DNAzyme Based Nanomachine for in Situ Detection of MicroRNA in

DNAzyme Based Nanomachine for in Situ Detection of MicroRNA in Living Cells ... Publication Date (Web): November 28, 2017 ... This target-responsive, ...
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A DNAzyme Based Nanomachine for in situ Detection of MicroRNA in living Cells Jing Liu, Meirong Cui, Hong Zhou, and Wenrong Yang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00710 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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A DNAzyme Based Nanomachine for in situ Detection of MicroRNA in living Cells Jing Liu,a, c Meirong Cui,a, b Hong Zhou*a, c and Wenrong Yang*a, c a

Shandong Provincial Key Laboratory of Detection Technology for Tumor Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, P. R. China.

b

Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Shandong Normal University, Jinan 250014, P. R. China. c

Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3217, Australia

ABSTRACT: The capability of in situ detection of microRNA in living cells with signal amplification strategy is of fundamental importance, and it will open up a new opportunity in development of diagnosis and prognosis of many diseases. Herein we report a swing DNA nanomachine for intracellular microRNA detection. The surfaces of Au nanoparticles (NPs) are modified by two hairpin DNA. We observe that one DNA (MB2) will open its hairpin structure upon partial hybridization with target miR-21 after entering into cells, and the other part of its hairpin structure could further react with the other hairpin DNA (MB1) to form a Zn2+ specific DNAzyme. This results in the disruption of MB1 through shearing action and the release of fluorescein Cy5. To provide an intelligent DNA nanomachine, MB2 is available again with the shearing action to bind with MB1, which provides effective signal amplification. This target-responsive, DNA nanomachine-based method showed a detection limit of 0.1 nM in vitro and we expected this approach could be an important step towards intracellular amplified detection and imaging of various analytes in living cells. KEYWORDS: microRNA, DNA nanomachine, Zn2+ -specific DNAzyme, signal amplification, functionalized gold nanoparticles

Deregulated microRNA (miRNA, approximately 19-25 nucleotides) levels have already been reliable markers for diagnosis and prognosis of many diseases.1, 2 Traditional techniques including standard PCR and northern blotting have been used for intracellular miRNA detection, in which complex RNA extraction steps are required, and these techniques has so far proven more difficult for in situ detection of intracellular targets. Therefore, in situ detection of miRNA expression at single cell level are urgently needed, which will provide a more complete spatial profile of RNA expression.3, 4 Among various in situ detection techniques, construction of nanomaterials based fluorescent probes (NFPs) is an important approach to monitor miRNA in living cells via fluorescence imaging. These NFPs demonstrate advantages including easily-transfection and its stability in cellular environments, however, these techniques lack sensitivity due to cellular local environments. Therefore, reliable and sensitive detection methods for intracellular miRNA are desirable.5, 6 Recently NFPs based optical imaging techniques for in vitro7-9 or even real-time sensitive detection of gene expression inside of cells have been developed.10-12 NFPs were synthesised mainly by fluorescein modified DNA probes. When NFPs entered into cells through endocytosis, NFPs are able to react with target molecular (for example miRNA, 13,14 mRNA15-18, ATP19, or telomerase20, 21) and thus result in the changes of fluorescence signal. These current intracellular

target imaging strategies provide intuitive results and better understanding of many diseases.22-25 And NFPs takes advantages of the unique properties of nanoparticle, including strong quenching efficiency, stability in bio system avoiding degradation, transfection agents-free endocytosis.26-30 However, there is a rigorous challenge in sensitivity because signal amplification in living cells is very difficult. Recently metal ion-specific DNAzyme have been developed for detection of vital metal ions and even biomolecules.31-34 And they showed broad prospects for in situ detection of intracellular analytes because additional enzyme is not required. Furthermore, DNAzyme based NFPs have been proposed for in situ gene detection. 35-39 Very recently a DNAzyme motor40 and a termed Au NP-based hairpin-locked-DNAzyme probe41 have been reported to sense intracellular miRNA. Both DNAzyme motor and the Au NP-based hairpin-locked-DNAzyme probe were responsive to a specific microRNA for amplified detection of the specific microRNA in individual cancer cells. However, these methods are limited as recycling target released to the cytochylema is much difficult to react with probes on Au NP and the designed probe was complex. Here, we report a new detection strategy through construction of a DNAzyme based DNA nanomachine. In this approach, two hairpin DNA (named MB1and MB2, both modified fluorescence Cy5 at the end of DNA) are functionalised on the surfaces of Au NPs. Initially we are not able to observe fluorescence signal of Cy5 due to the quenching effect between Au NPs and Cy5. After

