Amplified MicroRNA Detection and Intracellular Imaging based on an

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Amplified MicroRNA Detection and Intracellular Imaging based on an Autonomous and Catalytic Assembly of DNAzyme Lei Yang, Qiong Wu, Yuqi Chen, Xiaoqing Liu, Fuan Wang, and Xiang Zhou ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01000 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Amplified MicroRNA Detection and Intracellular Imaging based on an Autonomous and Catalytic Assembly of DNAzyme Lei Yang,‡a Qiong Wu,‡a Yuqi Chen,b Xiaoqing Liu,a Fuan Wang,*a Xiang Zhoub a Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, P. R. China. b Key Laboratory of Biomedical Polymers (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, P. R. China. Keywords: MicroRNA, Catalytic Hairpin Assembly, DNAzyme, Intracellular Imaging, Signal Amplification ABSTRACT: Abnormal microRNAs (miRNAs) expression is demonstrated to associate with various important biological processes, including tumorigenesis, metastasis, and progression. Given the low miRNA expression at the earlier stage of diseases, its amplified detection still requires more efforts. Inspired by the two-stage arithmetic amplifier of electric devices, we reported an autonomous and catalytic assembly of DNAzyme strategy by integrating DNAzyme biocatalyst and catalytic hairpin assembly (CHA) circuit. Here the catalytically inactive DNAzyme subunits were respectively grafted into these metastable CHA hairpin reactants that were kinetically impeded without false cross-hybridizations. Target catalyzed the nonenzymatic CHA-mediated successive assembly of dumbbell-like bis-DNAzyme nanostructures, leading to the efficient DNAzyme-mediated cleavage of fluorophore/quencher-modified substrate and to the generation of an amplified fluorescence signal. The present CHA-DNAzyme amplifier can be employed as a versatile and general sensing platform for analyzing other analytes, e.g., miRNA, by introducing a sensing module into the present system. Moreover, the homogenous CHA-DNAzyme method could realize the sensitive intracellular miRNA imaging in living cells, which is attributed to the inherently synergistic amplification property between DNAzyme and CHA reactions. Given the attractive analytical features of the autonomous CHA-DNAzyme system, the present strategy shows great promise for analyzing more different analytes in clinical research fields.

MicroRNA (miRNA) is a single-stranded endogenous small non-encoding RNA (19~23 nucleotides) that is mainly present in the cytoplasm.1 MiRNAs suppress the translation of their corresponding mRNAs, thus modulating a large variety of important biological processes, such as cell differentiation and proliferation.2, 3 An aberrant miRNAs expression is demonstrated to correlate with many fatal human diseases such as lung carcinoma and breast cancer.4, 5 Recently, miRNAs have been recognized as important tumor biomarkers and potential therapeutic targets. Thus, it’s of great significance to develop an accurate miRNAs assay for clinical application. However, miRNAs are usually low-expressed in cells, especially in cancerous cells of early stage,6 of which traditional non-amplified sensing schemes could not fulfil. Different enzyme-mediated signal amplification platforms, including polymerase chain reaction (PCR)7, 8 and rolling circle amplification (RCA),9-11 have thus been constructed. However, conventional PCR technique requires temperature cycling which is rather challenging in living cell analysis. The isothermal RCA method needs enzymes that may be susceptible to the surrounding complex cellular microenvironment. Therefore, further efforts are needed for developing new isothermal enzyme-free signal amplification methods, e.g., hybridization chain reactions (HCR),12-14 catalytic hairpin assembly (CHA),15-25 and DNAzyme catalytic reactions.26-40 Especially, most of the current techniques require the extraction of miRNA from cells prior to its analysis and thus are unable to perform real-time monitoring of

