A Programmed Dual-Functional DNA Tweezer for Simultaneous and

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A Programmed Dual-Functional DNA Tweezer for Simultaneous and Recognizable Fluorescence Detection of microRNA and Protein Wenting Yang, Yu Shen, Danyang Zhang, Chong Li, Ruo Yuan, and Wenju Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01266 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 28, 2019

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Analytical Chemistry

A Programmed Dual-Functional DNA Tweezer for Simultaneous and Recognizable Fluorescence Detection of microRNA and Protein Wenting Yang, Yu Shen, Danyang Zhang, Chong Li, Ruo Yuan*, and Wenju Xu* Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China * Corresponding author. Fax: +86 023 68253312. E-mail address: [email protected]; [email protected]

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ABSTRACT A programmed dual-functional DNA tweezer (DFDT) as signaling molecule is reported for the simultaneous and recognizable fluorescence detection of microRNA 21 (miRNA 21) and mucin 1 (MUC1). This unique DFDT is assembled from two Au NP-attached central strands (C1 and C2) and an arm strand (A) dually ended by fluorophores Cy3 and Cy5, which are spatially separated from Au NP in the originally opened state. Through the competitive affinity interaction between targets and their complementary and aptamer sequences tethered in two recognition strands (R1 and R2), miRNA 21 and MUC1 are respectively converted into two dependently displaced fuel strands (F1 and F2). The next hybridization with two pairs of unpaired segments overhung in open DFDT leads to its conformational closure, resulting in the approach of Cy3 and Cy5 to Au NP. Based on nanometal surface energy transfer scheme, the fluorescence emission of Cy3 or Cy5 is cooperatively quenched by Au NPs attached in C1 and C2. The significant variation of fluorescence intensity enables one-step, cost-effective and specific quantization of miRNA 21 and MUC1 with high sensitivity down to 32 fM and 2.6 fg·mL-1 (8.5 pM), respectively. The novel DFDT-based assay route of multiplex analytes is promising and has the potential for rapid and reliable diagnosis and treatment of cancer-related diseases. KEYWORDS: dual-functional DNA tweezer, recognizable fluorescence detection, multiplex targets, cooperative fluorescence quenching, miRNA 21, MUC1 Introduction Rapid, cost-effective, sensitive and recognizable quantitation of multiplex analytes

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is of great importance for biosensing, clinic medicine, human health etc. However, it is challenging to design and locate specific recognition and/or signaling molecules for selective signal translation and/or signal readout. To this end, many efforts have been paid to explore some specific recognition elements for distinguishable multiplex assay. The principles were mainly based on various affinity interactions between protein-aptamer,1 sequence-specific ssDNA,1,2 antigen- antibody,3,4 molecular beacon,5 or metal ion-dependent DNAzymes6,7. The multiplex targets were involved in two, three or more analytes of interest, such as oligonucleotides,8 proteins,1,9 small molecules,10 or even heavy metal ions.7 In fluorescence-based biosensing platforms, different recognition/signaling probes are separately tethered by various fluorescent dyes with non-overlapped emission spectra, fulfilling distinguishable signal readout via the fluorescence intensity change caused by energy transfer or absorbing between donor-acceptor pair.6,10 Also, some microsized or nanosized materials were utilized as matrices to conjugate and locate recognizable base sequences for the selective detection of multiplex miRNAs,11 however the sensitivity and specificity were low in differentiating similar miRNA sequences, along with complicated testing components and time-consuming operation.12 Notably, because of strong adsorption capability, low cost, easy modification and commercial accessibility, Au nanoparticle (Au NP) as energy acceptor has become a remarkable substitute of organic dye quenchers. As demonstrated, the nanometal surface energy transfer (NSET) scheme enables more effective fluorescence absorption over a longer distance than that of fluorescence resonance energy transfer,13,14 which is desirable and advantageous to acquire

