In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe

Analytica Chimica Acta 1079 (2019) 146e152

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe Kai Zhang a, b, Xue-Jiao Yang a, Ting-Ting Zhang a, Xiang-Ling Li a, **, Hong-Yuan Chen a, Jing-Juan Xu a, * a

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China

b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


A versatile probe for imaging and regulating of genetic molecules is proposed.
The well-designed probes showed high accuracy and specificity for premiRNAs in complex physiological microenvironment.
Highly effective inhibition of the Dicer-mediated cleavage process was achieved.
The LNA-MB probes owned potential for interfering disease-associated RNAs.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 April 2019 Received in revised form 6 June 2019 Accepted 8 June 2019 Available online 10 June 2019

A novel versatile locked nucleic acid modified molecular beacon probe (LNA-MB) was developed for imaging intracellular precursor miRNAs (pre-miRNAs) and disturbing Dicer-mediated cleavage process. The target recognition reaction between the smart probe and pre-miRNA can not only induce the conformational changes of probe and block the Dicer cleavage site, but also inhibit the cleavage process, and then achieve down-regulation of miRNA expression. Simultaneously, the target recognition reaction broke the fluorescence resonance energy transfer (FRET) between fluorophore donor FAM and acceptor TAMRA, which were labelled on the LNA-MB probe, further induced the relevant change of fluorescence signal, and then achieved imaging analysis of pre-miRNA and inhibition events in situ. Both in vitro and in single living cell studies showed that the versatile probes exhibited a remarkable performance in targeting with pre-miRNA-21, and nearly 65% downregulation of mature miRNA-21 was achieved with 100 nM probes. All investigations demonstrate that the proposed strategy represents a promising alternative for regulating and inhibiting endogenous disease-associated RNAs, then further for achieving therapeutic outcomes in personalized treatments. © 2019 Elsevier B.V. All rights reserved.

Keywords: microRNAs Locked nucleic acid Dicer-mediated cleavage process Regulation Imaging analysis

1. Introduction * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X.-L. Li), [email protected] (J.-J. Xu). https://doi.org/10.1016/j.aca.2019.06.016 0003-2670/© 2019 Elsevier B.V. All rights reserved.

MicroRNAs (miRNAs), short single-stranded noncoding RNAs of ~22-nucleotides, are critical regulators of many biological process,