intracellular miRNA. With advantages of simple, robust and thermal stable, these enzyme-free strategies provide a promising way for bioanalysis applications. Among these different enzyme-free amplification systems, CHA is a characteristic isothermal free-energy-driven reaction where the initiator catalyzes the cross-opening of two DNA hairpins and generates numerous duplex DNA (dsDNA) products.15 The remarkable features of CHA, including simple design and low background, enable its extensive applications for amplified detection of various nucleic acid (e.g., DNA16-18 and RNA19-21), small molecule (e.g., adenosine22) and protein (e.g., thrombin23) analytes. Traditional CHA reactions are mainly based on fluorophore or gold nanoparticle label, resulting in insufficient amplifications and poor sensitivities that need to be addressed for extensive and universal applications. This issue can be solved by integrating CHA with other different amplification schemes, such as HCR24 and RCA25 strategies. It is expected that the CHA system could be furtherly integrated with other nonenzymatic amplification schemes. Catalytic DNA molecules (DNAzymes) obtain growing interest as amplifying labels for biosensing events.26 The flexibility to encode DNA sequences with functional and structural information, together with the reduced nonspecific binding properties of nucleic acids, provide unique features for their wide applications ranging from bioanalysis to biomedicine fields.27 As compared with protein enzymes, the

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intrinsic merit of DNAzymes depends on their versatility of design and low cost. DNAzymes have been implemented to develop different electrical or optical metal-ion sensors through the aggregation or de-aggregation of gold nanoparticles.28, 29 Also, the DNAzyme was used for the amplified detection of various biologically important analytes. Especially, the amplified DNA detection was accomplished by the activation of an isothermal DNAzyme-based catabolism or anabolism process.30, 31 In contrast to these different cascaded DNAzyme amplifying schemes, an alternative isothermal enzyme-free approach was also developed, including the HCR-mediated assembly of long tandem DNAzyme nanochains.32, 33 DNAzyme has been extensively implemented for detecting nucleic acids,34 aptamer-substrate complexes,35 and metal ions,36 and following enzyme activities.37 These DNAzyme-involved biosensing achievements have been addressed in several recent comprehensive review articles.38-40 In spite of these achievement, more efforts are still desirable for integrating DNAzymes with other amplification approaches. Despite significant achievements have been made for miRNA sensing, most of these methods have been limited in routine in vitro miRNA detection. The further intracellular imaging of endogenous miRNAs still remains a challenge that needs to be explored. Recently, numerous in vivo hybridization-based techniques have been developed by using oligonucleotide probes, e.g., molecular beacons,41 spherical nucleic acids,42, 43 and photo-activated toehold-mediated strand displacement,44 facilitating the study of miRNA biology and the related diseases diagnosis. However, most of these probes convert the miRNA hybridization events into fluorescence signals in a stoichiometric reaction ratio. Given the short nature and lowlevel expression feature of miRNA, intracellular amplification strategies are thus imperative for real time miRNA imaging in living cells. Owing to recent advances of DNA nanotechnology, several nucleic acid amplification attempts have thus been made to improve the intracellular imaging methods. For example, HCR and CHA have been utilized for intracellular RNA imaging.45, 46 DNAzyme-driven nanomotors have also been constructed for efficient intracellular miRNA imaging.47, 48 DNAzyme-triggered CHA transduction has been furtherly used for intracellularly imaging the corresponding metal ion DNAzyme cofactors.49 Despite these isothermal methods have featured the ultrasensitive monitoring of lowly expressed miRNA, there is still urgent requirements for developing more effective enzyme-free cascaded DNAzyme approaches for amplified intracellular imaging researches. Herein, an enzyme-free isothermal CHA-DNAzyme system was developed for nucleic acid analysis by coupling the CHA circuit with a DNAzyme-mediated amplifier as well as transducer. In the present strategy, two hairpins were designed as CHA assembly constitutes and encoded with DNAzyme subunits that were partially caged in the stem regions. The hybridization of these functional hairpins was kinetically impeded. Target mediates the CHA-catalyzed hybridization of these functional hairpins to generate plenty of DNAzyme biocatalysts, resulting in an amplified readout signal. As a simple sensing strategy, the CHA-activated DNAzyme system enabled the sensitive DNA assay with a detection limit of 1 pM, which paved the way for high sensitive detection of other analytes, e.g., miRNA. The homogeneous CHA-DNAzyme system was adapted easily as a versatile sensing platform for analyzing miR-21, through incorporating an auxiliary hairpin.