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significantly varied signal in a more flexible and accessible manner. The dye-Au NP pair would provide a new paradigm to explore multi-functional biosensing features inside one probe molecule with expected convenience, rapidness, sensitivity and specificity.15,16 Owing to easy programming, precise incorporation of site-specific element, controllable mechanical movement, simple chemical synthesis and commercial availability,17-21 functional DNA nanostructures such as tweezers,22-26 walkers,27-29 gears,30 and nanocages31,32 are promising scaffolds to motivate desirable applications in biomolecule assembly, signal recognition, biosensing etc.33-36 Through the mechanical functions encoded in specific base sequences, diverse DNA architectures are powerful recognition elements for a large scope of targets in various biosensing systems including nucleic acid fragments, proteins, small molecules, metal ions etc.37 Specifically, as the simplest and most essential nanostructures, DNA tweezers (DT) are expected to show some close-to-reality behaviors like real tweezers to sense, capture, hold, and/or release molecular object(s) upon affinity interaction to trigger conformation switch between opening and closure,38-40 Structurally, the function of DT is activated by special energies (e.g. fuel strands) provided by one and/or two ssDNA overhang(s) tethered in the ends of DT. As previously reported, the opening and closure of programmed DT can be powered and regulated by specific ssDNA sequences or aptamers, pH changing, DNAzymes, or photonic stimuli, enabling unique flexibility, diversity, and recognition and/or signaling capability of functional DT.10,40-46 This would open up a new pathway for simplified, accessible and

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distinguishable multiplex assay, especially miRNA and protein in one homogenous system. Herein, we report a simple and programmed dual-functional DNA tweezer (DFDT) as signaling molecule for simultaneous and specific fluorescence detection of miRNA 21 and MUC1, whose over-expressions are closely associated with many types of cancers such as breast, lung, ovaries, colon and pancreas.28-30 This unique “W-type” DFDT is constructed by three elaborate single-stranded oligonucleotides: two Au NP-attached central strands (C1 and C2) and an arm strand (A) dually ended with fluorophores Cy3 and Cy5. Four unpaired segments overhung in the ends of DFDT hybridize with two fuel strands (F1 and F2), which are dependently displaced due to affinity combination between targets and two recognition strands (R1 and R2) encoded with the complementary sequence of miRNA 21 and the aptamer sequence of MUC1, respectively. This sequence-specific complementary hybridization strains the opened DFDT into closure, resulting in separated Cy3 and Cy5 proximal to two Au NPs labeled in C1 and C2 that cooperatively quench their fluorescence emission. The significantly varied fluorescence intensities are utilized for one-step, cost-effective, sensitive and selective quantization of miRNA 21 and MUC1. To the best of our knowledge, it is the first time to use this DFDT for simultaneous and distinguishable assay of multiplex miRNA and protein in one sample. EXPERIMENTAL SECTION Chemicals and Reagents. Sigma-Aldrich Chemical Co. (St. Louis, MO) provided us with carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), immunoglobulin G

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(IgG), prostate specific antigen (PSA), sodium citrate (1%), chlorauric acid (HAuCl44H2O, 1 mM) and hexanethiol (96%, HT). Magnetic Fe3O4 microspheres were purchased from BaseLine Chrom Tech Research Centre (Tianjing, China). Human mucin 1 (MUC1) was ordered from North Connaught Biotechnology (Shanghai, China). HPLC-purified oligonucleotides (see Table S1 in Supporting Information for their detailed base sequences) were synthesized by Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China), and miRNA 21, miRNA 141, miRNA 155 and Let-7a were provided by Takara Biotechnology Company Ltd. (Dalian, China). The stock solution of nucleotides and proteins were prepared by using 20 mM Tris-HCl buffer with 10 mM KCl and 15 mM MgCl2 (pH 7.4). In our experiments, phosphate buffer saline (PBS, pH 7.4) containing Na2HPO4, KH2PO4 and KCl with the same concentration of 0.1 M and ultrapure deionized water (DI water, ≥18.2 MΩ, Milli-Q, Millipore) were used. Apparatus. A Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) was used for all the fluorescence measurements. Nanomaterials and Au NP-modified oligonucleotides were characterized by using UV-vis spectrophotometer (Shimadzu, Tokyo, Japan) and Raman spectrometer (Renishaw Invia Raman spectrometer, Invia, U.K.) equipping a 633 nm line from a He-Ne laser (17 mW of power on 50×objective). The morphology of nanomaterials was tracked by scanning electron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan) at voltage of 20 kV and transmission electron microscopy (TEM, H600, Hitachi, Japan). The open and closed configuration of DFDT was recorded by dimension edge atomic force microscopy

