Biodegradable MnO2 Nanosheet-Mediated Signal Amplification in

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Biodegradable MnO2 Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA Jing Li, Daxiu Li, Ruo Yuan, and Yun Xiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13073 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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ACS Applied Materials & Interfaces

Biodegradable MnO2 Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA Jing Li, Daxiu Li, Ruo Yuan, and Yun Xiang* Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China E-mail: [email protected] (Y.X.). * Corresponding author. Tel.: +86-23-68252277 (Y. Xiang). ABSTRACT The monitoring of intracellular microRNAs plays important roles in elucidating the biological function and biogenesis of miRNAs in living cells. However, because of their sequence similarity, low abundance and small size, it is of great challenge to detect intracellular miRNAs, especially for those with much lower expression levels. To address this issue, we have developed an in cell signal amplification approach for monitoring down-regulated miRNAs in living cells based on biodegradable MnO2 nanosheet-mediated and target-triggered assembly of hairpins. The MnO2 nanosheets can adsorb and exhibit excellent quenching effect to the dye labeled hairpin probes. Besides, due to their biodegradability, the MnO2 nanosheets feature with highly reduced cytotoxicity to the target cells. Upon entering cells, the surface-adsorbed FAM- and Tamra (TMR)-conjugated hairpins can be released due to the displacement reactions by other proteins or nucleic acids and the degradation of the MnO2 nanosheets by cellular GSH. Subsequently, the down-regulated target miRNA-21 triggers cascaded assembly of the two hairpins into long dsDNA polymers, which brings the fluorescence resonance energy transfer (FRET) pair, FAM (donor) and TMR (acceptor), into close proximity to generate significantly enhanced FRET signals for detecting trace miRNA-21 in living cells. By carefully tailoring ACS Paragon Plus Environment 1


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the sequences of the hairpins, the developed method can offer new opportunities for monitoring various trace intracellular miRNA targets with low expression levels in living cells.

KEYWORDS: Manganese oxide nanosheets; Signal amplification; Self-assembly; MicroRNA; Living cells; INTRODUCTION MicroRNAs (miRNAs) are a type of small (18-25 nt in length), single stranded, endogenous and non-protein coding RNA molecules commonly encoded in plants, animals, and some viruses.1 MiRNAs can regulate gene expression through the promotion of the degradation or the inhibition of the translation of the target messenger RNAs,2 thereby playing important regulatory roles in cell differentiation, proliferation, apoptosis, gene expression and many other biological processes.1,3-7 Increasing research evidences have indicated that the change in expression of miRNAs is closely associated with the occurrence and development of different cancers and many other pathological conditions.8,9 For instance, miRNA-18b has shown to be up-regulated in the tumor samples of breast cancer patients10,11 while miRNA-143 is found to be down-regulated in breast and cervical cancers.12 Because of their regulatory roles in gene expression and variances in expression profiles, miRNAs have been extensively used as effective biomarkers for disease diagnosis and as therapeutic targets for disease treatment.13-16 Consequently, the development of robust methods for the identification and detection of miRNAs is expected to be of great clinical significance. Conventional methods for detecting miRNAs are mainly based on reverse transcription polymerase chain reaction,17 Northern blotting18 and microarray technologies.19 Although quantitative detection of different miRNAs can be achieved, these early developed assay methods still encounter several cost, time, sensitivity limitations, and this has triggered recent development of many new biosensor technologies for miRNA detection. These alternatives couple various signal amplification approaches, such as nanomaterials20 and enzymes21 with optical,22 electrochemical23,24 or electrochemiluminescent25 ACS Paragon Plus Environment 2