In addition, the CHA-DNAzyme system was successfully implemented for robust intracellular miRNA imaging. By taking advantage of the robust CHA amplifier and DNAzyme transducer, the CHA-DNAzyme procedure could be easily implemented for more biomedical applications.

MATERIAL AND METHODS Materials. H2O2, Hemin and 2, 2′-Azino-bis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS2−) were purchased from Sigma-Aldrich (Beijing, China). All of these oligonucleotides were synthesized and HPLC-purified by Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table S1). GelRed, fetal bovine serum (FBS) and Lipofectamine 3000 transfection reagent were purchased from Invitrogen (Carlsbad, CA). Dulbecco's Modified Eagle Medium (DMEM) was purchased from HyClone (Logan, Utah, USA). Trypsin was purchased from Genview (USA). MCF-7 and HeLa cells were bought from Shanghai Institutes for Biological Sciences (SIBS). Fluorescence Assay. Each functional hairpin (10 μM in 10 mM HEPES buffer containing 1 M NaCl, 50 mM MgCl2, pH 7.2) was heated to 95 °C for 5 min and then allowed to cool to room temperature (25 °C) for 2 h. The target DNA was added into the H1 (200 nM) + H2 (200 nM) + S (400 nM) mixture to catalyze the autonomous DNA-assembled DNAzyme reaction for 3 h unless specified in reaction buffer (10 mM HEPES, 100 mM NaCl, 20 mM MgCl2, pH 7.2). The successive fluorescence changes were monitored at a fixed emission wavelength of 520 nm upon a fixed excitation of 490 nm. Colorimetric Assay. All samples were prepared in 20 mM HEPES (pH 7.2, 200 mM NaCl). The as-prepared hairpins H4 (250 nM), H5 (250 nM), and H6 (50 nM) were incubated with miR-21 in HEPES for 1.5 h. Then, 1.1 μL of hemin (100 μM) was introduced and incubated for 0.5 h to form the hemin/Gquadruplex DNAzyme (100 μL). After then, the DNAzymecatalyzed oxidation reaction was implemented by furtherly adding 20 μL of ABTS2− (20 mM) and 20 μL of H2O2 (20 mM). UV-vis spectra were immediately collected after 5 min. Gel Electrophoresis Verification. The gel electrophoresis samples including H1, H2 (400 nM each) and DNA target (T1, 100 nM) were incubated in reaction buffer at 25 °C for 3 h. 10 μL of these samples were then mixed with 2 μL loading buffer and loaded into native polyacrylamide gel (9%). Electrophoresis was implemented at 120 V in 1×TBE buffer (89 mM Tris base, 89 mM boric acid, 2.0 mM EDTA, pH 8.3) for 2 h. After stained with GelRed, the gel was then imaged by FluorChem FC3 (Protein-Sample, USA) under 365 nm photoirradiation. Cell Culture and Imaging Analysis. All cells were cultured in DMEM including 10% FBS and 1% penicillin/streptomycin, and were routinely grown at 37 °C in 5% CO2 atmosphere. Cells were seeded into the small culture dishes with 1.0 mL DMEM medium for 24 h. The miR-21targeting CHA mixture including H3 + H1 + H2 + S (0.20 nmol each) was added to Opti-MEM (200 μL, solution I). Meanwhile, Lipofectamine 3000 was prepared in another Opti-MEM (200 μL, solution II). Then both of them were mixed for incubation of 10 min (solution III). Then the solution III was added into the dish with 50 μL FBS for 5 h. Before imaging, the cells were washed three times with PBS and added into freshly 1.0 mL DMEM medium. All fluorescence images were obtained from Leica TCS SP8 laser

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ACS Sensors scanning confocal microscopy. The FAM signal of green channel was collected from 500 nm to 600 nm upon an excitation of 488 nm.