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(AFM, Bruker Co., Germany). Gel Doc XR+ System (Bio-Rad, California, USA) was used to take images of gels. Dependent Releasing of Fuel Strands in Response to Multiplex Targets. We firstly prepared ca. 10 nm Au nanoparticles (Au NPs) and Au NPs-functionalized magnetic Fe3O4 microspheres (Au@Fe3O4) (see Figure S1, S2 and S3 in Supporting Information for characterizations). Next, 50 µL Au@Fe3O4 (1% in pH 7.4 PBS) was mixed with 5 µL of R1 (100 µM) and 5 µL of R2 (100 µM) at room temperature (RT). After 12 h, partially hybridized R1 and R2 were conjugated in Au@Fe3O4 surface through Au-N bond, followed by the addition of 20 µL HT (1 mM) to inhibit nonspecific binding and magnetic separation at least three times. The precipitates were incubated with 5 µL of F1 (100 µM) and F2 (100 µM) for 1.5 h, allowing for the hybridization of R1 and F1, and R2 and F2. After that, the introduction of 10 µL of miRNA 21 and 10 µL of MUC1 for 1.5 h initiated the specific recognition and affinity combination of R1 and miRNA 21, and R2 and MUC1, dependently displacing F1 and F2. Through magnetic separation, the supernatant containing released F1 and F2 was collected for the subsequent incubation with DFDT. Of note, the displacement of only F1 or F2 can be occurred in the presence of single miRNA 21 or MUC1. Assembly of Dual-Functional DNA Tweezer (DFDT). Firstly, NH2-labeled C1 and C2 were functionalized with a ca. 10 nm-sized Au NP through Au-N bond.47 Briefly, 10 µL of C1 or C2 (100 µM) was mixed with as-prepared Au NPs (480 µL) and stirred overnight, respectively. After aged in 1 mL PBS (pH 7.4) for 24 h, the

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resultant sediments, Au NP-attached C1 and C2 in 0.5 mL PBS, were introduced into 10 µL of A (100 µM) for 2 h. After centrifugation, this assembled DFDT was collected and re-dispersed in 100 µL PBS (pH 7.4). For the control experiments, two similar DNA tweezers (DFDT’ and DFDT’’) were also prepared by using intact C2 or C1 without Au NP labeling, respectively. Fluorescence Measurements. Before and after the treatment of the as-assembled DFDT (10 µL, 2 µM) with the dependently displaced F1 and F2 at 37 °C for 1.5 h, the fluorescence spectra were recorded on the spectrophotometer with 5 nm excitation and emission slits and 950 V of PMT voltages. With the excitations of Cy3 at 530 nm and Cy5 at 635 nm, the fluorescence emission spectra were collected in the range of 500~650 nm for Cy3, and 600~760 nm for Cy5, respectively. The emission spectrum peaks of Cy3 at 565 nm and Cy5 at 665 nm, corresponding to miRNA 21 and MUC1, respectively, were used to determine the experimental parameters and analytical performances of the DFDT-based multiplex fluorescence method. Moreover, we also investigated the emission spectra of DFDT in response to single target, miRNA 21 or MUC1. Polyacrylamide Gel Electrophoresis (PAGE). In the gel electrophoresis assay of the oligonucleotide hybridizations, different tested solution containing 2 μL 6×loading buffer was placed into 16% gel electrophoresis in a Model DYCP-31E electrophoretic device (Wo De Life Sciences Instrument Co. Ltd., Beijing, China). By using 1×TBE buffer, all the prepared gels were run at 100 V for 90 min. After that, the obtained gels were stained with ethidium bromide for 20 min, and the FR-980A gel image analysis