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entering into cells, MB2 opens its hairpin structure upon partly hybridization with target microRNA 21(miR-21). The remainder of the opened MB2 further react with MB1, which is also modified on surfaces of Au NPs, and thus forms Zn2+-specific DNAzyme. In presence of Zn2+, DNAzyme, which works as a DNA nanomachine, is able to disrupt MB1 from the middle and Cy5 is released, and then fluorescence is recovered. After this shearing action, MB2 is available again to bind with another MB1 around it as a DNA nanomachine. It turn out that one target binding could result in a few times of shear actions, which enable effective signal amplification. (Scheme 1). Moreover, with the appropriate proportion of MB2 (also as a DNAzyme strand) and MB1 (as DNAzyme substrate strand) on the surface of Au NPs, optimal amplification is able to be obtained. This strategy is effective to improve the sensitivity of imaging which can produce more fluorescence signals upon one target miRNAs inside cells.

Scheme 1. Preparation of DNAzyme assistant DNA nanomachine and its application for in situ miR-21 imaging

EXPERIMENTAL SECTION Materials and Reagents. HeLa cells were obtained from Key-GEN biotechnology Company (Nanjing, China), HepG2 cells (Human hepatocellular carcinoma cell line) and Lipofectamine 2000 were purchased from Shanghai Bio leaf Biotechnology Company (Shanghai, China), L-02 cells (human normal hepatocytes) were from Silver Amethyst Biotech. Co. Ltd. (Beijing, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) and sulfhydryl PEG were obtained from Sigma Chemical Company. Au NPs with an average diameter of 15 nm were purchased from Ted Pella, Inc. All other reagents were of analytical grade and used without purification. All aqueous solutions were prepared using DEPC treated ultrapure water from a Milli-Q system (Millipore, USA). The DNA sequences used in this paper were purchased from Sangon Biotech Co., Ltd. (Shanghai China). RNA sequences were synthesized and purified by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The sequences are as follows: MB1: 5’-Cy5-CCA CCA CTT TTT ACC CAC TAT rA GGA AGT CTG AAT TTT GTG GTG GTT TTT TTT -SH-3’

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MB2: 5’-Cy5-TA TCA GAC TTC TCC GAG CCG GTC GAA ATA GTG GGT TTT TTT TTT TTT TCA ACA TCA GTC TGA TAA GCT ATT TT-SH-3’ MiR-21 mimics： 5’-UAGCUUAUCAGACUGAUGUUGA-3’ Anti-miR-21： 5’-TCAACATCAGTCTGATAAGCTA-3’ One-base mismatch DNA： 5’-TAGCTTATCAGGCTGATGTTGA-3’ Control DNA：5’-CACAGCCGGACTACTCCTAGTG-3’ Synthesis of NFPs. NFPs was synthesized according to previous report with mirror modifications.42 Gold nanoparticles (Au NPs, 15 nm in diameter, 2.4 nM) were stored at 4ºC. MB2 and MB1 were added to a solution of Au NPs with the ranging ratio from 5:100, 10:100, 20:100, 35: 100 to 50:100 (MB2/MB1) and stirred overnight. The following day, 0.1 mL SH-PEG (10 µM) was added and incubated for 12 h in the dark. 2 M sodium chloride was added to the solution to achieve a final concentration of 0.1 M to stabilize the probe through eight times. After incubation at room temperature for another two days, the solution was centrifuged (13,000 rpm, 20 min) and resuspended in 10 mM Tris-HCl (pH 7.4) solution. Then it was stored at 4 °C prior to use. In-Vitro MiR-21 Detection. NFPs (100nM) and Zn2+ (5µM, or without Zn2+ for control group) were incubated with target miR-21 mimics of different concentrations and two control DNA respectively. The concentrations of target miR-21 mimics were 0.1 nM,1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 800 nM, and 1µM). After incubation for 1 h at 37°C, the fluorescence was monitored at 649 nm. Real-Time Fluorescence Tracking in Single Cell. First, HeLa cells were cultured in DMEM for 24 h. Then, the cells were incubated with 5 µM Zn2+ at 37 °C in 5 % CO2 for 60 min. Next, the cells were washed three times and incubated with NFPs (1 nM) for 0h, 2h, 3 h, 4h, 5h and 6h. Finally the cells were washed three times with PBS for imaging. Fluorescence images were acquired on a confocal laser scanning microscope and were recorded by Cy5 in red channel with 638 nm excitation. HepG2 cells and L-02 cells were also employed for comparison under the same experimental conditions. Cell Viability Assay. Cell viability was measured by MTT assay. To investigate the cytotoxicity of the NFPs, a MTT assay was carried out when the NFPs existed. HeLa cells were dispersed within replicate 96-well microtiter plates to a total volume of 200 µL well−1. Plates were maintained at 37 °C in incubator for 24 h. After the original medium was removed, Hela cells were incubated with unmodified Au NPs (1 nM), NFPs (1 nM) for 6 h, 12 h, 24 h and 48 h. Then, 100 µL MTT solutions (0.5 mg mL−1 in PBS) were added to each well. After 4 h of incubation, the remaining MTT solution was removed, and 150 µL of DMSO was added to each well to dissolve the