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including regulation of gene expression, cell proliferation, differentiation and survival, and maintaining homeostasis of the cell [1,2]. Researchers have revealed that the aberrant expression of miRNAs has been linked to various physiological abnormalities and pathological conditions, and often results in various diseases in humans [3e5]. Due to the great importance of biological miRNAs, quite a few relevant works have been established for analysis of miRNAs [6e10] and thorough investigation of miRNAs functional mechanism [5,11e14]. During the miRNAs biogenesis, the maturation of miRNAs should undergo a highly coordinated series of enzyme cleavage activities. The primary miRNAs (pri-miRNAs), formed via the RNA polymerases II in the cell nucleus, is cleaved by RNase III enzyme complex to produce the intermediate premature shorter stem-loop ~80-nucleotide RNA hairpin, which is termed as precursor miRNAs (pre-miRNAs) [15,16]. The pre-miRNA is subsequently exported from the nucleus to the cytoplasm and processed into 21e24 base pair miRNA duplexes in Dicer-mediated cleavage process, which have been revealed as the core step in the whole miRNAs biogenesis [17]. Relevant works have demonstrated that in the field of RNA related disease, specific down-regulation of miRNAs expression is one of the focuses in genetic therapies. Interfering the Dicermediated miRNAs maturation process by various small molecules [18e20] or nanoparticle based inhibitors [21], is a smart approach to regulate gene expression levels. Nevertheless, to inhibit the Dicer process via RNA mimics binding with target pre-miRNAs is rare. Therefore, the development of novel and versatile probes for effectively regulating miRNAs levels via blocking Dicer cleavage is expected to be of great clinical significance. Recently, locked nucleic acid (LNA) has attracted considerable attention due to its unique structure. The 20 -O atom and 40 -C atom for LAN are connected by a methylene bridge, which locks the sugar pucker to the 30 -C endo conformation (Fig. S1) [22,23]. The constraint on the sugar moiety results in higher affinity and higher melting temperature (Tm) value for the nucleic acid duplex. Intrinsic limitations of the low Tm in molecular beacon affinity for target miRNA can be overcome by the incorporation of LNA backbone into molecular beacon design [24]. Therefore, these unique structural features make LNA an ideal tool for constructing nucleic acid probe in complex physiological microenvironment. Herein, we designed a novel locked nucleic acid modified molecular beacon (termed as LNA-MB) probe for imaging and regulating genetic biomolecules, which overcomes the inherent shortcomings of oligonucleotide formed probes, such as nonspecificity and instability. The Dicer-mediated process of miRNA21 mature was employed in our strategy. MiRNA-21, one of the most potent miRNAs that block apoptosis in human cancer, is upregulated in various cancers and has oncogenic activity [25e27]. It is anticipated that our well-designed MB probe can specifically hybridize to the target pre-miRNA-21, then block the pre-miRNA cleavage site of Dicer due to the recognition reaction of LNA-MB probe and pre-miRNA-21, thus inhibit conversion of pre-miRNA21 into mature miRNA-21 and lower the expression level of miRNA-21, therefore induce apoptosis of cancer cells. During the inhibiting process, the LNA-MB probe used fluorescence resonance energy transfer (FRET) fluorophore pairs for signal output because of the advantages of FRET technology: a) excellent selectivity by effectively eliminating homogeneous solution fluctuation, b) more accurate detection since the simultaneous change of the donor fluorescence signal and acceptor fluorescence signal. Based on the excellent properties of the LNA-MB probe, our probe can achieve simultaneously imaging of pre-miRNAs in situ, inhibiting the Dicermediated miRNAs maturation and regulating the expression level of miRNAs.

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2. Experimental section 2.1. Materials and apparatus Dicer was purchased from Diazyme Laboratories (La Jolla, CA, USA). Nuclease inhibitor RNasin, and diethypyrocarbonate (DEPC) were ordered from Promega. Other chemicals were all analytical grade. All solutions were prepared with Milli-Q (Branstead) purified double distilled water having specific resistance of >18 MU cm. All oligonucleotide samples were prepared with phosphate buffer (20 mM phosphate, 10 mM magnesium acetate, 1 unit mL1 RNasin, 0.1% DEPC, pH 7.0). The tips and tubes are RNase-free and do not require pretreatment to inactivate RNases. Oligonucleotides were purchased from Genscript Biotechnology Co., Ltd (Nanjing, China) and listed in Table S1. The fluorescence spectra were obtained on a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). The RT-qPCR was performed on ABI 7500 Real-Time PCR instrument (Applied Biosystems, USA). The cell images were obtained on a TCS SP5 laser scanning confocal microscope (Leica, Germany).

2.2. Pre-miRNA-21 detection Different concentrations of pre-miRNA-21 or control pre-miRNA were added into the reaction solution containing LNA-MB probes (500 nM), and the reaction mixture was incubated at room temperature for 30 min. Then, the fluorescence emission spectra of the obtained solution were directly recorded.

2.3. Polyacrylamide hydrogel electrophoresis Nondenaturing polyacrylamide gel electrophoresis (PAGE) (15%) was carried out at a 100 V constant voltage for 1 h in 1  TBE buffer by using Bio-Rad electrophoresis analyser. The gel was taken photograph under UV light on the Bio-Rad ChemDoc XRS (USA) after staining with GelRed for 10 min. The concentration of each strand is 1 mM.