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transduction means to achieve the detection of miRNA targets with high selectivity and sensitivity. With no doubt, the miRNA assay methods have been significantly advanced. However, most of such methods can only detect miRNAs extracted from cancer cells, and the detection of intracellular miRNAs in living cells, which can elucidate the biological function and biogenesis of miRNAs in vivo, has been rarely reported.26 Fluorescence in situ hybridization (FISH)27,28 and locked nucleic acid-based FISH29,30 assays have been the dominant imaging technologies for the detection of intracellular miRNA molecules. However, FISH-based imaging methods require the fixation of cells and are not amenable to be applied to living cells. Besides, the sensitivity and selectivity for differentiating analogous miRNAs among family members is limited.31 Recently, the employment of nanomaterials for detecting intracellular miRNAs in living cells has attracted intensive attentions due to the unique optical, electrical and thermal properties of such materials.32 Of particular interest is the application of the carbon-based (e.g., graphene, carbon dots and carbon nanotubes) and gold-based (e.g., gold nanoparticles and nanorods) materials for intracellular sensing purposes.33,34 The Ju group35 employed the nucleic acid adsorbed carbon nitride nanosheets for detecting intracellular miRNA-18a in living HepG2 cells while Huh and co-workers36 used hyaluronic acid-based nanocontainers containing miR-34a beacons as probes for imaging miR-34a in MCF-7 cancer cells. These nanomaterial-based methods have indeed advanced the monitoring of up-regulated or overexpressed intracellular miRNAs, yet the detection of down-regulated miRNAs in cells remains a major challenge due to the very low amount of these miRNAs. 37 With an effort to address this issue, Li38 and Chu31 groups, respectively, have integrated rolling circle amplification (RCA) with FISH to achieve sensitive detection of intracellular miRNAs. However, the involvement of enzymes (phi29 DNA polymerase) in RCA and the requirement of cell fixation prevent the application of such methods from monitoring miRNAs in living cells. Although very recently, a cascade hybridization reaction method was built as a in cell signal amplification means to image low amount miRNA in living cells,39 the use of liposome-based transfection agents encounter the issues of low transfection efficiency, toxicity, limited loading, and complicated preparation steps.40 ACS Paragon Plus Environment 3


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In response to current challenges in monitoring trace miRNA in living cells, we report here a biodegradable MnO2 nanosheet-mediated hybridization chain reaction (HCR) strategy for detecting miRNAs expressed at very low levels in living cells. MnO2 nanosheets are graphene structure analogous nanomaterial that exhibit efficient fluorescent quenching ability to organic dye-linked ssDNAs.41 In addition, MnO2 nanosheets offer diverse bio-functionality and can be degraded by cellular species such as glutathione (GSH) to significantly reduce their toxicity to cells, enabling them promising delivery and sensing intermediates for living cells.41-43 HCR, first reported by Pierce and colleagues,44 involves the self-assembly formation of long dsDNA polymers between two hairpin DNAs triggered by ssDNA strands. Such a hairpin assembly process is isothermal and can offer complete enzyme-free signal amplification for amplified sensing of different nucleic acid targets at very low levels.45-47 In our design, two hairpin DNAs separately labeled with fluorescence resonance energy transfer (FRET) organic dyes are delivered into living cells by MnO2 nanosheets, and upon entering cells the hairpin DNAs can be released due to the displacement reactions by other proteins or nucleic acids48 and the degradation of the MnO2 nanosheets by cellular GSH. Consequently, the down-regulated miRNAs present in the cells can trigger the hairpins to assemble into dsDNA polymers and bring the FRET pairs into close proximity, leading to significantly amplified FRET signals for detecting these trace miRNAs in living cells. The intracellular sensing approach developed herein thus offers three obviously advantages over other reported ones. First, the use of biodegradable MnO2 nanosheets reduces their cytotoxicity to the cells. Second, our FRET-based approach can minimize false positive signals, which occurs in common quencher/dye sensing systems due to probe accumulation or degradation.49 Third, in cell HCR signal amplification enables enzyme-free detection of trace levels of intracellular miRNAs. EXPERIMENTAL SECTION Chemicals and Materials: Manganese chloride tetrahydrate (MnCl2·4H2O), tetramethylammonium hydroxide (TMA·OH), L-glutathione reduced (GSH), dimethyl sulfoxide (DMSO), CaCl2, MgCl2, hydrogen peroxide (H2O2, 3 wt %) and Tris-HCl were purchased form Sinopharm Chemical Reagent ACS Paragon Plus Environment 4

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Co.,


Ltd.

(Shanghai,

China).