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crystals. The absorbance of MTT at 490 nm is dependent on the degree of activation of the cells. Then cell viability was expressed by the ratio of absorbance of the cells incubated with the NFPs to that of the cells incubated with culture medium only. Instruments. UV-vis absorption spectra were recorded using a Cary 60 UV-vis spectrometer (Agilent Technologies Co. Ltd., USA). Transmission electron microscopy (TEM) was conducted on a JEOL JEM-2100 at an accelerating voltage of 200 kV equipped with EDX (Energy-dispersive analysis of Xrays). Fluorescence imaging was performed using a Leica TCS SP8 inverted confocal microscope (Leica, Germany). Fluorescence images were acquired on a confocal laser scanning microscope and were recorded by Cy5 in red channel with 638 nm excitation. All fluorescence measurements were carried out on a F4600 fluorometer (Hitachi, Japan). The emission spectra were obtained by exciting the samples at 649 nm.

fied at the end of two types of hairpin DNA closely attached to the Au NPs surface, thus resulting high-efficiency fluorescence quenching effect from the Au NPs. It is well-known that Zn2+ is an important factor for the activity of DNAzyme. In order to study the feasibility of this design, we next verified the formation of DNAzyme on Au NPs using the fluorescence signal of the NFPs after the reaction of miR-21 mimics with or without the treatment of Zn2+.

RESULTS AND DISCUSSION Here Au NPs with a diameter of 15 nm were used for the synthesis of NFPs due to their capabilities of efficiently quenching fluorophores and easily being modified with oligonucleotides. In our experiments, the ratio of two types of hairpin DNA, which were modified on surfaces of Au NPs, plays an important role for obtaining maximum amplified fluorescence signal. As showed in Figure 1a, a series of molar ratios of MB2 (as a DNAzyme strand) and MB1 (as DNAzyme substrate strand) ranged from 5:100 to 50:100 were employed during the preparation of NFPs. The fluorescence results were recorded after above NFPs (with different molar ratio of MB2/MB1) were treated with target miR-21 mimics in vitro. Figure 1b showed that changing the molar ratio of MB2/MB1, fluorescence signal was increased first and then declined, showing its maximum value at 1:10 (MB2/MB1). Then this optimal molar ratio was used in the following experiments.

Figure 1. a) Scheme of the preparation of NFPss (with different molar ratio of MB2/MB1 on Au NPs); b) Fluorescence response of the NFPs with different molar ratio of MB2/MB1 for target miR-21 mimics detection in vitro.

Transmission electron microscopy (TEM) image indicated the average size of Au NPs was about 15 nm. Au NPs-NFPs, showed a characteristic DNA peak at 260 nm in the UV/Vis spectrum, confirming the modification of NFPs on surfaces of Au NPs (Figure 2b). Here, fluorescein Cy5 which was modi-

Figure 2. a) TEM images of Au NPs (left) and NFPs (right, Au NPs modified with MB2/MB1); b) UV-Vis spectra of Au NPs and NFPs. Insert is enlarged region of DNA adsorption from NFPs.