2.4. Cell culture and imaging analysis Human cervical cancer cells (HeLa cells) were cultured with cell culture medium containing DMEM mixed with 10% fetal calf serum (FCS, Invitrogen), penicillin (100 mg mL1) and streptomycin (100 mg mL1). And cells were propagated at 37  C with 5% CO2 atmosphere in a standard cell incubator. Pre-miRNA-21 plasmid (PCMV-miR21, Addgene plasmid # 20381) transfected into HeLa cells were performed by using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA). Prior to transfection, the cells were seeded in a 6-well plate (for RT-qPCR and Western blotting analysis) or confocal dish (for confocal fluorescence assay). Firstly, pre-miRNA-21 plasmid and Lipofectamine 3000 Reagent were diluted by Opti-MEM Medium reduced serum media according to the manufacturer's instructions. Then diluted pre-miRNA-21 plasmid was added into to the diluted Lipofectamine 3000, and the mixture was incubated at 37  C for 15 min. After the incubation, the mixture was added to the cells, and then the cells were cultured at 37  C for 20 h. After the transfection of pre-miRNA-21 plasmid, the LNA-MB probes were transfected into pre-miRNA-21 plasmid treated HeLa cells with similar operations. Hence, the pre-treated HeLa cells were incubated for another 2 h for the confocal fluorescence assay, 4 h for RNA extraction or 48 h for Western blot analysis, respectively.

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2.5. Quantitative reverse transcription PCR (RT-qPCR) assay Total RNA was extracted from HeLa cells using Trizol reagent (Invitrogen, Beijing, China) following the manufacturer's instructions. The integrity of total RNA was assessed by gel electrophoresis. RT-qPCR analysis of miRNA was performed with UltraSYBR One Step RT-qPCR Kit (CWbio. CO. Ltd, Beijing, China) on an ABI 7500 Real-Time PCR instrument. GAPDH was used as internal control. The primer sequences used were as follows: GAPDH: 50 - ACCACAGTCCATGCCATC-30 and 50 -TCCACCACCCTGTTGCTGTA3’. The poly (A) tails was added to the miRNAs by E. coli Poly (A) polymerase (Fermentas, USA). The cDNA samples were then prepared using Hifi-MMLV first Strand cDNA Synthesis Kit (CWbio. CO. Ltd, Beijing, China). The amount of miRNA was determined via comparative threshold (Ct) method (2DDt method) after normalizing target miRNA's Ct values to those for GAPDH. 2.6. Western blotting analysis PDCD4 protein expression was measured by Western blotting analysis. Briefly, total proteins were isolated using RIPA Lysis buffer containing protease inhibitors cocktail, separated in 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. Each membrane was incubated for 1 h in blocking solution containing 5% bovine serum albumin (BSA) in PBS-Tween solution at

room temperature. The membranes were incubated with primary antibody and secondary antibody conjugated with horseradish peroxidase and then visualized by chemiluminescent (ECL) substrate. GAPDH was used as internal control. 3. Results and discussion 3.1. Optimization and feasibility of LNA-MB probes The inhibition of Dicer-mediated miRNA maturation by the LNAMB probe is schematically depicted in Scheme 1. The MB probe employed in our strategy contains three functional regions: FRET region with FAM as a donor and TAMRA as the acceptor, stem region, and hairpin loop region which formed with ribonucleic acid instead of deoxyribonucleotide to avoid the degradation of DNA sequences in cell cytoplasm. In the hybridization assay format, emission intensity was monitored by exciting at 470 nm, where FAM was powerfully excited while TAMRA was scarcely excited. The loop of LNA-MB probes was a perfect complement of the target molecule pre-miRNA-21. In the absence of target, the probes are in the natural conformation with intermolecular FRET, and own low fluorescence signal ratio R, which is attributed to the close proximity of FRET donor and accepter. In the presence of pre-miRNA-21, the probes hybridized with the loop of the pre-miRNA-21, which is the cleavage site of Dicer [28,29], and then induced the change of

Scheme 1. (A) The structure of LNA-MB probe. Yellow bases indicate LNA, and other bases are RNA. (B) The crystal structure of the Dicer/pre-miRNA complex (PDB number: 6BU9). (C) Schematic illustration of the LNA-MB probe to regulate miRNA expression in living cells by inhibiting the Dicer-mediated cleavage process.