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system (Shanghai, China) was used to scan the gel images. RESULTS AND DISCUSSION Design of Dual-Functional DNA Tweezer (DFDT). It is attractive and challenging to sensitively and recognizably determine multiplex targets in one homogenous sample, whereas it is crucial to design multifunctional recognition and signaling probes. In this work, a simple, novel and programmed DFDT is constructed for the first time to distinguish and detect multiplex miRNA 21 and MUC1. For proof-of-concept, this unique DFDT is assembled by using two ca. 10 nm-sized Au NP-labeled central strands (C1 and C2), and an arm strand (A) dually ended with fluorophores Cy3 and Cy5 as signaling tags. As designed, both C1 and C2 contain one 8-nt domain and two 12-nt domains flanked by the middle 3-nt spacer of TGA (C1) and GAC (C2), respectively. Two 8-nt and two 12-nt domains of A are separated by the middle 8-thymine (T8) bases as a flexible linker. The complementary hybridization among C1, C2 and A guides the formation of a “W-type” DFDT architecture described here, where three helixes with 12-base-pair (bp) are hinged by two 3-nt bases and a T8 to stabilize DFDT in open state and facilitate the configuration switch. In open DFDT, both Cy3 and Cy5 are spatially separated to Au NP, giving the most significant fluorescence signal.5 When four unpaired segments overhung in the ends of DFDT hybridize with two fuel strands (F1 and F2) as external stimuli, the open DFDT is strained into closure, bringing Cy3 and Cy5 proximal to Au NP. This cooperative quenching of Cy3 and Cy5 by two Au NPs labeled in C1 and C2 is supposed to be in accordance with the scheme of nanometal

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surface energy transfer (NSET) over a longer distance than FRET.13,14 Figure S4 in Supporting Information depicts the detailed base sequence of DFDT in open and closed states. Also, in the presence of miRNA 21 and MUC1, the successful configuration switch of DFDT from opening to closure is well supported by the AFM images (Figure S5 in Supporting Information).

Scheme 1. Schematic illustration of the programmed “W-type” DFDT for recognizable fluorescence detection of miRNA 21 (Cy3) and MUC1 (Cy5). The gray domains in C1, C2 and A are complementary each other to form three 12-bp stiff helixes of DFDT. The two pairs of unpaired segments of C1 and A (in blue), and C2 and A (in red) hybridize with F1 and F2, respectively, enabling the open DFDT switched into closure to bring both Cy3 and Cy5 spatially proximity to Au NPs for cooperative fluorescence quenching. 10


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Principle of DFDT as Signaling Probe. The two units of the open “W-type” DFDT can separately respond to external stimulus F1 or F2, achieving the recognizable fluorescence detection of miRNA21 and MUC1. To this end, by using magnetic Au@Fe3O4 as conjugating substrates to minimize the background signal through step-by-step magnetic separation, two NH2-labeled R1 and R2 encoding with complementary sequence of miRNA 21 and aptamer sequence of MUC1 are conjugated in the Au@Fe3O4 surface by Au-N bond. The resultant DNA complex contains a 6-bp ds-helix and two unpaired segments (see Scheme 1). The hybridization of F1 with R1, and F2 with R2 forms another two 16-bp ds-helixes. When introducing miRNA 21 and MUC1, the distinguishable affinity interaction between R1 and miRNA 21, and R2 and MUC1 is activated to dependently displace F1 and F2. Using them as external stimuli to treat the as-prepared DFDT, two pairs of unpaired segments concurrently hybridize with F1 and F2. The closure of open DFDT brings both Cy3 and Cy5 approaching to Au NP. The significantly decreased fluorescence intensity is indirectly dependent on miRNA 21 and MUC1. Notably, the presence of single target miRNA 21 or MUC1 only strains one unit of open DFDT, leading to fluorescence decreasing of single dye. From this point of view, this unique DFDT acts as a biresponsive molecule element for both specific recognition and signal readout, enabling one-step, sensitive, simultaneous and recognizable detection of multiplex targets in one sample. The PAGE image of different samples is shown in Figure S6 in Supporting Information. Fluorescence Response of DFDT as Signaling Probe. The response capability of