RESULTS AND DISCUSSION The principle of isothermal integrated CHA-DNAzyme system. Here, we report on an isothermal enzyme-free nucleic acid signal amplified detection based on CHA-mediated DNAzyme assembly. As illustrated in Figure 1A, the present sensing system is based on the design of two appropriate hairpin nanostructures that each includes DNAzyme subunits in a “caged” inactive configuration. The system consists of the two hairpins H1 and H2 and a ribonucleobase (rA)-containing DNAzyme substrate S. The substrate S is labelled at its 5'- and 3'-ends with a fluorophore/quencher (F/Q) pair (where F = 6carboxy fluorescein, FAM; Q = Black Hole Quencher-1, BHQ-1), resulting in the quenching of the luminescence of the fluorophore through an effective fluorescence resonance energy transfer (FRET) process. Each of the two hairpins includes two different domains that correspond to the two complement subunits of the Mg2+-dependent RNA-cleaving DNAzyme. The partial participation of the DNAzyme subunits into the stems of hairpins H1 and H2 blocks the assembly of a catalytically active DNAzyme structure. H1 includes the sequence b-c-d that is complementary to the DNA analyte T1. The stem domain d* of H1 is complementary to the toehold region d of H2. H1 also contains an elongated domain d*-c* for assisting the formation of hairpin structure. H2 is encoded with a partially complementary sequence d-e*-d*-c* of H1 that is further elongated with a locking sequence e. Both of H1 and H2 are functionalized at their 5'- and 3'-ends with the DNAzyme subunits I and II, respectively. Although the hybridization between H1 and H2 is thermostatically favored, yet is kinetically impeded without target. T1 opens hairpin H1 via a toehold-mediated strand displacement, leading to the formation of T1-H1 hybrid. The newly exposed sticky sequence d* of H1 docks to the toehold d of H2, and then opens H2 via branch-migration, leading to the assembly of H1H2 duplex DNA (dsDNA) and the regeneration of analyte T1 for CHA reaction. The resulting dumbbell-like H1-H2 hybrid includes two catalytically active DNAzyme units at each end of the duplex DNA (nominated as doublet CHA-DNAzyme system). Thus, target T1 stimulates the efficient CHA reaction, leading to the successive hybridizations between hairpins H1 and H2, and continuously generating H1-H2 product. In the presence of Mg2+ ions, the DNAzyme recognizes and cleaves its substrate S. While the fluorophore is quenched in intact substrate S, the DNAzyme-cleaved S leads to the generation of high fluorescence readout. Validation of integrated CHA-DNAzyme system. The two hairpin components of our CHA-DNAzyme system were theoretically optimized (by an open-access online software, NUPACK50) to dissipate a potential signal leakage without initiator while to maintain a high signal gain for analyzing initiator (details see Figure S1). Firstly, native polyacrylamide gel electrophoresis (PAGE) was used to investigate the feasibility of our CHA-DNAzyme approach, Figure 1B. No new band emerged for CHA-DNAzyme reactants in the absence of their corresponding trigger (lane 3), indicating the hairpins mixtures coexist stably without forming H1-H2 hybrid without initiator. With the addition of DNA target to the CHADNAzyme mixture, a bright new band with lower electrophoretic mobility was revealed (lane 5), which

corresponds to the CHA-generated H1-H2 complex. It is clear that the robust CHA-DNAzyme system can only be specifically triggered by its initiators with high signal gains and negligible background. These electrophoresis experiments demonstrate the successful operation of our CHA-DNAzyme platform.

Figure 1. (A) Scheme of the nonenzymatic integrated CHADNAzyme circuit for amplified DNA detection. (B) Native polyacrylamide gel electrophoresis characterization of the CHA reaction as depicted in Figure 1A: (1) H1; (2) H2; (3) mixture of H1+H2; (4) mixture of H1+T1; (5) mixture of H1+H2+T1. Control experiments of singlet and doublet CHA-DNAzyme system as presented by (C) the time-dependent fluorescence changes (at λ=520 nm) and (D) the fluorescence intensity ratio (ΔFt/ΔFb): (a) H1T+H2T+T1+S; (b) H1T+H2+T1+S; (c) H1+H2T+T1+S; and (d) H1+H2+T1+S. ΔFb and ΔFt refer to the fluorescence intensity changes without and with DNA target, respectively. Error bars were derived from n = 5 experiments.