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stem-loop structure of probes (Fig. S2). Hence, the MB probes opened up and the FRET pairs separated from each other, leading to an increased fluorescence signal ratio R. In theory, the probe elicits a signal only upon direct hybridization to the complementary premiRNA loop sequence. Therefore, pre-miRNA assessment can be performed in one step without the need for qPCR or amplification steps, and excess probe does not have to be removed prior to measurement. The stem length of LNA-MB probes was optimized to achieve the most significant signal change during the analysis process. Optimization for the preparation of LNA-MB probes (MB: MB2, MB3, MB4, MB5, MB6, MB7 and MB8, Table S1) was based on measuring the change of fluorescent signal ratio R (FFAM/FTAMRA) before and after adding the target molecule pre-miRNA-21. Before adding the target molecule, the FRET-MB probe should own high efficient fluorescence resonance energy transfer and low signal ratio R. As shown in Fig. 1A, the fluorescence signal ratio gradually decreased with the prolongation of the stem spacer length (red column), and reached a platform when the length was 7 base pairs (MB7). This phenomenon may be attributed to the stability differences of hybridized stem part among the series of MB probes. The MB2, MB3 and MB4 with less complementary base pairs may not form the hairpin structure, which leaded the MB existing single-stranded form, then the two fluorescence dyes far away from each other, and owned the high fluorescence signal ratio R. Afterwards, the target molecule pre-miRNA-21 was added to these MB probes solution, respectively. During the hybridization, the MB7/pre-miRNA-

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21 duplex showed the most significant signal change. As the hybridization between MB7 and pre-miRNA-21 formed rigid double strands, then the FRET in MB probes between FAM and TAMRA was broken, and the system displayed high signal ratio R (green column and black point). Based on these results, it was found that MB7 was the optimal probe to achieve analysis of target molecule in this study. Moreover, the differences in the detection performance between the LNA-MB probes and traditional MB probes (no LNA modified probes, MB7’ Table S1) were also studied. The results demonstrated that while detection of the pre-miRNA, the fluorescence signal ratio change in the group of LNA-MB probes was much higher than that of traditional MB group (Fig. S3). The phenomenon was attributed to that the LNA-MB probes owned stronger affinity to target molecules and can formed much steadier MB/pre-miRNA duplex than that of the traditional MB probes [30,31]. To understand the time-dependent signal change of the fluorescence probe during analysis, kinetic studies have been performed to optimize the reaction time for achieving the best signal responsive performance. As can be seen in Fig. S4A, the fluorescence signal ratio R rapidly changed in the first 10 min, and then reached a maximum in 30 min. When the reaction time was longer than 30 min, the fluorescence signal barely changed. Therefore, we chose the 30 min reaction time for further investigation. In addition, a higher concentration of pre-miRNA-21 produced a higher signal ratio value, which formed the basis for quantitative analysis of pre-miRNA. Based on the optimal assay conditions, the well-designed probe