The

cell

culture

reagents

and

3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Dingguo Biological Technology Co., Ltd. (Chongqing, China). The MCF-7 and HeLa cell lines were obtained from the cell bank of the type culture collection of the Chinese Academy of Sciences (Shanghai, China). Lipofectamine○ R 3000 and all synthetic oligonucleotides with the sequences listed in Table 1 were synthesized by Invitrogen Biotechnology Co., Ltd. (Shanghai, China). Table 1 Sequences of the synthetic oligonucleotides Name H1-FAM

H2-TMR

H3 miRNA-21 let-7a (control miRNA)

Sequence from 5’ to 3’ (FAM)-CAGAC TGATG TTGAC GTGAA ACTCA ACATC AGTCT GATAA GCTA GTTTC ACGTC AACAT CAGTC TGT-(TAMRA) AG CTTAT CAGAC TGATG TTGA (FAM)-CAGAC TGATG TTGAC GTGAA ACTCA ACATC AGTCT-(Dabcyl) GATAA GCTA UAGCU UAUCA GACUG AUGUU GA UGAGG UAGUA GGUUG UAUAG UU

miRNA-21

mUmCmAmAmCmAmUmCmAmGmUmCmUmGmAmUmAmAmGmCmU

inhibitor

mA (m: 2’-O-methyl)

Apparatus: All fluorescence signals were obtained on a RF-5301PC ﬂuorescence spectrophotometer (Shimadzu, Tokyo, Japan). The JPK NanoWizard 3 Bioscience atomic force microscope (AFM) with the tap 300Al-G silicon probe (Budget Sensors, Bulgaria) was used to characterize the morphology of the nanosheets at a resonant frequency of 320 kHz and force constant of 42 N/m. Gel electrophoresis analysis was carried out on a DYY-8C electrophoretic apparatus (Beijing WoDeLife Sciences Instrument Co., Ltd). Confocal fluorescence images were obtained on a confocal laser scanning

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fluorescence microscopy with an Olympus FV1000 confocal scanning system with an objective lens (20×). The MTT assay was performed on a RT 6000 microplate reader. Synthesis and Characterization of MnO2 Nanosheets: MnO2 nanosheets were prepared according to previous reports with slight modifications.50 In brief, the mixture (20 mL) of TMA·OH (0.6 M) and H2O2 (3 wt %) was reacted with MnCl2·4H2O (10 mL, 0.3 M) solution within 15 s. After the formation of a dark brown suspension, the resulting mixture was stirred overnight at room temperature, centrifuged at 2000 rpm for 10 min, washed with deionized water and methanol, and then dried at 60 ºC. Subsequently, 10 mg dried crude product was dispersed in 20 mL deionized water and ultrasonicated for another 10 h. Finally, the dispersion was centrifuged at 2000 rpm for 30 minutes, and the supernatant was used in the experiments. The concentration of the MnO2 nanosheets was determined by inductively coupled plasma emission spectrometer. Fluorescence Quenching and Recovery Experiments: H1-FAM and H2-TMR were first heated to 90 ºC for 5 min separately and cooled down to room temperature for 1 h before use. Next, various concentrations of MnO2 nanosheets (0 to 20 μg mL-1) were added into the mixture of H1-FAM (25 nM) and H2-TMR (25 nM) in 200 μL Tris-HCl buffer (100 mM, 25 mM MgCl2, 5 mM CaCl2, pH 7.5). After 20 minutes, the fluorescence intensities of the solutions were recorded. For the fluorescence recovery experiment, GSH was added to the H1-FAM/H2-TMR/MnO2 nanosheets (MnO2: 20 μg mL-1; H1-FAM and H2-TMR each at 25 nM) in Tris-HCl buffer at a final concentration of 1 mM to reduce MnO2 for 5 min, and then the fluorescence emission was recorded. In Vitro HCR amplified FRET Detection: MnO2 nanosheets (20 μg mL-1) were added to the mixture of H1-FAM (25 nM) and H2-TMR (25 nM) in Tris-HCl buffer to prepare the H1-FAM/H2-TMR/MnO2 probe solution. After that, GSH (1 mM) was added to the solution for 5 min. This was followed by the addition of the target miRNA-21 (2.5 nM) or the control let-7a miRNA (2.5 nM), and the solution was incubated at 37 ºC for 4 h to allow miRNA-triggered HCR between H1-FAM and H2-TMR. Finally, the reaction mixture was directly transferred into 200 μL quartz cuvette for fluorescence measurement. The