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this DFDT and the cooperative quenching efficiency of Au NPs were demonstrated by investigating the fluorescence signal of the intact DFDT, DFDT’ and DFDT’’ in the presence of single miRNA 21 or MUC1. From Figure 1A, the intact DFDT displays the most significant fluorescence emission of Cy3 (curve a), while the presence of miRNA 21 results in decreased fluorescence intensity (F) of DFDT’ without Au NP labeling in C2 (curve b), which is smaller than that of the proposed DFDT (curve c). From Figure 1B, similar results for Cy5 responding to MUC1 (curve a’) are observed for the DFDT’’ without Au NP labeling in C1 (curve b’) and the proposed DFDT (curve c’). Thus, more than 30% F variation of this DFDT than DFDT’ and DFDT’’ would suggest that two Au NPs modified in C1 and C2 can cooperatively quench the fluorescence emission of either Cy3 or Cy5 over a relatively longer distance,13,14,26 in spite of one unit closure of DFDT in response to miRNA 21 or MUC1.

Figure 1. Fluorescence spectra of DFDT, DFDT’ (A) and DFDT’’ (B) in the absence of miRNA 21 (curve a) and MUC1 (curve a’) and in the presence of 0.2 μM miRNA 21 (curve b and c) or 50 pg·mL-1 MUC1 (curve b’ and c’). As shown in the upper, 12


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curve b and b’ represent DFDT’ and DFDT’’ by using intact C2 or C1 without Au NP labeling, respectively. Based on the above observations, we further studied the fluorescence spectra of the proposed DFDT in the absence and presence of miRNA 21 and/or MUC1, and the obtained results are displayed in Figure 2. In the absence of miRNA 21 and MUC1, almost the same F as that of intact DFDT is observed (curve a and b in Figure 2A). However, the simultaneous introduction of miRNA 21 and MUC1 results in significantly decreased F of both Cy3 and Cy5 (curve b in Figure 2B). From Figure 2C, the treatment of miRNA 21 without MUC1 leads to significantly decreased F of Cy3 and almost unchanged F of Cy5 (curve b), while the presence of only MUC1 without miRNA 21 gives rise to similar results for Cy5 and Cy3 (Figure 2D). This indicates that the closure of two units of DFDT can be separately regulated in response to single or two target(s), and the efficiently cooperative quenching of two Au NPs labeled in C1 and C2 in the closed DFDT can occur over a longer distance. These results well support the feasibility and reliability of this DFDT for the simultaneous and distinguishable detection of multiplex targets in one sample.

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Figure 2. Fluorescence spectra of the programmed DFDT (A) in the absence (curve a) of miRNA 21 and MUC1 and in the presence (curve b) of (B) miRNA 21 and MUC1, (C) miRNA 21 without MUC1, (D) MUC1 without miRNA 21 in one tested solution. The concentration of miRNA 21, MUC1 and oligonucleotides was 0.2 μM, 50 pg·mL-1 and 1 nM, respectively. Analytical Performance of DFDT as Signaling Probe. To evaluate the detection sensitivity and linear range of the DFDT-based strategy, the fluorescence spectra were measured in the presence of miRNA 21 and MUC1 with different concentrations. Figure 3 shows the results obtained under the optimal experimental conditions (see Figure S7 in Supporting Information). Obviously, the F of Cy3 and Cy5 gradually decreased with the increasing of the concentration of miRNA 21 from 0 nM to 10 nM and MUC1 from 0 pg·mL-1 to 100 pg·mL-1 (curve a to j in Figure 3A). This may be 14


