to the achievement of the application in biosensors. Herein, we demonstrate a novel DNA three-way junctions (TWJs) with three functional moieties, which integrates the electrochemical, fluorescent and colorimetric properties. Upon addition of external stimuli including DNA, thrombin and ATP, the specific interactions between targets and sensing elements could induce strand displacement reaction

and

conformation

transformation,

resulting

in

the

integration

of

G-quadruplex/hemin complex as electrochemical probe, lighting up the fluorescence of DNA/Ag nanoclusters, and enhancing the catalytic activity of DNAzyme to catalyze H2O2-TMB system to generate colorimetric signal. Such a functional DNA nanostructure actually serve as a biosensing platform, showing high sensitivity and selectivity for DNA, thrombin and ATP detection. Furthermore, We also shown that this novel sensing platform can be utilized to detect three different kinds of targets independently and simultaneously. Our intergrated concept provides a paradigm for exploring the potential of TWJs in analytical applications.

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INTRODUCTION DNA as an excellent biomaterial has been used for construction of numerous two- and three-dimensional self-assembling nanostructures and devices 1-6 due to the highly specific interactions.7,8 It is a remarkable fact that DNA nanostructures are yet of limited applicability since DNA itself lacks the optical, electronic, or chemical functionality. To date, severals approaches have been explored for the functionalization of DNA nanostructures, which usually based on some typical systems, such as nucleic acid hybridization,9,10 antigen-antibody binding,11,12 or biotin-streptavidin interaction.13-15 However, these reported approaches usually require labeling an appropriate functional group, which leads to a high cost of operation and potentially complex processes. To realize multifunctional applications in the sensing and nanotechnology, it is therefore of great importance to explore new methods for the decoration of DNA scaffolds with diverse functional moieties with unique chemical and physical properties. Junctions arise when three or more helices meet at a single point in DNA or RNA nanostructures. In such nanostructures, the notable feature is their branched, which is benefit for the construction of versatial nanoscale assemblies.26-20 In general, branched structures not only provide a unique window into nucleic acids nanotechnology, but also are propitious to use as building blocks in nanotechnology applications.21,22 The ability to attach different functional moieties to a molecular building block could allow for the development of applications in different fields. Construction of functional nanostructures has demonstrated its power in realizing high sensitivity and selectivity for the detection of biomolecules.23

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However, there is rare reports about the development of multifunctional platforms based on junction DNA nanostructures for applications in intelligent sensing. Herein, we for the first time develop a targets-induced grafting technique to engineer the DNA three-way junctions (TWJs) model structure with diverse functional moieties, endowing the DNA nanostructures with multifunctional catalytic and fluorescent properties. In particular, we have demonstrated that these nanoarchitectures can be used as sensing platform for the sensitive and selective detection of various biomilecules. Scheme 1 shows the sensing concept for the detection of different biomolecules. We start with one TWJs containing three branched DNA duplex strand for developing three kinds of biosensors. (A) In part 1, in the presence of target DNA, target DNA can hybridize with DNA TWJs to release E1 through strand displacement reaction (SDR).24,25 Then, the released E1, containing part of sequences of G-quadruplex, hybridized with E2 that immobilized on an gold electrode surface. This could produce the G-quadruplex structure, which provides a structural basis for specific binding to hemin with high affinity.26,27 The assembly architecture could be electrochemically characterized by taking hemin as an electroactive probe, giving out the electrochemical signal.28 (B) In part 2, silver nanoclusters protected by DNA (DNA/Ag NCs) have been developed as a new class of fluorophores for biosensors.29-31 One branch of our designed TWJs (Scheme 1) contains the template sequences (orange) for the synthesis of DNA/Ag NCs. Under the “off” state, the prepared DNA/Ag NCs show very weak fluorescence. However, when thrombin was added into the solution, the aptamer could combine with thrombin to form G-quadruplex structure, and then released from nanostructure. Then, the Ag-En easily hybridized with DNA TWJs to light up