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fluorescence emission spectrum in the region from 500 to 640 nm was recorded by using the excitation wavelength of 488 nm. Native Polyacrylamide Gel Electrophoresis (PAGE): PAGE was carried out to verify the HCR events. In a typical experiment, H1 (2 μM) and H2 (2 μM) were incubated with miRNA-21 or control let-7a for 4 h at 37 ºC. All samples were then loaded into the 16 % polyacrylamide gel matrix and run in 1×TBE buffer at 100 V for 80 min. After ethidium bromide (EB) staining, gels were visualized using a digital camera (EOS 550D) to obtain the images under 365 nm UV irradiation. Cell Culture and Confocal Fluorescence Imaging: HeLa cells and MCF-7 cells were provided by the Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) solution containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin in a humidified atmosphere of 5% CO2 was used to culture cells at 37 ºC for 24 h in the culture dishes. Subsequently, the living cells were treated with H1-FAM/H2-TMR/MnO2 nanosheets in culture medium at 37 ºC for 4 h. After that, PBS (0.1 M, pH 7.4) was used to wash cells for three times and the fresh medium containing 10% fetal bovine serum was added. Then, cells were visualized under an Olympus FV1000 confocal scanning system with an objective lens (20×). FAM fluorescence image was recorded in the green channel and FRET fluorescence image was recorded in red channel. Uptake of the MiRNA-21 Inhibitor by HeLa Cells: The miRNA-21 inhibitor was transfected into HeLa cells using the Lipofectamine○ R 3000 reagent at final concentration of 500 nM for 6 h according to the instructions from the manufacturer. HeLa cells were then incubated with fresh culture medium containing the H1-FAM/H2-TMR/MnO2 nanosheets for another 4 h, followed by washing cells with PBS and recording fluorescence emission images on the confocal scanning system. Cytotoxicity Assay: HeLa cells (1×105 cells/well) were seeded in 96-well microtiter plates with 200 μL cell medium containing 10% fetal bovine serum and cultured in 5% CO2/95% air at 37 ºC for 24 h. After replacing with fresh medium, various concentrations of MnO2 nanosheets (0~60 μg mL-1) were added into the cell medium for 24 h incubation. Then, the MTT solution (0.5 mg mL -1) was further ACS Paragon Plus Environment 7


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incubated with the cells in each well for another 4 h. Subsequently, after removing the remaining MTT medium, DMSO (150 μL) was added to each well for 30 min. The cytotoxicity of MnO2 nanosheets was determined by measuring the absorbance at 488 nm using a RT 6000 microplate reader. RESULTS AND DISCUSSION

Scheme 1. Illustration of MnO2 nanosheet-mediated in cell HCR signal enhancement for sensitively detecting miRNA-21 in living cells. MnO2 nanosheets can adsorb and transport the hairpin DNA probes into cells. Intracellular GSH degrades the MnO2 nanosheets to release the free hairpins, which can assemble into dsDNA polymers upon binding to the target miRNA-21. Amplified FRET signals are generated to realize high sensitivity. Our signal amplified intracellular miRNA sensing principle is illustrated in Scheme 1. The two hairpin DNA probes, H1-FAM and H2-TMR, are elaborately designed to avoid spontaneous self-assembly in the absence of the target miRNA-21. The 5’-terminus of H1 is labeled with FAM (donor) while TMR (acceptor) is conjugated to the stem region of H2 in order to position the two dyes into proximity upon the assembly of H1-FAM and H2-TMR. Both H1-FAM and H2-TMR can be easily adsorbed on the basal plane of MnO2 nanosheets via van der Waals force.51 Due to their excellent quenching ability and good biocompatibility, MnO2 nanosheets can thus efficiently quench the


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fluorescence of FAM and TMR and can deliver the two hairpins into cells via endocytosis to protect them from enzymatic degradation. Upon entering cells, the MnO2 nanosheets can be immediately reduced to Mn2+ dispersed in cytoplasm by intracellular GSH, and the adsorbed hairpins are then released with the combination effect of the displacement reactions by other proteins or nucleic acids. When the target miRNA-21 is present in the cells, it can specifically hybridize with the sticky end of H1-FAM (the purple sequence in H1-FAM) and open the hairpin structure to unlock a single stranded region (the blue sequence in H1-FAM) to hybridize with and unfold H2-TMR. The unfolded H2-TMR can again hybridize with H1-FAM to initiate HCR between H1-FAM and H2-TMR, leading to the self-assembly formation of dsDNA polymers. As a result, the fluorescent dyes FAM and TMR are brought into close position, resulting in significantly amplified FRET signals from FAM to TMR for highly sensitive detection of intracellular miRNA-21.