Two types of hairpin DNA (MBI and MB2), functionalized with the fluorophore Cy5 at the 5’end and thiol at the 3’ end, were modified on the surface of Au NPs. MB2 consists of two essential sections, a target binding sequence and a Zn2+ dependent DNAzyme sequence. Here, fluorescence is efficiently quenched due to the vicinity between Cy5 and Au NP. While upon target miR-21, target binding sequence of MB2 hybridized with miR-21, and the remaining DNAzyme sequence further bond with MB1 around it, resulting in the formation of DNAzyme structure. In the presence of Zn2+, DNAzyme was “active” and cleaved MB1 at the middle into two shorter fragments. Then the fragment modified with Cy5 was released to the solution with expected greatly enhance fluorescence. From Figure 3a we observed that much higher fluorescence signal appeared from NFPs incubated in the presence of Zn2+ with 1µM target miR-21 mimics than that without Zn2+. It is clear that the changes of fluorescence signal was mainly from the formation of “active” DNAzyme and sheared MB1 DNA, which resulted in the release of Cy5. Because of the resulting far-away distances of Cy5 to the surfaces of Au NPs, the fluorescence signal enables recover and be observed obviously. Partly hybridization between MB2 and target miR-21 is critical for the formation of a DNAzyme nanomachine. We then used one-base mismatch DNA and a control DNA (not com-

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plementary with MB2) to conduct the following experiments. Figure 3b showed extremely low fluorescent compared with using target complementary DNA (miR-21 mimics), which indicated the excellent specificity and selectivity of this method. When increased amount of target miR-21 mimics (0.1nM1µM, Figure 3c), we observed the enhancement of the fluorescence, which further indicated that the increased fluorescence response was only from the target miR-21.

Figure 3. a) Fluorescence spectra of the NFPs incubated with 1µM target miR-21 mimics. Red curve is under the condition of 5µM Zn2+ in solution and black curve is without Zn2+ addition for control group; b) Fluorescence spectra of the NFPs incubated with 1µM target miR-21 mimics, one-base mismatch DNA and control DNA (not complementary with MB2); c) Fluorescence results of the sensor under different concentrations of miR-21 mimics (0.1 nM, 1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 800 nM, 1µM).

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NFPs modified with only MB2 was used in the experiment. The reason is that target miR-21 inside the cells could bind with MB2 modified on the surface of Au NPs and opened its hairpin structure. The opened MB2 made fluorescein Cy5 (modified on the end of MB2) away from Au NPs and resulted in the obvious fluorescence signal. However, since the target miR-21 is not able to directly react with MB1, the MB1 modified NFPs was not able response to the intracellular target miR-21. And we also observed significant signal reduction in the absence of the designed nanomachine on the NFPs, suggesting that the designed nanomachine is important in signal amplification. Meanwhile, there was little fluorescence signal when we used the NFPs which was only modified with MB1 under the same experimental conditions. In order to find out whether the introduction of this nanomachine would make an impact on the cell vability, we then evaluated the toxicity of the designed NFPs by MTT tests. It has been reported that the absorbance of MTT (490 nm) is dependent upon the degree of activation of the cells.45 Then the ratio of the absorbance of MTT treated with HeLa cells solution which were incubated with the NFPs (or bare Au NPs) to that of MTT treated with the cells incubated with only culture medium for control was studied to show the cell viability. Figure 4b showed high cell viabilities (more than 90%) when incubated with bare Au NPs and the NFPs for a long time, which indicated little cytotoxicity or side effects of NFPs for living cells.