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the DNA/Ag NCs and enhance the fluorescence intensity.32,33 (C) In Part 3, the branched structure includes a bulge-loop structure that contains G-quadruplex sequence. In the initial state, a part of G-quadruplex sequence can form a stable duplex structure, resulting in the sequence is in a inactive state to eliminate the formation of G-quadruplex/hemin complex. But when adenosine triphosphate (ATP) was incorporated into the loop domain of TWJs on the basis of the binding between ATP and its aptamer, which can open duplex structure, and further facilitate the released sequences form the G-quadruplex/hemin complex. The obtained

complex

can

catalyze

the

H2O2-mediated

oxidation

of

3,3′,5,5′-tetramethylbenzidine sulfate (TMB) to generate colorimetric signal.34 Thus, our rational design on engineering DNA nanostructure with multifunctional moieties allows for the development of a sensing platform for analysis of DNA, thrombin and ATP with the output of electrochemical, fluorescent and colorimetric signals, respectively. EXPERIMENTAL SECTION. Preparation of DNA nanostucture: 100 µL of DNA (1.0 µM) solution in Tris-Ac buffer (20 mM, 1.0 mM Mg2+, pH 7.4) were kept at 88 °C for 10 min, and then cooled to room temperature (RT) gradually. DNA oligonucleotides used in this study were purchased from Shanghai Sangon Biological Engineering Technology (China) and the sequences were listed in Table S1. Electrochemical DNA Assays: In DNA assay, Y1, Y2, Y3 and E1 (1.0 µM, respectively) in Tris-Ac buffer (20 mM, 1.0 mM Mg2+, pH 7.4) were kept at 88 °C for 10 min, and then cooled gradually to RT. Subsequently, target DNA was added and incubated for 30 min. E2 (1.0 µM) containing 50 µM TCEP in Tris-Ac buffer (20 mM, 1.0 mM Mg2+, pH 7.4)

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were kept at 88 °C for 10 min, and cooled to RT. The above solution was dropped on the Au electrode and kept for 4.0 h. Subsequently, the electrode was rinsed thoroughly with pure water, and dried with a nitrogen stream. After that, 10 µL of 1.0 mM MCH was droped on the electrode and kept for 5 min to reduce the nonspecific adsorption. Finally, 10 µL of obtained cumulated solution was added onto the electrode and incubated for 12 h before the measurement of the DPV signal. Synthesis of DNA/Ag Nanoclusters: AgNO3 solution (6.0 µM) was added into 1.0 µM oligonuleotides in phosphate buffer (20 mM, 5.0 mM Mg2+, pH 7.4). After incubated for 30 min, NaBH4 (6.0 µM) was added, the mixture was allowed for another 4.0 h, and then Ag-En (1.0 µM) was added into the solution at RT to form Ag nanoclusters with high fluorescence. Fluorescent Thrombin Assays: In thrombin assay, Y1, Y2, Y3 and aptamer (1.0 µM, respectively) in phosphate buffer (pH 7.4, 20 mM, 5.0 mM Mg2+) was kept at 88 °C for 10 min, and drop gradually to RT. Subsequently, thrombin was added and incubated at 37 °C for 40 min, and then 6.0 µM of AgNO3 was added in the mixture. 30 min later, NaBH4 (6.0 µM) was added to keep another 4.0 h, and finally, Ag-En (1.0 µM) was added into the solution and incubated for overnight in the dark before measuring the fluorescence intensity. Colorimetric ATP Assays: In ATP assay, Y1, Y2 and Y3 (0.1 µM, respectively) in Tris-Ac buffer (20 mM, 1.0 mM Mg2+, pH 7.4) was heated at 88 °C for 10 min. Subsequently, ATP was added and incubated at 37 °C for 2 h, and then an equal volume of 20 mM Tris-Ac buffer (pH 7.4, 40 mM K+, 150 mM Na+) and 10 µL hemin (5.0 µM) were added and the mixture was further incubated for 1.0 h to form the hemin–G-quadruplex DNAzymes. Colorimetric analysis was performed in the TMB-H2O2 system. In the experiment, 385 µL