Figure 1. (A) AFM image, (B) Height profile of the section in (A), and (C) UV-Vis absorption spectrum of MnO2 nanosheets. Inset of (A): The enlarged view of the white line circled area. The morphology of the as prepared MnO2 nanosheets was first characterized by atomic force microscopy (AFM). As shown in Figure 1A and 1B, the MnO2 nanosheets display a two-dimensional sheet structure with a size of height profile ~1.3 nm and ~200 nm in width. Furthermore, the UV-Vis spectroscopy of the MnO2 nanosheets exhibits a maximum absorption peak at ~370 nm. These experimental results indicate the successful preparation of the MnO2 nanosheets.



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Figure 2. (A) Fluorescence spectra of the mixtures of H1-FAM (25 nM) and H2-TMR (25 nM) with the addition of different concentrations of MnO2 nanosheets. From a to i: 0, 5, 8, 10, 12, 14, 16, 18, 20 μg mL-1 of MnO2 nanosheets. The mixtures were excited at 488 nm to record the emission from FAM. Inset: Changes in fluorescence intensity vs. the concentration of the MnO2 nanosheets (the mixtures were exited at 488 nm and 540 nm, respectively, to record the emissions from FAM and TMR). (B) Fluorescence recovery of H1-FAM/H2-TMR/MnO2 in the presence of 1 mM GSH. Fluorescence spectra of FAM were recorded with the excitation wavelength of 488 nm. Next, the fluorescence quenching capability of the prepared MnO2 nanosheets was evaluated by mixing H1-FAM (25 nM) and H2-TMR (25 nM) with different concentrations of MnO2 nanosheets. It can be seen in Figure 2A that the H1-FAM hairpins show strong fluorescence emission in the absence of the MnO2 nanosheets when excited at 488 nm, and the fluorescence intensity of H1-FAM and H2-TMR gradually decreases with increasing concentration of MnO2 nanosheets from 0~20 μg mL-1 with complete fluorescence quenching at the concentration of 20 μg mL-1 (Inset of Figure 2A). This observation reveals the fluorescence of the hairpin monomers can be efficiently quenched by the MnO2 nanosheets.

However,

the

addition

of

GSH

(1

mM)

to

the

completely

quenched

H1-FAM/H2-TMR/MnO2 solution leads to significant restoration (89.8%) of FAM emission (Figure 2B), owing to the reduction of the MnO2 nanosheets to Mn2+ by GSH and the release of the hairpins. Such results further confirm that the MnO2 nanosheets can be efficiently degraded by GSH.