MiR-21 has been reported to be overexpressed in most malignant tumor cells.43,44To assess the target-response feasibility and efficiency of the designed NFPs for miR-21 in living cells, we used confocal laser scanning microscopy (CLSM) to monitor the fluorescence response of the NFPs which was delivered into HeLa cells (miR-21 over expressed) via endocytosis. Figure S1 showed the real-time monitoring of the NFPs intervening HeLa cells (previous treated with Zn2+) during the first 6h. Initially we were not able to see fluorescence signal, and as time goes on, red fluorescence signal is able to be observed and become increasingly clear from 2h to 6h, which indicated that the designed NFPs was effective during the in situ detection of miR-21, which the DNA nanomachine was activated. By using nanomachine, we expected the fluorescence signal should be amplified effectively and competent to highly sensitively detect miR-21. Subsequently, the control experiments were performed. We synthetized a control NFPs which was modified with only one type of hairpin DNA (only MB1, and only MB2). The CLSM images from Figure 4a exhibited that there was little fluorescence signal when we used NFPs only modified with MB1 during the treatment of Hela cells for 6h. While a very weak fluorescence signal was observed when a

Figure 4. a) Fluorescence images of HeLa cells (pretreated with 5 µM Zn2+ for 1h) cultured with control NFPs (modified with only MB1 or MB2); little fluorescence from MB1 probe was observed and indicated there is no reaction occurred between MB1 and target; The weak signal from MB2 suggested that MB2 reacted with target miR-21 and opened its hairpin structure, generating a

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small amount of fluorescence without signal amplification strategy from DNAzyme b) Cell viability of HeLa cells using 1 nM NFPs and 1 nM bare Au NPs. Error bars represent the standard deviation of three replicates.

We demonstrated that the intracellular miR-21-responsed NFPs was able to effectively work in HeLa cells as scissors to realize fluorescence recovery of Cy5 and signal amplification. And what would be happen if we pre-treat Hela cells with miR 21 mimics or anti-miR 21 transfection? We then used Hela cells transfected with miR 21 mimics and anti-miR 21 for target miR-21 imaging, and Hela cells without treatment were also used as control group. Figure 5 showed that increased and decreased fluorescence signal could be observed respectively when using miR 21 mimics and anti-miR 21 transfected Hela cells. Additionally, we used HepG2 cells (hepatocellular carcinoma cell line) and L02 cells (human normal hepatocytes) instead of HeLa cells in the following experiments. From the results of Figure 6, we observed that carcinoma cells including HepG2 cells showed obvious fluorescent signal, but there is no clear fluorescence in human normal hepatocytes L02, which indicated that this method could evaluate different miR-21 levels.

Figure 6. CLSM images of NFPs after incubation for 4 h with HepG2 cells and L02 cells for target miR-21 imaging.

In summary, we developed a smart NFPs modified with two types of hairpin DNA which could response to intracellular miR-21 and work as a nanomachine inside the cells. MB2 DNA which was modified on the NFPs could specifically and partly bind with target miR-21, open its hairpin structure and further form DNAzyme upon the hybridization with MB1. In the presence of Zn2+, DNAzyme worked as a nanomachine, resulting in the substrate strands abscission and the fluorescence signal recovery of Cy5. More importantly, during the whole process, DNAzyme substrate strand (MB1) was snipped to release the fluorescein, while DNAzyme strand (MB2, probe for target) was not damaged or consumed, which is able to continue to work as a nanomachine. Therefore, one targetmiR-21 binding could result in a few times of shear action which is effective for signal amplification. Furthermore, this in situ sensing strategy is easy to operate and extracted miRNA from rupturing the cells is not required compared with other techniques including real-time quantitative PCR or single-cell northern blotting. Based on the overexpression of miR-21 in cancer cells, we hope this miRNA-responsive nanomachine model provides a simple but effective way to distinguish cancer cells from normal cells, and further evaluate other important targets during intracellular amplified detecting and imaging.

ASSOCIATED CONTENT Supporting Information Available: The following files are available free of charge. Supporting information. Real-time monitoring of fluorescence images of HeLa cells cultured with NFPs.

AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected]. Figure 5. CLSM images of NFPs after incubation for 4 h with Hela cells without treatment and with the treatment of miR-21 mimics transfection and anti-miR-21 transfection.

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT Financial support was provided by the National Natural Science Foundation of China (Grant Nos.: 21505065, 21675074, 21675075), the “Innovation Team Development Plan” of the Ministry of Education Rolling Support (IRT_15R31) and the Taishan Scholar Program of Shandong Province.