Fig. 1. (A) Effect of the length of stem base pairs on the fluorescence signal ratio R of FAM to TAMRA (FFAM/FTAMRA) for LNA-MB only (red column) and LNA-MB treated with 300 nM pre-miRNA-21 (green column). The difference value of green columns and red columns are marked by black points. (B) Fluorescence intensity change in green (522 nm) and red (572 nm) emission wavelengths upon addition of different target molecule pre-miRNA-21 concentrations to LNA-MB probes (a to g: 0 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM). Excitation wavelength was 470 nm. (C) Specificity test, the fluorescence signal ratio of reaction solution containing LNA-MB probes and the pre-miRNA-21 or other pre-miRNAs. Concentrations of all the pre-miRNAs are 500 nM. (D) Box plots reflecting the distribution (n ¼ 10) of added pre-miRNA-21 and calculated concentration (a to e: 50 nM, 100 nM, 200 nM, 300 nM and 500 nM). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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was employed to analyze pre-miRNA in solution. In the absence of target molecules, FRET pairs in the LNA-MB probes was intact, they displayed dual emission peaks at 522 nm (FAM) and 572 nm (TAMRA) with excitation at 470 nm (Fig. 1B). Then to evaluate the analytical performance of the developed probes for pre-miRNA detection, different concentrations of pre-miRNA from one stock were added to the probes solution. With the increasing concentration of pre-miRNA, the fluorescence intensity of FAM at 522 nm gradually increased and the fluorescence intensity of TAMRA at 572 nm gradually decreased, which was attributed to that the target recognition reaction induced the formational alteration of the MB probes and then the separation of the FRET pair. Moreover, the LNA-MB probe owned the ability to react with pre-miRNA in a dose-response manner, which can be utilized for quantitative detection of the analyte. The fluorescence signal ratio exhibited a linear correlation to the concentration of pre-miRNA from 50 to 500 nM, and with a linear regression equation Y ¼ 0.00388X (nM) þ0.273, where Y is the signal intensity ratio and X is the concentration of pre-miRNA (R2 ¼ 0.9936). The detection limit of this assay, three times the standard deviation of the signal from control test, was calculated as 48.2 nM (Fig. S4B). To determine the specificity of molecular beacons for targeting to pre-miRNA-21, analogs (pre-miRNA-122, pre-miRNA-126, and pre-miRNA-141) were examined under the same conditions. As depicted in Fig. 1C, only pre-miRNA-21 induced a significant fluorescence signal change, conversely there was negligible fluorescent change in the presence of other analogs. The results demonstrated the excellent selectivity of this probe applied in pre-miRNA-21 detection. The stability of the designed LNA-MB probes, as a significant challenge for practical analyses in a complex physiological system, was evaluated in the following experiment. The box data presented that the LNA base formed probe exhibits high stability in real-samples (Fig. 1D). Moreover, the recoveries of pre-miRNA-21 in spiked serum samples were high up to 98.71e103.52%, revealing the proposed method owned excellent accuracy and significant potential in the application of intracellular analysis (Table 1).

3.2. Inhibitory property of the designed LNA-MB probes for miRNA21 Based on these motivating results of the LNA-MB probes, we further investigated the inhibitory effect of the probes on Dicermediated pre-miRNA-21 cleavage process. Specifically, the synthetic pre-miRNA-21 was incubated with Dicer and different amount of LNA-MB probes (0 mM, 1 mM). Then the miRNA maturation level was determined utilizing a 15% denaturing polyacrylamide gel. As shown in Fig. 2, while pre-miRNA-21 was just incubated with Dicer, high content of mature miRNA-21 obtained by cleaving pre-miRNA-21 via Dicer (lane 2). While incubated premiRNA-21 with the versatile LNA-MB probes and Dicer, the content of matured miRNA obviously decreased (lane 4), indicating that LNA-MB probes bind to pre-miRNA-21 and thereby hinder Dicer mediated miRNA maturation processing. All these results suggested

Fig. 2. PAGE data. Lane 1, pre-miRNA-21; Lane 2, pre-miRNA-21 treated with Dicer; Lane 3, pre-miRNA-21/LNA-MB duplex; Lane 4, pre-miRNA-21 treated with 1 mM LNAMB and Dicer.

that the LNA-MB probes can selectively target to pre-miRNA, induce structural changes, achieve signal switch and further interfere the cleavage process of the pre-miRNA. 3.3. Regulation of miRNA-21in living cells We further tested both the feasibility of imaging pre-miRNA-21 and inhibitory effect of Dicer-mediated process with the LNA-MB probes in living cells. First, HeLa cells were seeded in a 20 mm confocal dish for 24 h, then incubated with pre-miRNA-21 plasmid (Fig. S5) for the expression of pre-miRNA-21. Subsequently LNA-MB probes were added into cell cultured solution before confocal observation. Then the stability of the probes in complex physiological microenvironment was investigated before the imaging assay. The outcomes showed that the fluorescence signal of LNAMB probes was steady and no remarkable signal change was observed even after incubating the probes in salts or protein-riched cell lysate solution for 6 h (Fig. S6), revealing that LNA modified molecular beacon probes owned excellent stability and without degradation in physiological microenvironment. During the imaging observation, the fluorescence intensity of FAM and that of TAMRA were measured at different time points. As shown in Fig. 3, at time t ¼ 0 min, intense red fluorescence signal of TAMRA were visible in the cytoplasm region, indicating most FRET pairs were still adjacent and own high efficient energy transfer. With increasing incubation time, a significant increase in the green fluorescence intensity and a corresponding decrease in the red fluorescence intensity were observed in these target cells. After 3.5 h, extremely bright green fluorescence emission with negligible red fluorescence emission was observed. This was consistent with the results of detection in vitro (Fig. 1B), as target-responsive reaction induced the formational alteration of the MB probes and lead to the dissociation of FRET pair, thereby inducing the consistent fluorescent signal change. All results demonstrated that the smart