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Figure 3. (A) PAGE demonstration of miRNA-21-triggered HCR assembly of H1 and H2. Lane 1: H1 (2 μM); Lane 2: H2 (2 μM); Lane 3: H1 (2 μM) and H2 (2 μM); Lane 4: H1 and H2 in the presence of the control miRNA let-7a (100 nM); Lane 5: H1 and H2 in the presence of miRNA-21 (100 nM); Lane 6: H1 and H2 in the presence of miRNA-21 (250 nM). (B) In vitro FRET analysis of different solutions. Black curve: H1-FAM (25 nM); Red curve: H2-TMR (25 nM); Blue curve: the mixture of H1-FAM (25 nM) and H2-TMR (25 nM) with the addition of MnO2 nanosheets (20 μg mL-1); Green curve: GSH/H1-FAM/H2-TMR/MnO2 with the addition of GSH (1 mM); Purple curve: miRNA-21 (2.5 nM)/GSH/H1-FAM/H2-TMR/MnO2; Cyan curve: let-7a (2.5 nM)/GSH/H1-FAM/H2-TMR/MnO2. All solutions were excited at 488 nm. To verify that HCR self-assembly of H1 and H2 can be triggered by miRNA-21, native polyacrylamide gel electrophoresis (PAGE) experiments were performed (Note: the H1 and H2 used in PAGE were free of any dye labels). As shown in Figure 3A, H1, H2 and the mixture of H1 and H2, respectively, exhibit a single band with similar electrophoretic mobility (Lane 1 to lane 3), indicating that H1 and H2 are metastable and can co-exist without the presence of the target miRNA-21 trigger. Moreover, the incubation of H1 and H2 with a control miRNA (let-7a) leads to neglect changes in electrophoretic mobility (Lane 4 vs. Lane 3), suggesting that the control miRNA is unable to initiate the HCR reaction. However, once the miRNA-21 (100 nM) is introduced to the mixture of H1 and H2, consecutive bands with much lower mobility (Lane 5) can be observed, and the increase of the target miRNA-21 concentration to 250 nM results in the decrease in assembly formation of longer dsDNA polymers, which is a typical phenomenon of HCR.42 The results shown here indicate that the two hairpins can be self-assembled into dsDNA polymers by the miRNA-21 trigger. ACS Paragon Plus Environment 11


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To demonstrate that the introduction of the target miRNA-21 to GSH/H1-FAM/H2-TMR/MnO2 can trigger the HCR between H1-FAM and H2-TMR and further activate the FRET signals, series tests were performed in vitro. As shown in Figure 3B, when excited at the wavelength of 488 nm, H1-FAM exhibits very strong fluorescence emission at 520 nm (Black curve) while very weak fluorescence emission spectra of H2-TMR at 570 nm (Red curve) is observed due to the significantly minimized FRET between FAM and TMR. With the addition of MnO2 nanosheets, the fluorescence of the hairpin mixture is almost completely quenched (Blue curve) because of the significant quenching effect of the MnO2 nanosheets to the surface-adsorbed hairpins, and the introduction of GSH to the mixture leads to remarkable recovery of the fluorescence of FAM (Green curve) as discussed previously. Importantly, the incubation of the target miRNA-21 to GSH/H1-FAM/H2-TMR/MnO2 results in apparent FRET phenomenon with the decrease and increase of fluorescence intensity at 520 nm and 570 nm (purple curve), respectively, with the excitation wavelength of 488 nm. Such FRET is basically due to the miRNA-21-triggered assembly formation of the H1-FAM/H2-TMR dsDNA polymers (demonstrated in Figure 3B), which bring FAM and TMR into close proximity to facilitate the corresponding FRET. A control experiment was also performed by incubating the control miRNA (let-7a) with GSH/H1-FAM/H2-TMR/MnO2 and no FRET signals can be observed (Cyan curve), indicating that the control miRNA is unable to trigger HCR. Such comparison reveals the high selectivity and the great potential of this approach for sensitive FRET detection of the target miRNA-21.

Figure 4. Cytotoxicity assay of HeLa cells incubated with different concentrations of MnO2 nanosheets for 24 h.


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As the first step for the application of the MnO2 nanosheets in detecting intracellular miRNA-21, MTT assays were used to evaluate the cytotoxicity of the MnO2 nanosheets by assessing cell viability. HeLa cells were incubated with MnO2 nanosheets at different concentrations from 0~60 μg mL-1. As shown in Figure 4, the cell viability slightly decreases with increasing concentration of MnO2 nanosheets, and there is insignificant viability loss of the cells with MnO2 nanosheets concentration below 20 μg mL-1. Moreover, 86% cells remain alive when cells were treated with 60 μg mL-1 MnO2 nanosheets. Compared to other commonly used grahene oxide cell imaging nanoprobes,52,53 the MnO2 nanosheets exhibit some improvement on cell viability. These results indicate that MnO2 nanosheets have low toxicity and desirable biocompatibility for probe transporting. To ensure high cell viability, 20 μg mL-1 of the MnO2 nanosheets was used for subsequent experiments.