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originated from that increased concentrations of miRNA 21 and MUC1 result in target-dependent displacement of more F1 and F2 due to the specific affinity combination of miRNA 21/MUC1 with their recognition elements encoded in R1 and R2, respectively. Upon treatment with more released F1 and F2, more open DFDT architectures are configurationally switched into closure through the hybridization with two pairs of unpaired segments overhung in DFDT. As such, the spatial proximity of both Cy3 and Cy5 to Au NPs leads to significant fluorescence quenching. As a result, the F of Cy3 and Cy5 linearly responds to the logarithm of miRNA 21 concentration ranging from 0.1 pM to 10 nM (Figure 3B) and MUC1 from 0.01 pg·mL-1 to 100 pg·mL-1 (Figure 3C), respectively. The corresponding linear equations are F=1732-692.8lgcmiRNA

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with a correlation coefficient (R) of 0.9931 and

F=1646-511lgcMUC1 with 0.9908 of R. Based on the rule of three standard deviations (3sB/m, where sB and m refer to the standard deviation of the blank solution and the slope of the above-obtained linear equations, respectively),48,49 the limit of detections (LOD) of miRNA 21 and MUC1 were evaluated to be 32 fM and 2.6 fg·mL-1 (8.5 pM), respectively. Thus, the sensitive and recognizable fluorescence assay for multiplex targets is successfully achieved in a wide linear range. Table S2 in Supporting Information summarized the analytical performances of this method in comparison with other reported methodologies for single target detection, miRNA 21 or MUC1.

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Figure 3. (A) Fluorescence spectra of this DFDT responding to miRNA 21 (left, curve a to j) and MUC1 (right, curve a to j) under the optimized experiment conditions. The resultant linear relationship (B and C) plotted by the fluorescence intensity (F) vs the concentrations and their logarithm (inset) of miRNA 21 from 0.1 pM to 10 nM and MUC1 from 0.01 to 100 pg·mL-1. Error bars: standard deviation (s), n=5. Specificity of DFDT as Signaling Probe. The recognizable specificity of the DFDT-based strategy was evaluated by separately measuring the fluorescence spectra of miRNA 21 (1 nM) against miRNA 141, miRNA 155 and let-7 with the same concentration of 10 nM, and MUC1 (50 pg·mL-1) against CEA, AFP, PSA and IgG with the same concentration of 500 pg·mL-1. As shown in Figure 4, miRNA 21, MUC1 and their mixtures yield the most significant decreasing of F of Cy3 (Figure 16


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4A) and Cy5 (Figure 4B) over the tested interferents with 10 times higher concentration than miRNA 21 and MUC1, indicating desirable selectivity of this programmed DFDT in response to the matched target in a homogenous solution. This may be attributed to the specific affinity capability of two recognition probes, as well as the effective and cooperative fluorescence quenching of Au NPs in the closed DFDT. Importantly, this proposed one-step and enzyme-free detection route also exhibit additional convenience and cost-effectiveness, endowing it great promise for the recognizable and simultaneous assay of multiplex targets of interest.

Figure 4. Specificity of this DFDT in response to (A) miRNA 21 (1 nM) against miRNA 141 (10 nM), miRNA 155 (10 nM) and let-7a (10 nM) and their mixture, and (B) MUC1 (50 pg·mL-1) against CEA (500 pg·mL-1), AFP (500 pg·mL-1), PSA (500 pg·mL-1) and IgG (500 pg·mL-1) and their mixture. Error bars: s, n=5. Preliminary Application of DFDT as Signaling Probe. To verify the practicability and applicability of this DFDT, we evaluated miRNA 21 in MCF-7 cell lysis solution and MUC1 in MDA-MB-231 cell lysis solution. The cell lysis solutions were prepared by taking the total RNA extraction solutions from the human cancer cell lines (MCF-7 and MDA-MB-231), respectively. From Figure 5A, the F of Cy3