The proof-of-concept demonstration of the proposed CHADNAzyme amplification strategy was further examined. The high signal amplification capacity of our doublet CHADNAzyme approach was based on an efficient generation of Mg2+-dependent DNAzyme that was attached to each end of the CHA-involved dsDNA product (H1-H2), resulting in a successive cleavage of DNAzyme substrate and a concomitant generation of high fluorescence readout. To further validate the mechanism as described in Figure 1A, the CHA hairpins H1 and H2 were respectively converted to H1T and H2T where the DNAzyme subunits of CHA hairpins were replaced with poly-thymine (T) sequences. The reconstituted hairpins H1T and H2T were introduced to trigger the CHA-DNAzyme procedure. Accordingly, the autonomous CHA-mediated H1TH2T cross-hybridizations cannot produce DNAzyme amplifiers while each H1-H2T or H1T-H2 pair hybridization event produces only one DNAzyme unit in CHA process (nominated as singlet CHA-DNAzyme system, Figure S2). Unsurprisingly, no obvious fluorescence increasement was observed for H1T+H2T mixture after CHA transduction, data a of Figures 1C and 1D. The slight increasement is attributed to

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the non-specific interactions difference between substrates and H1T-H2T hybrid product even if no DNAzyme is formed. Meanwhile, a moderate fluorescence enhancement was observed for singlet CHA-DNAzyme system consisting of H1T+H2 or H1+H2T mixture, data b and c of Figures 1C and 1D, respectively. However, this fluorescence change was still much lower (only 25% ~ 50%) than that of intact doublet CHA-DNAzyme system, data d of Figures 1C and 1D. A different signal amplification capacity of these H1T- and H2Tsubstituted singlet CHA-DNAzyme systems is might attributed to their different DNAzyme-generating capacities and varied DNAzyme microenvironments. Obviously, the CHA-motivated successive cross-opening of these hairpin reactants leads to an effective DNAzyme generation. All of these results could be easily explicated as follows. The generation of DNAzyme occurs in linear amplification with a multiple reaction ratio (1:N) in the amended H1T- or H2Treplaced singlet CHA-DNAzyme system, while one target generates a doubled signal gain (1:2N) in the present intact doublet CHA-DNAzyme method. The theoretical analysis is consistent with the fluorescence measurements, demonstrating the amplification feature of the current CHA-DNAzyme amplifier.

analyte T1 was introduced into the aforementioned CHADNAzyme system, a tremendously elevated fluorescence was revealed and it reached to a plateau after ca. 3 h (bar b of Figure 2A). Accordingly, a fixed time-interval of 3 h was chosen as the most suitable CHA-DNAzyme reaction time for acquiring fluorescence spectra subsequently. After ensuring an efficient operation of the CHA-DNAzyme strategy, the optimized system was applied for quantitative detection of DNA analyte. The fluorescence spectra were obtained after the CHA-DNAzyme mixture was incubated with different concentrations of target DNA for 3 h (Figure 2B). Obviously, the fluorescence change (∆F) increased with the elevated DNA concentration, indicating that the triggering of CHADNAzyme system is highly analyte-dependent (Figure 2C). Figure 2D depicts a linear correlationship between the fluorescence change (λ = 520 nm) and DNA concentration ranged from 5 pM to 1 nM (R2=0.990). The detection limit was acquired to be 1 pM based on the conventional 3σ calculation strategy. The performance of our CHA-DNAzyme amplifier is comparable to most of these fluorescent nonenzymatic detection platforms (Table S2), which is originated from a synergistic amplification effect between CHA and DNAzyme constitutes.

Figure 2. (A) Fluorescence (at λ = 520 nm) monitoring the CHADNAzyme system as described in Figure 1A, in the absence (a) and presence (b) of DNA target T1. (B) Fluorescence spectra of the CHA-DNAzyme system upon analyzing different concentrations of DNA analyte after a fixed time-interval of 3 h. (C) Resulting calibration curve. (D) Resulting linear correlationship curve. Error bars were derived from n = 5 experiments.