Figure 5. HeLa cells incubated with H1-FAM/H2-TMR/MnO2 nanosheets for different time scales from 1 h to 4 h. Scale bar = 20 μm. The

time-dependent

fluorescence

imaging

experiments

were

performed

by

incubating

H1-FAM/H2-TMR/MnO2 with HeLa cells for different time scales to monitor FRET. As shown in Figure 5, no apparent FRET can be observed with the incubation of H1-FAM/H2-TMR/MnO2 with HeLa cells for 1 h. This is probably due to the relatively short HCR reaction time to develop the FRET



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signal. Further extension of the incubation time to 4 h results in the delivery of more H1-FAM and H2-TMR into the cells for miRNA-21 triggered HCR, and increasing FRET intensity can be obtained.

Figure 6. Confocal fluorescence images of miRNA-21 in HeLa cells incubated with different probes. (a) Normal HeLa cells without probes; (b) H3 (FAM and Dabcyl labeled molecular beacon)/MnO2 nanosheets;

(c)

H1-FAM/H2-TMR/MnO2

nanosheets;

(d)

H2-TMR/MnO2

nanosheets;

(e)

H1-FAM/H2-TMR. All samples were incubated at 37 ºC for 4 h. Scale bar = 50 μm. To validate our amplified approach for the detection of intracellular miRNA-21, HeLa cells known to express lower levels of miRNA-2154 were used and incubated with different probes. HeLa cells were first incubated with a conventional fluorescently quenched hairpin probe (H3, with similar sequence to H1 but labeled with the FAM and Dabcyl pair) adsorbed on the MnO2 nanosheets. As displayed in Figure 6b, almost no green emission from FAM is visible due to the low-expression levels of miRNA-21 and the traditional 1:1 stoichiometric ratio of the molecular beacon to the target miRNA-21. However, when HeLa cells were treated with H1-FAM/H2-TMR/MnO2 nanosheets, both clear green and red emissions could be observed (Figure 6c). Such observation can be ascribed to the following reasons. Upon entering the cells, the intracellular GSH degrades the MnO2 nanosheets and the proteins or nucleic acids displaces the adsorbed hairpins to release H1-FAM and H2-TMR, and the target miRNA-21 triggers the assembly of the liberated H1-FAM and H2-TMR into dsDNA polymers and ACS Paragon Plus Environment 14

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brings FAM and TMR into close proximity to generate efficient FRET, leading to red fluorescence emission. While, the green emission can be owing to the un-assembled H1-FAM. The comparison between Figure 6b and 6c thus clearly indicates the in cell signal amplification capability of our approach by miRNA-21 triggered HCR between H1-FAM and H2-TMR for detecting down-regulated miRNAs in living cells. Two control experiments were also performed by incubating the cells with H2-TMR/MnO2 nanosheets (Figure 6d) and the mixture of H1-FAM and H2-TMR without using MnO2 nanosheets (Figure 6e). Accordingly, no fluorescence emissions can be observed due to either the lack of the FRET donor (FAM, Figure 6d) or insufficient delivery of H1-FAM and H2-TMR into the cells without using the MnO2 nanosheet carrier (Figure 6e).

Figure 7. Confocal fluorescence images of (a) MCF-7 cells, (b) and (c) HeLa cells treated without and with the miRNA-21 inhibitor sequences (500 nM), respectively. Cells were incubated with H1-FAM/H2-TMR/MnO2 nanosheets for 4 h. Scale bar = 20 μm. To investigate the capability of the developed approach for differentiating the miRNA-21 expression levels in various cancer cells, the human breast cancer (MCF-7) and HeLa cells were tested. It is well documented that the MCF-7 cells have a relatively high expression level of miRNA-21 while the HeLa cells feature with a relatively low endogenous content of miRNA-21 compared with MCF-7 cells.55 After incubating the cells with H1-FAM/H2-TMR/MnO2 nanosheets at 37 ºC for 4 h, the confocal ACS Paragon Plus Environment 15