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buffer (pH 5.0, 20 mM KCl, 26.6 mM citrate, 51.4 mM Na2HPO4), 5.0 µL TMB (0.5% (w/v)) and 10 µL H2O2 (3% (w/v)) were added and the resulting assay solution was kept for 5.0 min before taking the photos and collecting UV-vis spectra. RESULTS AND DISCUSSION For the demonstration of such sensing concept, agarose gel electrophoresis (AGE) was performed to demonstrate the formation of the DNA TWJs as each DNA strand was added (Figure 1A). A distinct band shift was observed, indicating the effectiveness of DNA assembly. From the results, we can conclude that Y1, Y2, Y3, E1 and aptamer bind together to form the DNA TWJs (lane 4). Every branched strand of the formed DNA TWJs was decorated with functional component, endowing the DNA nanostructures with special properties. AGE was also used to identify the strand displacement mechanism (Figure 1B). The band of lane 1 represents DNA TWJs, after the addition of target DNA, because the total bases of target DNA are 5 nucleotides shorter than that of E1, the mobility of the band became faster (lane 2), revealing the occurence of SDR. Subsequently, in order to validate the released E1 could hybridze with the E2 immobilized on the gold electrode, electrochemical impedance spectroscopy (EIS), sensitive to the charge transfer resistance, was utilized to characterize the hybridization process on the gold electrode. The hybridization between the E2 DNA fixed on the gold and the input strand (E1) make gold surface have more insulating molecules, leading to the impedance increase. As shown in Figure 1C, a very small semicircle domain (representing the Rct) was observed for the bare Au electrode (curve a). However, after the modification of strand E2 on the gold surface, the Rct signal increases obviously due to the introduction of insulating E2 (curve b). And then,

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the Rct signals continuously increased with the addition of 6-mercaptohexanol (MCH, blocking agent) (curve c) and input E1 (curve d), proving the input E1 successfully hybridized with E2 to form the stable duplex DNA on the gold electrode. The differential pulse voltammetry (DPV) technique was then utilized to monitor our sensing process. We observe no and an obvious DPV signal, ascribed to the oxidation reaction of the electroactive probe (hemin), in the absence (curve a) and presence (curve b) of target DNA, respectively (Figure 2A), indicating the effectiveness of our part 1. Figure 2B shows the DPV curves of the assembled gold electrode in the presence of different concentrations of target DNA. Obviously, the peak current increase with the increase of target DNA concentration. This is because higher concentration of target DNA will displace more E1, which induces the formation of more abundant G-quadruplex structure, further binding with the hemin and giving stronger electrochemical signals. Figure 2C shows the calibration curve between i/i0 and the concentration of target DNA, where i and i0 are defiend as the peak current detected after the addition of each target DNA and the blank peak current, respectively. It is found that a good linear range from 5.0 to 100 nM (R = 0.989) and the limit of detection is 5.0 nM in the present TWJs-based electrochemical sensing system. For specificity test, other DNA containing mismatched nucleotides (Table S1) were investigated. Figure 2D shows the peak current changes in the presence of target DNA T1 (1.0 µM), other mismatched DNA (2.0 µM), ATP (4.0 µM) and thrombin (4.0 µM). It can be seen that the peak current changed obviously in the presence of T1, this is because T1 can effectively hybridize with DNA TWJs to release E1. Whereas others of mismatched DNA and ATP and thrombin make peak current a little change. These results clearly demonstrate

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that our TWJs-based eletrochemical sensor shows a very high selectivity for DNA dectection. In order to demonstrate the multifunction of present proof-of-concept sensing system, a homogeneous model was adopted for fluorescent thrombin analysis (part 2 of Scheme 1). The initially formed DNA/Ag NCs show very weak fluorescence upon excitation (curve a, Figure 3A). After the hybridization of Ag-En with DNA TWJs, the fluorescence intensity has been greatly enhanced (curve b and c, Figure 3A). When aptamer was first hybridzed with DNA TWJs, followed by the addition of Ag-En, there is no enhancement on the fluorescence intensity (curve d and e, Figure 3A), demonstrating that the aptamer can effectively prevent the Ag-En to improve the fluorescence. However, after the addition of thrombin, the fluorescence intensity of Ag nanoclusters could enhance obviously (curve f, Figure 3A). This indicates that the thrombin combined with its aptamer,35 and the formed composite was

released from TWJs. The Ag-En could further hydridize with DNA TWJs

to improve the fluorescence. The above formation process was further identified by AGE (Figure 1B). Through comparing lane 1 and lane 3, we found the TWJs band had lower mobility after the addition of thrombin and strand Ag-En. This should be attributed to the fact that the bases of strand Ag-En are 16 nucleotides longer than that of aptamer. This result indicates that strand Ag-En could hybridze with DNA TWJs in the presence of thrombin. The fluorescence spectra were recorded in the presence of different concentarions (from 10 to 100 nM) of thrombin. In Figure 3B, We can find that the fluorescence intensity gradually enhanced as the increase of thrombin. Figure 3C shows the calibration curve of fluorescence intensity with the increasing concentration of thrombin. The detection range is