Figure 3. (A) Illustration of the isothermal enzyme-free integrated miR-21-targeting CHA-DNAzyme circuit by furtherly incorporating a “helper” hairpin. (B) Fluorescence spectra of the extended CHA-DNAzyme system with different concentrations of miR-21. (C) Resulting calibration curve. Inset: linear calibration curve. (D) Fluorescence intensity changes (λ = 520 nm) of the extended CHA-DNAzyme system with 50 nM of different miRNAs: (a) let-7a miRNA, (b) miR-155, (c) miR-429, (d) miR141, and (e) miR-21. Error bars were derived from n = 5 experiments.

Fluorescence assay of DNA analyte in vitro. The reaction time-interval of the system was explored to achieve an optimized analyzing performance. The H1+H2 mixture shows weak fluorescence change (bar a of Figure 2A), indicating that these hairpin mixtures are metastable enough, due to the efficient formation of an energetically favorable hairpin structure. All of these hairpins are kinetically impeded without obvious signal leakage (spontaneous cascaded CHADNAzyme reactions) without initiator. However, when the

Fluorescence assay of miR-21 in vitro. It should be noted that the analyte (DNA) and the transduction (Mg2+-dependent DNAzyme) elements of the CHA-DNAzyme system can be easily extended to miRNA target and hemin/G-quadruplex HRP-mimicking DNAzyme, respectively. The optimized CHA-DNAzyme amplifier can also be utilized as a general sensing strategy for detecting other targets, e.g., miR-21,

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ACS Sensors without alternating any of these hairpin constitutes. Abnormal miR-21 has been recognized as an important oncogenic miRNA and is associated with many cancer diseases.4-6 As illustrated in Figure 3A, a “helper” hairpin H3 was designed to recognize miR-21 target, releasing the versatile trigger sequence (T1) to motivate the CHA-DNAzyme process and to generate a significant amplified fluorescence readout signal. The auxiliary “helper” hairpin H3 was theoretically (by NUPACK50) and experimentally optimized to obtain lower background and higher signal gains (details see Figure S3). A moderate stem length of hairpin H3 ensures the high detection performance of our miR-21-targeting CHA-DNAzyme system. The optimized H3 was then introduced into the CHADNAzyme platform for analyzing miR-21. Figure 3B shows the fluorescence spectra of the miR-21-targeting system upon analyzing varied concentrations of miR-21 for 5 h. The fluorescence change (∆F) increased substantially with increasing concentrations of miR-21 analyte from 10 pM to 50 nM (Figure 3C), which is consistent with that of DNA assay. Meanwhile, there exists a good linear correlationship between fluorescence changes (∆F) and the concentration of miR-21 with a fine correlation coefficient R2 = 0.984, as shown in Figure 3C inset. Based on the CHA-DNAzyme amplifier, the detection limit of miR-21 was estimated to be 10 pM. Thus the CHA-DNAzyme system can be used as a versatile amplification module for more general sensing applications with the help of an auxiliary sensing module. The performance of the CHA-DNAzyme system not merely relies on its high amplifying capacity, but also on its high selectivity. The selectivity of the CHA-DNAzyme-amplified miR-21 assay was then investigated by challenging the updated CHADNAzyme system with a series of interfering nucleic acids: let-7a miRNA, miR-155, miR-429 and miR-141. As shown in Figures 3D and S4, no obvious fluorescence change was obtained for these interfering miRNAs (curves a, b, c and d), while only miR-21 produced a high fluorescence readout (curve e), demonstrating a high selectivity of the newly constructed miR-21-sensing system. Colorimetric assay of miR-21 in vitro. The present RNAcleavage DNAzyme-based fluorescent sensing platform needs internal modification of RNA (chimeric RNA) and tedious fluorophore/quencher-labelled substrates. It is expected the CHA-DNAzyme system can be furtherly extended to a labelfree approach by adapting a different DNAzyme catalyst. The implementation of other high catalytic DNAzymes enables a more facile transduction of the CHA-DNAzyme process. This was exemplified by the introduction of hemin/G-quadruplex HRP-mimicking DNAzyme that allows the catalyzed colorimetric transduction of various sensing events. Accordingly, the CHA-amplified detection of miR-21 target was enabled for generating colorimetric readout signal via the DNAzyme-catalyzed oxidation of ABTS2– substrate to the colored ABTS–• product by H2O2. As illustrated in Figure 4A, this label-free colorimetric CHA-DNAzyme system was composed of two functional CHA hairpins (H4 and H5), and an auxiliary miR-21-rocogniton hairpin H6 was similarly introduced to recognize miR-21 analyte. Here the Mg2+dependent DNAzyme subunits of H1 and H2 were respectively replaced with the HRP-mimicking DNAzyme subunits to generate H4 and H5. Thus, miR-21 initiated the successive cross-hybridization of hairpins H4 and H5 and to produce H4H5 hybrids decorating with two G-quadruplex structures. And, in the presence of hemin, these hemin/G-quadruplex