Table 1 Results of the recovery test of pre-miRNA-21 in 10-fold diluted human serum. pre-miRNA-21 sample

added (nM)

found (nM)

Recovery ± SD (%) (n ¼ 3)

1 2 3 4 5

50 100 200 300 500

51.57 103.52 198.46 296.14 505.68

103.14 ± 3.26 103.52 ± 2.78 99.23 ± 3.96 98.71 ± 3.28 101.14 ± 2.98

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Fig. 3. Time course of confocal images of HeLa cells incubated with LNA-MB probe. The merged image represents overlay of the FAM channel and TAMRA channel. Scale bar 25 mm.

LNA-MB probes integrated target molecule recognition and FRET signal change functions in one, which can be used for noninvasive and monitoring of intracellular pre-miRNAs. Motivated by the results of the LNA-MB probes affecting miRNAs maturation via hindering Dicer process in vitro, the feasibility of the probes served as miRNA regulator was further verified in living cells. Attractively, recent reports have demonstrated that the expression level of miRNAs was closely related to the downstream proteins. Programmed cell death 4 (PDCD4), as a tumor suppressor protein [32], is one of the main targets of miRNA-21 [33]. In particular, decrease in the levels of miRNA-21, would correspond to higher expression of PDCD4 and vice versa. Then the quantitative reverse transcription PCR (RT-qPCR) analysis and western blotting analysis were performed to ascertain the extent of down-regulation of miRNA-21 and up-regulation of PDCD4, respectively. Specifically, HeLa cells were pre-treated with pre-miRNA-21 plasmid and different concentrations of LNA-MB probes. Then cells were washed with PBS and cultured in fresh growth medium.

Subsequently, cellular RNA or protein was isolated, and RT-qPCR or western blotting analysis was performed to investigate the regulation extent of the LNA-MB probes. Firstly, RT-qPCR analysis exhibited a corresponding decrease trend of the expression level of mature miRNA-21 with the increased concentrations of the probes (Fig. 4A). While compared to untreated control set, nearly 65% downregulation of mature miRNA-21 was achieved in cancer cells at 100 nM of the LNA-MB probes. Further calculation revealed the downregulation of miRNA-21 level induced by the LNA-MB probes exhibited a clear concordant effect with the probe concentrations, and with an IC50 value of 63.62 nM in this assay (Fig. 4B). As expected, the endogenous level of PDCD4 protein of the test group treated with the potential inhibitor LNA-MB probe was high up to 600%, compared to the untreated control group (Fig. 4C). Collectively, these outcomes illustrated that the LNA-MB probe targeting to the pre-miRNA-21 owned excellent inhibitory potential inside the living cells. Meanwhile the target-recognition reaction between the probe and pre-miRNA can successfully prevent Dicer progress,

Fig. 4. (A) RT-qPCR analysis of mature miRNA-21 expression levels in the HeLa cells treated with different concentration LNA-MB. (B) Relative miRNA-21 levels vs. amount of LNAMB. (C) Representative image of Western blotting analysis of PDCD4 levels in HeLa cells treated with different amount of LNA-MB.