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REFERENCES (1) Calin, G. A.； Croce, C. M. MicroRNA Signatures in Human Cancers. Nat. Rev. Cancer. 2006, 6, 857-866. (2) Croce, C. M. Causes and Consequences of MicroRNA Dysregulation in Cancer. Nat. Rev. Genet. 2009, 10, 704-714. (3) He, L.；Thomson, J. M.； Hemann, M. T.； Hernando-Monge, E.； Mu, D.； Goodson, S.； Powers, S.； Cordon-Cardo, C.； Lowe, S. W.； Hannon, G. J.； Hammond, S. M. A MicroRNA Polycistron as a Potential Human Oncogene. Nature 2005, 435, 828833. (4) Lee, H.； Park, J. E.； Nam, J. M. Bio-Barcode Gel Assay for MicroRNA. Nat.Commun. 2014, 5, 3367-3343. (5) Visvader, J. E. Cells of Origin in Cancer.Nature. 2011, 469, 314322. (6) Degliangeli, F.； Pompa, P. P.； Fiammengo, R. Nanotechnology-Based Strategies for the Detection and Quantification of MicroRNA. Chem. Eur. J. 2014, 20, 9476-9492. (7) Duan, R. X.； Zuo, X. L.； Wang, S. T.； Quan, X. Y.； Chen, D. L.； Chen, Z. F.； Jiang, L.； Fan, C. H.； Xia, F. Lab in a Tube: Ultrasensitive Detection of MicroRNAs at the Single-Cell Level and in Breast Cancer Patients Using Quadratic Isothermal Amplification. J. Am. Chem. Soc. 2013, 135, 4604-4607. (8) Wang, L. D.； Deng, R. J.； Li, J. H. Target-Fueled DNA Walker for Highly Selective MiRNA Detection. Chem. Sci. 2015, 6, 67776782. (9) Degliangeli, F.； Kshirsagar, P.； Brunetti, V.； Pompa, P. P.； Fiammengo, R. Absolute and Direct MicroRNA Quantification Using DNA–Gold Nanoparticle Probes. J. Am. Chem. Soc. 2014, 136, 22642267. (10) Medley, C. D.； Drake, T. J.； Tomasini, J. M.； Rogers, R. J. ； Tan, W. H. Simultaneous Monitoring of the Expression of Multiple Genes Inside of Single Breast Carcinoma Cells. Anal. Chem. 2005, 77, 4713-4718. (11) Santangelo, P. J.； Nix, B.； Tsourkas, A.； Bao, G. Dual FRET Molecular Beacons for mRNA Detection in Living Cells. Nucleic Acids Res. 2004, 32, e57. (12) Zheng J. Li, C.； Cansiz, S.； Wu, C.； Xu, J.； Cui, C.； Liu, Y.； Hou, W.； Wang, Y.； Zhang, L.； Teng ，I. T.； Yang, H. H.； Tan, W. Self-assembly of DNA Nanohydrogels with Controllable Size and Stimuli-Responsive Property for Targeted Gene Regulation Therapy. J. Am. Chem. Soc. 2015, 137, 1412-1415. (13) Hemphill, J.； Deiters, A. DNA Computation in Mammalian Cells: MicroRNA Logic Operations. J. Am. Chem. Soc. 2013, 135, 10512-10518. (14) Zhang, P. H.； He, Z. M.； Wang, C.； Chen, J. N.； Zhao, J. J.； Zhu, X. N.； Li, C. Z.； Min, Q. H.； Zhu, J. J. In Situ Amplification of Intracellular MicroRNA with MNAzyme Nanodevices for Multiplexed Imaging, Logic Operation, and Controlled Drug Release. ACS nano. 2015, 9, 789-798. (15) Jayagopal, A.； Halfpenny, K. C.；. Perez, J. W； Wright, D. W. Hairpin DNA-Functionalized Gold Colloids for the Imaging of mRNA in Live Cells. J. Am. Chem. Soc. 2010, 132, 9789-9796. (16) Li, X. L.； Zhang, Z. L.； Zhao, W.； Xia, X. H. ； Xu, J. J.； Chen, H. Y. Oriented Assembly of Invisible Probes: Towards Single mRNA Imaging in Living Cells. Chem. Sci, 2016, 7, 3256-3263. (17) Zhou, Q. M.； Ma, Y.； Wang, Z. H.； Wang, K.； Liu, R. N. ； Han, Z. H.； Zhang, M.； Li, S. W.； Gu, Y.Q. Optimized Ultra-