DNAzymes are formed to catalyze the oxidation of ABTS2− by H2O2 to colored product ABTS•− (λ = 420 nm). The label-free CHA reaction was executed after 1.5 h (Figure 4B) and then the hemin/G-quadruplex DNAzyme-catalyzed oxidation of ABTS2− was carried out for an optimized time-interval of 5 min (Figure S5) to obtain the absorbance spectra. Figure 4C depicts the resulting absorbance spectra of the CHADNAzyme system upon analyzing variable concentrations of miR-21. A noteworthy increasement of the absorbance was observed as the increasing concentration of miR-21. Control experiments show that only minute absorbance change was observed without miR-21 target (bar a of Figure 4B), implying that the colorimetric readout of the system, indeed, originates from the successive cross-hybridization of these hairpins, and the efficient generation of the active hemin/Gquadruplex DNAzyme units (bar b of Figure 4B). The calibration curve of miR-21 is depicted in Figure 4D, revealing that miR-21 could be analyzed with high sensitivity.

Figure 4. (A) Scheme for the label-free CHA-mediated hemin/Gquadruplex DNAzyme system for amplified miR-21 assay. (B) Time-dependent CHA-DNAzyme reaction as revealed by the absorbance changes of ABTS2− (λ = 420 nm) for non-triggered (a) and miR-21-triggered (b) system. (C) Absorbance spectra of the CHA-DNAzyme system with different concentrations of miR-21. (D) Resulting calibration curve. Inset: linear calibration curve. Error bars were derived from n = 5 experiments.

CHA-DNAzyme-mediated intracellular miR-21 imaging. The merit of our CHA-DNAzyme system was not only reflected by its general (target) design and modular (DNAzyme) transduction, but also on its real time sensing applicability in complex biological environment. The CHADNAzyme amplifier has been demonstrated for accurately quantifying miR-21 analyte in vitro, we then expected this system could be utilized for real-time monitoring of intracellular miRNA under in vivo conditions. The fluorescent doublet CHA-DNAzyme system was then investigated for intracellular monitoring miR-21 in different mammalian cells by confocal laser scanning microscopy (CLSM). The CHADNAzyme reactants were phosphorothioate-functionalized to ensure their moderate biostabilities in complex cell culture