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fluorescence images were recorded and displayed in Figure 7. From this figure, we can see significantly increased red fluorescence (due to FRET) intensity from the MCF-7 cells than that of the HeLa cells (Figure 7a vs. 7b), indicating more miRNA-21 content in the MCF-7 cells. Such observation is in well agreement with the fact that the miRNA-21 level is over-expressed in MCF-7 cells other than HeLa cells.56 Besides, further experiment was performed by the transfection of the miRNA-21 inhibitor sequences into HeLa cells prior to the incubation of the cells with the H1-FAM/H2-TMR/MnO2 nanosheets. The inhibitor, which could hybridize with miRNA-21, was methoxylated to prevent the digestion from nuclease present in the cells. As expected, no red fluorescence emission can be observed (Figure 7c) because the over amount of the inhibitor sequences hybridize with the miRNA-21 target and suppress subsequent HCR between H1-FAM and H2-TMR. The results shown here thus demonstrate the signal enhancement and low background of the method for monitoring intracellular miRNA-21. CONCLUSIONS In conclusion, we have demonstrated the detection of down-regulated and low content of miRNA-21 in living cells with MnO2 nanosheet-mediated HCR amplification of FRET signals. The MnO2 nanosheets can readily delivery the hairpin probes into the cells. The degradation of the MnO2 nanosheets by intracellular GSH and displacement reactions by proteins or nucleic acids lead to the release of the free hairpins for HCR, resulting in significantly amplified FRET signals for detecting trace miRNA-21 in living cells. With the advantages of low cytotoxicity, reduction of false positive signals and significant in cell signal amplification, the demonstrated method thus offers new opportunities for monitoring various trace amount of RNA species in living cells for accurate and early diagnosis of different caners and for therapeutic evaluation as well. ASSOCIATED CONTENT Supporting Information. Materials including amplified FRET in vitro detection of miRNA-21 and dynamic light scattering characterization of the MnO2 nanosheets. This information is available free of charge via the Internet at http://pubs.acs.org/. ACS Paragon Plus Environment 16

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AUTHOR INFORMATION Corresponding Author *Tel./Fax: +86-23-68252277; E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (Nos. 21275004 and 21675128). REFERENCES (1) He, L.; Hannon, G. J. MicroRNAs: Small RNAs with a Big Role in Gene Regulation. Nat. Rev. Genet. 2004, 5, 522–531. (2) Valencia-Sanchez, M. A.; Liu, J.; Hannon, G. J.; Parker, R. Control of Translation and mRNA Degradation by MiRNAs and SiRNAs. Genes Dev. 2006, 20, 515–524. (3) Dong, H.; Lei, J.; Ding, L.; Wen, Y.; Ju, H.; Zhang, X. MicroRNA: Function, Detection, and Bioanalysis. Chem. Rev. 2013, 113, 6207–6233. (4) Ambros, V. The Functions of Animal MicroRNAs. Nature 2004, 431, 350–355. (5) 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, 828–833. (6) Selbach, M.; Schwanhäusser, B.; Thierfelder, N.; Fang, Z.; Khanin, R.; Rajewsky, N. Widespread Changes in Protein Synthesis Induced by MicroRNAs. Nature 2008, 455, 58–63. (7) Bartel, D. P. MicroRNAs: Target Recognition and Regulatory Functions. Cell 2009, 136, 215–233. (8) Ruan, K.; Fang, X.; Ouyang, G. MicroRNAs: Novel Regulators in the Hallmarks of Human Cancer. Cancer Lett. 2009, 285, 116–126. (9) Tsitsiou, E.; Lindsay, M. A. MicroRNAs and the Immune Response. Curr. Opin. Pharmacol. 2009, 9, 514–520. (10) Chen, J. F.; Murchison, E. P.; Tang, R.; Callis, T. E.; Tatsuguchi, M.; Deng, Z.; Rojas, M.; Hammond, S. M.; Schneider, M. D.; Selzman, C. H.; Meissner, G.; Patterson, C.; Hannon, G. J.; Wang, D. Z. Targeted Deletion of Dicer in the Heart Leads to Dilated Cardiomyopathy and Heart Failure. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2111–2116. (11) Yin, J. Q.; Zhao, R. C.; Morris, K. V. Profiling MicroRNA Expression with Microarrays. Trends in Biotechnol. 2008, 26, 70–76. (12) Iorio, M. V.; Ferracin, M.; Liu, C. G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; Menard, S.; Palazzo, J. P.; Rosenberg, A.; Musiana, P.; Volinia, S.; ACS Paragon Plus Environment 17


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