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lower the level of miRNA maturation and further regulate the downstream endogenous protein, which can be potential nucleic acid-based therapeutics for beating most malignant tumor. 4. Conclusion In summary, we have presented a smart and versatile LNA-MB probe based on the integration of imaging genetic molecules and disturbing Dicer-mediated cleavage process all in one. The hybridization assay format between the smart probes and premiRNAs can not only achieve imaging of biomolecules in situ, but also inhibit the maturation of miRNAs in vitro and in living cells, hinting that the attractive probes can potentially serve as a novel class of miRNAs regulator. It is the hope that in near future, more improved similar probes on the basis of these functions will be developed as promising tools in the research of disease-associated RNAs, clinical approaches and even personalized medicine. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Conflicts of interest The authors declare no competing financial interest. Acknowledgement We gratefully acknowledge the support from the National Natural Science Foundation of China (Grants 21327902, 21605072, 21705061) of China. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.06.016. References [1] D. Baek, J. Villen, C. Shin, F.D. Camargo, S.P. Gygi, D.P. Bartel, The impact of microRNAs on protein output, Nature 455 (2008) 64e71. [2] W. Filipowicz, S.N. Bhattacharyya, N. Sonenberg, Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9 (2008) 102e114. [3] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215e233. [4] A. Lujambio, S.W. Lowe, The microcosmos of cancer, Nature 482 (2012) 347e355. [5] J.T. Mendell, E.N. Olson, MicroRNAs in stress signaling and human disease, Cell 148 (2012) 1172e1187. [6] L. Wang, R. Deng, J. Li, Target-fueled DNA walker for highly selective miRNA detection, Chem. Sci. 6 (2015) 6777e6782. [7] S. Bi, Y. Cui, L. Li, Dumbbell probe-mediated cascade isothermal amplification: a novel strategy for label-free detection of microRNAs and its application to real sample assay, Anal. Chim. Acta 760 (2013) 69e74.