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determined to be from 10 to 100 nM (R = 0.990) with a detection limit of 10 nM. Figure 3D shows the fluorescence changes in the presence of thrombin (1.0 µM), other proteins (4.0 µM), T1(4.0 µM) and ATP (4.0 µM). We found that thrombin could induce an obvious fluorescence change whereas other proteins and ATP and T1 show nearly negligible fluorescence change, indicating the excellent selectivity of our TWJs-based fluoresenct sensor. As a third demonstration of the general applicability and multifunction of present proof-of-concept sensing system, we attempted to detect ATP based on colorimetric methods (Part 3 of Scheme 1). To demonstrate the feasibility of this strategy, we first test the catalytic ability of the designed probe under different conditions (Figure 4A). Photograph a is the background signal of the H2O2-TMB system and curve a is the corresponding UV-visible absorption. In the presence of DNA TWJs, the color of the solution became a little bit blue (photograph b), and the corresponding absorption enhanced a little (curve b). However, after the addition of ATP, the color of the solution became obvious blue (photograph c), and the corresponding absorption enhanced greatly (curve c). This is because that ATP combined with its aptamer, generates the formation of the Gquadruplex/hemin complex, resulting in an oxidation of TMB to generate a color change. AGE was used to verify the above colorimetric sensing process.We got the same mobility of the band no matter in the absence or presence of ATP (lane 1 and 4, Figure 1B). This is because the structure of DNA TWJs does not change even if ATP has bound with its aptamer. In order to better understand the above sensing process, we used circular dichroism (CD) to monitor the conformation change of TWJs in the presence and absence of ATP. As shown in Figure S1, the CD

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spectra of DNA TWJs with ATP has a negative band at 245 nm and a positive band near 265 nm (curve a). While in the presence of ATP, the negative and positive bands of CD spectra exhibit red shift (curve b). This should be induced by the formation of antiparallel G-quadruplex structure when ATP binds with its aptamer.36, 37 From the above results, we could conclude that ATP indeed binded with its aptamer, which opened duplex structure and then released the caged G-quadruplex sequence. As for ATP colorimetric analysis, the photographs of solution were recorded in the presence of different concentrations of ATP (Figure 4B). With increasing the concentration of ATP, the color became deep (the inset of Figure 4B), and absorbance of the oxidation of TMB increased gradually. Figure 4C shows the calibration curve between absorbance at 650 nm and DNA concentration, and the calibration curve for quantitative analysis of ATP is shown in the inset. Our colorimetric sensor can determine the ATP with the concentration from 0.04 to 0.4 µM (R = 0.987) and a limit of detection is 40 nM. For the selectivity test, GTP, CTP and UTP, analogues of ATP, were chosen as contrast. As shown in Figure 4D, we can observe that 4.0 µM ATP can induce an obvious color change, while the analogues (4.0 µM), T1 (4.0 µM) and thrombin (4.0 µM) showed the negligible color change. These results prove that our TMJs-based colorimetric sensing platform shows excellent selectivity for ATP dectection. It is always a challenge for many sensing platform to detect different targets simultaneously due to non-specific binding, cross-reactivity between probes and targets, and probes with chemically and optically unique properties. This prompts us to explore the feasibility of the constructed sensing platform for targets detection simultaneously. Figure 5

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shows the results of the sensing platform toward different targets combinations. We can clearly observe that the addition of T1 gives only electrochemical signal. However, the target combination of T1 and thrombin gives electrochemical and fluorescent signal, respectively. While, in the presence of T1, thrombin and ATP target combination, three strong different signals can be observed, respectively. On the basis of all the obtained results, we can draw the conclusion that this constructed sensing platform can be used to detect three targets independently and simultaneously. In summary, we have demonstrated the possibly to assembly diverse functional component onto DNA junction nanostructures, endowing them with multifunction and anisotropy, which provides a general approach to develop sensors for the sensitive and selective detection of various biomolecules. The specific interaction between targets and sensing elements could result in the integration of G-quadruplex/hemin complex as electrochemical probe, lighting up the fluorescence of DNA/Ag NCs, and inducing the formation of DNAzyme to catalyze H2O2-TMB to generate colorimetric signal. Through those three strategies, we could achieve to detect DNA, thrombin and ATP with the output of electrochemical, fluorescent and colorimetric signals, respectively. Furthermore, We also demonstrated that this novel sensing platform can respond to three analyzes independently and simultaneously. It is expected that our sensing based on TWJs provides a paradigm for the possibilities of creating DNA junction nanostructures with more multiple functionalities for the development of other biosensors with high sensitivity and selectivity. ACKNOWLEDGEMENT Thanks for the support of National Natural Science Foundation of China (Grant Nos. 12