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sound Conditions for Enhanced Sensitivity of Molecular Beacons in the Detection of MDR1 mRNA in Living Cells. Anal. Chem. 2016, 88, 2808-2816. (18) Chen, T.；Wu, C. S.；Jimenez, E.；Zhu, Z.；Dajac, J. G.； You, M.； Han, D.；Zhang, X.；Tan, W. DNA Micelle Flares for Intracellular mRNA Imaging and Gene Therapy. Angew. Chem. Int. Ed. 2013, 52, 2012-2016. (19) Yang, Y. J.；Huang, J.；Yang, X. H.； Quan, K.； Wang, H.； Ying, L.；Xie, N. L.； Ou, M.；Wang, K. M. Aptazyme–Gold Nanoparticle Sensor for Amplified Molecular Probing in Living Cells. Anal. Chem. 2016, 88, 5981-5987. (20) Qian, R. C.； Ding, L.； Yan, W. L.；Lin, M. F.；Ju, H. X. A Robust Probe for Lighting up Intracellular Telomerase via Primer Extension To Open a Nicked Molecular Beacon. J. Am. Chem. Soc. 2014, 136, 8205-8208. (21) Ma, Y.； Wang, Z. H.； Zhang, M.； Han, Z. H.；Chen, D.； Zhu, Q. Y.； Gao, W. D.； Qian, Z. Y.； Gu, Y. Q. A TelomeraseSpecific Doxorubicin-Releasing Molecular Beacon for Cancer Theranostics. Angew. Chem. Int. Ed. 2016, 55, 3304-3308. (22) Pan, W.； Zhang, T. T.； Yang, H. J.；Diao, W.；Li, N.； Tang, B. Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four-Color Nanoprobe. Anal. Chem. 2013, 85, 10581-10588. (23) Zang, Y. D.；Wei, Y. C.；Shi, Y. J.；Chen, Q.；Xing, D. Chemo/Photoacoustic Dual Therapy with mRNA-Triggered DOX Release and Photoinduced Shockwave Based on a DNA-Gold Nanoplatform. Small, 2016, 12, 756-769. (24) Choi, C. K. K.；Li, J. M.；Wei, K. C.；Xu, Y. J.；Ho, L. W. C.； Zhu, M. L.；To, K. K. W.；Choi, C. H. J.；Bian, L. M. A Gold@Polydopamine Core–Shell Nanoprobe for Long-Term Intracellular Detection of MicroRNAs in Differentiating Stem Cells. J. Am. Chem. Soc. 2015, 137, 7337-7346. (25) Dong, H. F.；Lei, J. P.；Ju, H. X.；Zhi, F.；Wang, H.；Guo, W. J.；Zhu, Z.；Yan, F. Target-Cell-Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe. Angew. Chem. Int. Ed. 2012, 51, 4607-4612. (26) Li, N.；Chang, C.；Pan, W.；Tang, B. A Multicolor Nanoprobe for Detection and Imaging of Tumor-Related mRNAs in Living Cells. Angew. Chem. Int. Ed. 2012, 51, 7426-7430. (27) Prigodich, A. E.；Randeria, P. S.；Briley, W. E.； Kim, N. J.； Daniel, W. L. ； Giljohann, D. A. ； Mirkin, C. A. Multiplexed Nanoflares: mRNA Detection in Live Cells. Anal. Chem. 2012, 84, 2062-2066. (28) Seferos, D. S.；Giljohann, D. A.；Hill, H. D.；rigodich, A. E. P.； Mirkin, C. A. Nano-Flares:  Probes for Transfection and mRNA Detection in Living Cells. J. Am. Chem. Soc. 2007, 129, 1547715479. (29) He, Z, M.; Zhang, P. H.; Li, X.; Zhang, J. R.; Zhu, J. J. A Targeted DNAzyme-Nanocomposite Probe Equipped with Built-in Zn2+ Arsenal for Combined Treatment of Gene Regulation and Drug Delivery. Sci. Rep.2016, 6, 22737. (30) Qian, R. C.；Cao, Y.； Long, Y. T. Binary System for MicroRNA-Targeted Imaging in Single Cells and Photothermal Cancer Therapy. Anal. Chem. 2016, 88, 8640-8647. (31) Li, L.；Feng, J.；Fan, Y. Y.；Tang, B. Simultaneous Imaging of Zn2+ and Cu2+ in Living Cells Based on DNAzyme Modified Gold Nanoparticle. Anal. Chem. 2015, 87, 4829-4835.