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and Cy5 gradually decreases with rising of cell numbers in the obtained lysates, suggesting that miRNA 21 and MUC1 were highly expressed in MCF-7 and MDA-MB-231 cells, respectively. The resultant linear calibration curves plotted by the corresponding F v.s. the cell concentration are displayed in Figure 5B. In addition, by using the standard addition method, we investigated the recovery of miRNA 21 and MUC1 in healthy human serum samples, which were kindly provided by Xinqiao Hospital, Army Medical University (Chongqing, China). From Table S3, the recoveries of miRNA 21 and MUC1 range from 95.3% to 98.2% with relative standard deviation (RSD) of 0.85 to 2.3%, and 92% to 108% with RSD of 0.25 to 1.1%, respectively. These above observations demonstrate the desirable potential of this proposed method for the recognizable determination of miRNAs and proteins in a homogenous system, which would open interesting and attractive paths for biosensing and bioanalysis.

Figure 5. (A) Fluorescence spectra of the detection system when adding different concentrations of MCF-7 cancer-cell lysates (left, a to f): 1×104, 2×104, 4×104, 6×104, 8×104 and 10×104 cells·mL-1, and MDA-MB-231 (right, a to f) 2×104, 4×104, 6×104, 8×104, 10×104 and 12×104 cells·mL-1. (B) The resultant linear calibration curves

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plotted by the F of Cy3 and Cy5 v.s. the concentration of MCF-7 and MDA-MB-231 cancer-cell lysates. Error bars: s, n=5. CONCLUSIONS In summary, a novel and programmed “W-type” DFDT is reported for simple, cost-effective, simultaneous and recognizable fluorescence assay of miRNA 21 and MUC1. This unique DFDT contains two Au NP-labeled central strands and an arm strand dually ended with two fluorescent dyes, in which three stiff ds-helixes are formed to stabilize its open conformation and facilitate the flexible configuration switch. In response to miRNA 21 and MUC1, two dependently displaced fuel strands hybridize with the unpaired overhangs in DFDT for its closure, bringing two originally separated dyes approach to Au NPs. The Au NPs-quenched fluorescence emission is utilized for one-step quantization of multiplex targets with high sensitivity and good reliability. To the best of our knowledge, this is the first use of a functional DFDT as recognition element and signaling probe in the fluorescence system, which has three specific features: i) Based on the scheme of nanometal surface energy transfer over a longer distance than FRET, the cooperatively quenching efficiency of Au NPs is more significant for the fluorescence intensity variation. ii) The specific competitive combination of targets enables high responsiveness to single target without overlapped interference. iii) Using easily prepared Au@Fe3O4 magnetic composites as substrates, the stepwise magnetic separation greatly minimized the background signal. The unique advantages of this method would open a new pathway for the wide application of multifunctional DNA nanodevices in biosensing and

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bioanalysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS publications website. The preparation of Au NPs and Au NPs-functionalized magnetic Fe3O4 microspheres (Au@Fe3O4); SEM, TEM, UV-Vis and Raman characterizations of Au NPs and Au@Fe3O4; The detailed base sequences and AFM images of open and closed DFDT; The PAGE Characterization; The optimization of experiment conditions; The comparison of analytical performances of this method with those of other reported methodologies for miRNA 21 and MUC1 detection; The recovery of miRNA 21 and MUC1 in human serum samples. ACKNOWLEDGMENTS We deeply appreciate the National Natural Science Foundation of China (21775123 and 21775124) and the Natural Science Foundation Project of Chongqing (cstc2018jcyjAX0214) for the financial support to this work.

REFERENCES (1) Canoura, J.; Wang, Z. W.; Yu, H. X.; Alkhamis, O.; Fu, F. F.; Xiao, Y. No structure-switching required: A generalizable exonuclease-mediated aptamer-based assay for small-molecule detection. J. Am. Chem. Soc. 2018, 140, 9961-9971.

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A Programmed Dual-Functional DNA Tweezer for Simultaneous and

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