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21190040, 91227114). ASSOCIATED CONTENT Supporting Information. Detailed information about materials, apparatus and agarose gel electrophoresis, DNA sequences and circular dichroism characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure captions Scheme 1 Schematic illustration of the TWJs-based sensing platform used for the DNA, thrombin and ATP analysis, respectively.

Figure 1 (A) Agarose gel electrophoresis: Lane 1: Y3; lane 2: Y3+Y2; lane 3: Y3+Y2+Y1; lane 4: Y3+Y2+Y1+Aptamer+E1. (B) Agarose gel electrophoresis: Lane 1: Y3+Y2+Y1+ Aptamer+E1; lane 2: Y3+Y2+Y1+Aptamer+E1+target T1; lane 3: Y3+Y2+Y1+Aptamer +E1+Thrombin+Ag-En; lane 4: Y3+Y2+Y1+Aptamer +E1+ATP. (C) The EIS responses of (a) bare Au electrode, (b) E2/Au electrode, (c) MCH/E2/Au electrode and (d) E1/MCH/E2/Au electrode.

Figure 2 (A) The DPV responses of MCH/E2/Au electrode under different conditions: (a) in the absence of target T1 and (b) the presence of target T1 (1.0 µM). (B) The DPV responses of MCH/E2/Au electrode in the presence of different concentrations of T1 (from a to g): 0, 5, 20, 50, 100, 500, 1000 nM. (C) The relationship between the i/i0 and the concentration of T1. The inset shows a linear relationship of i/i0 as a function of T1 concentration. (D) Selectivity of TWJs-based eletrochemical sensor for analyzing target T1, other DNA with one (T2), two (T3) and four (T4) mismatched nucleotides.

Figure 3 (A) The fluorescence spectra of DNA/Ag NCs in the absence of (a) reagent, and presence of (b, excitation spectra) and (c, emission spectra) Ag-En (1.0 µM), (d) aptamer (1.0 µM), (e) aptamer (1.0 µM) and Ag-En (1.0 µM), and (f) aptamer (1.0 µM), Ag-En (1.0

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µM) and thrombin (4.0 µM). (B) Fluorescence emission spectra of DNA/Ag NCs in the presence of thrombin with different concentrations (from a to h): 0, 10, 20, 50, 100, 200, 500, 1000 nM. (C) The relationship between the fluorescence intensity and the concentration of thrombin. The inset shows a linear relationship of fluorescence intensity as a function of thrombin concentration. (D) Selectivity analysis for thrombin.

Figure 4 (A) The photographs (inset) and UV-vis absorption spectra of (a) TMB-H2O2 system, (b) DNA TWJs + TMB-H2O2, (c) DNA TWJs + TMB-H2O2 + 4.0 µM ATP. (B) The photographs (inset) and UV-vis absorption spectra of colorimetric sensor analyzing different concentrations of ATP (from a to h): 0, 0.04, 0.1, 0.2, 0.4, 1.0, 2.0, 4.0 µM. (C) The relationship between the absorption at 650 nm and the concentration of ATP. The inset shows a linear relationship of absorbance as a function of ATP concentration. (D) Selectivity analysis for ATP.

Figure 5 The results of the sensing platform toward different targets combinations: (A) in the presence of T1 (1.0 µM), (B) in the presence of T1 (1.0 µM) and thrombin (1.0 µM), (C) in the presence of T1 (1.0 µM), thrombin (1.0 µM) and ATP (4.0 µM).

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Scheme 1

Figure 1

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Figure 2

Figure 3

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Figure 4

Figure 5

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For TOC only

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