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(32) Wu, W. P.；Hwang, K.；Lan, T.；Lu, Y. A DNAzyme-Gold Nanoparticle Probe for Uranyl Ion in Living Cells. J. Am. Chem. Soc. 2013, 135, 5254-5257. (33) Lu, C. H.；Wang, F.；Willner, I. Zn2+-Ligation DNAzymeDriven Enzymatic and Nonenzymatic Cascades for the Amplified Detection of DNA. J. Am. Chem. Soc. 2012, 134, 10651-10658. (34) Zhang, X. B.；Wang, Z. D.；Xing, H.； Xiang, Y.；Lu, Y. Catalytic and Molecular Beacons for Amplified Detection of Metal Ions and Organic Molecules with High Sensitivity. Anal. Chem. 2010, 82, 5005-5011. (35) Jimmy Huang, P. J.；Liu, J. An Ultrasensitive Light-up Cu2+ Biosensor Using a New DNAzyme Cleaving a PhosphorothioateModified Substrate. Anal. Chem. 2016, 88, 3341-3347. (36) Liu, J.； Cao, Z.；Lu, Y. Functional Nucleic Acid Sensors. Chem. Rev. 2009, 109, 1948-1998. (37) Sigel, R. K. O.；Sigel, H. A Stability Concept for Metal Ion Coordination to Single-Stranded Nucleic Acids and Affinities of Individual Sites. Acc. Chem. Res. 2010, 43, 974-984. (38) Cui, L.；Peng, R. R.；Fu, T.；Zhang, X. B.；Wu, C. C.； Chen, H. P.； Liang, H.；Yang, C. J.；Tan, W. H. Biostable LDNAzyme for Sensing of Metal Ions in Biological Systems. Anal. Chem. 2016, 88, 1850-1855. (39)Yang, J. M.；Dou, B. T.；Yuan, R.；Xiang, Y. Proximity Binding and Metal Ion-Dependent DNAzyme Cyclic Amplification-

Integrated Aptasensor for Label-Free and Sensitive Electrochemical Detection of Thrombin. Anal. Chem. 2016, 88, 8218-8223. (40) Peng, H. Y.; Li, X. F.; Zhang, H. Q.; Le, X. C. A MicroRNAInitiated DNAzyme Motor Operating in Living Cells. Nat. Commun. 2017, 8, 14378-14390. (41) Yang,Y. J.; Huang, J.; Yang, X. H.; He, X. X.; Quan, K.; Xie, N. L.; Ou, M.; Wang, K. M. Gold Nanoparticle Based Hairpin-LockedDNAzyme Probe for Amplified miRNA Imaging in Living Cells. Anal. Chem. 2017, 89, 5850-5856 (42) Zhang, P. H.； Cheng, F. F.; Zhou, R.； Cao, J. T.； Li, J. J.； Burda, C.；Min, Q. H.；Zhu，J. J. DNA-Hybrid-Gated Multifunctional Mesoporous Silica Nanocarriers for Dual-Targeted and MicroRNA-Responsive Controlled Drug Delivery. Angew. Chem. Int. Ed. 2014, 53, 2371-2375. (43) Zhang, H.Q.; Lai, M.; Zuehlke, A.; Peng, H.; Li, X.-F.; Le, X. C. Excellent Stability of a Lithium-Ion-Conducting Solid Electrolyte upon Reversible Li+/H+ Exchange in Aqueous Solutions. Angew. Chem. Int. Ed. 2015, 54, 1- 6. (44) Krichevsky, A. M. ； Gabriely, G. MiR-21: A Small Multifaceted RNA. J. Cell. Mol. Med. 2009, 13, 39-53. (45) Dong, H. F.； Lei, J. P.； Ding, L.； Wen, Y. Q.； Ju, H. J.； Zhang, X. J. MicroRNA: Function, Detection, and Bioanalysis. Chem. Rev. 2013, 113, 6207-6233.

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