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medium. MCF-7 cells, known with high miR-21 expression, were chosen as a model to probe the performance of the intracellular miR-21-targeting CHA-DNAzyme imaging system. An obvious fluorescence signal was observed for the CHA-DNAzyme-mediated miR-21 assay (sample a of Figures 5A and S6A), demonstrating the successful implementation of CHA-DNAzyme imaging system in living cells. The average loading of the produced CHA units was roughly estimated to be 3.8×10-19 mol/cell in MCF-7 cells (detailed experimental procedures and calculations, see Figure S7 and the accompanying description). This implies a high miR-21 expression in MCF-7 cells and an exact intracellular location of miR-21 analyte. Meanwhile, an improved amplification efficiency of the present CHA-DNAzyme-imaging was indirectly demonstrated by substituting one of the CHA hairpin reactants from the system (details see Figure S8). Unsurprisingly, the fluorescence readout of the singlet CHADNAzyme imaging system (H1T- or H2T-substituted CHADNAzyme system, samples b and c of Figure S8) was obviously lower than that of doublet CHA-DNAzyme system in MCF-7 cells (sample a of Figure S8), which is consistent with those of fluorescence experiments. This furtherly demonstrated the highly efficient amplification feature of the CHA-DNAzyme system even in intracellular conditions. Moreover, no fluorescence imaging signal was observed for the H1T/H2T-substituted CHA-DNAzyme system (as a merely CHA control, sample d of Figures S8). It is reasonable since no DNAzyme catalyst was assembled even the CHA reaction was successful implemented. This, on the other hand, demonstrated that the fluorescence activation is indeed originated from the DNAzyme-mediated cleavage of substrate that generates the high fluorescence readout. Meanwhile, an anti-miR-21 antisense inhibitor oligonucleotide was designed to eliminate the intracellular miR-21, and was then utilized to prove that the observed fluorescence signal was generated exactly from the intracellular miR-21 rather than other miRNAs. A negligible fluorescence signal was observed in the antisense inhibitortreated MCF-7 cells (sample b of Figures 5A and S6A), implying that the CHA-DNAzyme system can discriminate miRNA of varied expression levels in living cancer cells. The versatile of our CHA-DNAzyme imaging system was further verified by monitoring a relatively low expression of miR-21 in HeLa cells, where a comparably reduced fluorescence was revealed (sample c of Figures 5A and S6A). Obviously, our CHA-DNAzyme imaging system could easily discriminate varied cells based on their distinct miRNA expressions. Figure 5B shows a statistical histogram analysis of the CHADNAzyme imaging system that was collected from large quantities of their corresponding living cells. Clearly, the CHA-DNAzyme imaging system indeed offers a modular and robust analytical toolbox for amplified miRNA detection in living cells. With the aid of multiplexed fluorophore-labels, this homogeneous CHA-DNAzyme amplifier offers new ways for early cancer diagnosis through the simultaneous quantification of multiple RNA targets.

CONCLUSIONS In conclusion, we have constructed an enzyme-free paradigm for isothermal nucleic acid detection by integrating the high amplifying capabilities of CHA and DNAzyme reactions. Two DNAzyme subunits were respectively grafted into the CHA hairpin reactants, which were kinetically impeded from false

cross-hybridization. Target could efficiently catalyze the hybridization of these two hairpin reactants to properly connect these DNAzyme subunits, leading to the assembly of a large amount of DNAzyme biocatalysts. The feasibility and capability of our CHA-DNAzyme strategy were systematically investigated and demonstrated in ideal buffer and complicated intracellular environments. These versatile fluorescent and colorimetric sensing platforms were constructed by integrating respectively RNA-cleavage DNAzyme and HRP-mimicking DNAzyme into the doublet CHA-DNAzyme system. Both of the fluorescent and colorimetric CHA-DNAzyme sensing methods were simple and relied only on autonomous DNA hybridization reactions, without additional enzymes and particular instruments. It is also an inherently scalable amplification module for sensitive and selective detection of other clinically important target, e.g., miRNA, upon its facile integration with an auxiliary “helper” hairpin. More importantly, the molecularly engineered CHA-DNAzyme system was successfully implemented for analyzing miRNA in complex biological environment. Upon its integration with other powerful affinity probes, e.g., aptamers, the present CHA-DNAzyme sensing strategy is expected to play an important role in monitoring other metabolite targets inside living cells.

Figure 5. (A) Intracellular analysis of miR-21 by CHADNAzyme-imaging system. CHA-DNAzyme-mediated imaging of miR-21 in (a) MCF-7 living cells, (b) MCF-7 living cells treated with an anti-miR-21 inhibitor oligonucleotide, and (c) HeLa cells by CLSM. All scale bars correspond to 40 μm. (B) Statistical histogram analysis of these above cell samples. Error bars were derived from n = 50 living cells.

ASSOCIATED CONTENT Supporting Information. DNA sequences, optimizing and control experiments of CHA-DNAzyme system, and comparison of the performance of present system with others. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

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ACS Sensors * E-mail: [email protected].

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors sincerely acknowledge the financial support from Natural Science Foundation of China (21503151, 21874103 and 81602610), National Basic Research Program of China (973 Program, 2015CB932601), Jiangsu Provincial NSFC (BK20161248, BK20160381), Wu-han Youth Science and Technology Plan (No. 2016070204010131) and Fundamental Research Funds for the Central Universities (No. 2042018kf0210).

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