[8] Y. Han, F. Zhang, H. Gong, C. Cai, Multifunctional G-quadruplex-based fluorescence probe coupled with DNA-templated AgNCs for simultaneous detection of multiple DNAs and MicroRNAs, Anal. Chim. Acta 1053 (2019) 105e113. [9] L. Liu, Q.M. Rong, G.L. Ke, M. Zhang, J. Li, Y.Q. Li, Y.C. Liu, M. Chen, X.B. Zhang, Efficient and reliable microRNA imaging in living cells via a FRET-Based localized hairpin-DNA cascade amplifier, Anal. Chem. 91 (2019) 3675e3680. [10] M. Hong, H.X. Sun, L.D. Xu, Q.L. Yue, G.D. Shen, M.F. Li, B. Tang, C.Z. Li, In situ monitoring of cytoplasmic precursor and mature microRNA using gold nanoparticle and graphene oxide composite probes, Anal. Chim. Acta 1021 (2018) 129e139. [11] R. Garzon, G. Marcucci, C.M. Croce, Targeting microRNAs in cancer: rationale, strategies and challenges, Nat. Rev. Drug Discov. 9 (2010) 775e789. [12] C.C. Pritchard, H.H. Cheng, M. Tewari, MicroRNA profiling: approaches and considerations, Nat. Rev. Genet. 13 (2012) 358e369. [13] J. Elmen, M. Lindow, S. Schutz, M. Lawrence, A. Petri, S. Obad, M. Lindholm, M. Hedtjarn, H.F. Hansen, U. Berger, S. Gullans, P. Kearney, P. Sarnow, E.M. Straarup, S. Kauppinen, LNA-mediated microRNA silencing in nonhuman primates, Nature 452 (2008) 896e899. [14] M. Ha, V.N. Kim, Regulation of microRNA biogenesis, Nat. Rev. Mol. Cell Biol. 15 (2014) 509e524. [15] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature 411 (2001) 494e498. [16] S. Kadener, J. Rodriguez, K.C. Abruzzi, Y.L. Khodor, K. Sugino, M.T. Marr II, S. Nelson, M. Rosbash, Genome-wide identification of targets of the droshapasha/DGCR8 complex, RNA 15 (2009) 537e545. [17] N. Jafari, H.P. Dogaheh, S. Bohlooli, G.G. Oyong, Z. Shirzad, F. Alibeiki, S.H. Asl, S.J. Zargar, Expression levels of microRNA machinery components Drosha, Dicer and DGCR8 in human (AGS, HepG2, and KEYSE-30) cancer cell lines, Int. J. Clin. Exp. Med. 6 (2013) 269e274. [18] H. Yan, U. Bhattarai, Z.-F. Guo, F.-S. Liang, Regulating miRNA-21 biogenesis by bifunctional small molecules, J. Am. Chem. Soc. 139 (2017) 4987e4990. [19] C.M. Connelly, R.E. Boer, M.H. Moon, P. Gareiss, J.S. Schneekloth, Discovery of inhibitors of microRNA-21 processing using small molecule microarrays, ACS Chem. Biol. 12 (2017) 435e443. [20] D. Lim, W.G. Byun, J.Y. Koo, H. Park, S.B. Park, Discovery of a small-molecule inhibitor of protein-microRNA interaction using binding assay with a sitespecifically labeled Lin28, J. Am. Chem. Soc. 138 (2016) 13630e13638. [21] K. Zhang, X.-J. Yang, W. Zhao, M.-C. Xu, J.-J. Xu, H.-Y. Chen, Regulation and imaging of gene expression via an RNA interference antagonistic biomimetic probe, Chem. Sci. 8 (2017) 4973e4977. [22] I. Yildirim, E. Kierzek, R. Kierzek, G.C. Schatz, Interplay of LNA and 2 '-0-Methyl RNA in the structure and thermodynamics of RNA hybrid systems: a molecular dynamics study using the revised AMBER force field and comparison with experimental results, J. Phys. Chem. B 118 (2014) 14177e14187. [23] C. Zhou, J. Chattopadhyaya, Intramolecular free-radical cyclization reactions on pentose sugars for the synthesis of carba-LNA and carba-ENA and the application of their modified oligonucleotides as potential RNA targeted therapeutics, Chem. Rev. 112 (2012) 3808e3832. [24] X. Olson, S. Kotani, B. Yurke, E. Graugnard, W.L. Hughes, Kinetics of DNA strand displacement systems with locked nucleic acids, J. Phys. Chem. B 121 (2017) 2594e2602. [25] X. Pan, Z.-X. Wang, R. Wang, MicroRNA-21: a novel therapeutic target in human cancer, Cancer Biol. Ther. 10 (2010) 1224e1232. [26] Z.-X. Wang, H.-B. Bian, J.-R. Wang, Z.-X. Cheng, K.-M. Wang, W. De, Prognostic significance of serum miRNA-21 expression in human non-small cell lung cancer, J. Surg. Oncol. 104 (2011) 847e851. [27] R. Kumarswamy, I. Volkmann, T. Thum, Regulation and function of miRNA-21 in health and disease, RNA Biol. 8 (2011) 706e713. [28] S. Gu, L. Jin, Y. Zhang, Y. Huang, F. Zhang, P.N. Valdmanis, M.A. Kay, The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo, Cell 151 (2012) 900e911. [29] M.D. Shortridge, M.J. Walker, T. Pavelitz, Y. Chen, W. Yang, G. Varani, A macrocyclic peptide ligand binds the oncogenic microRNA-21 precursor and suppresses dicer processing, ACS Chem. Biol. 12 (2017) 1611e1620. [30] B. Vester, J. Wengel, LNA (Locked nucleic acid): high-affinity targeting of complementary RNA and DNA, Biochemistry 43 (2004) 13233e13241. [31] M.B. Baker, G. Bao, C.D. Searles, In vitro quantification of specific microRNA using molecular beacons, Nucleic Acids Res. 40 (2012) e13. [32] F.J. Sheedy, E. Palsson-McDermott, E.J. Hennessy, C. Martin, J.J. O'Leary, Q. Ruan, D.S. Johnson, Y. Chen, L.A.J. O'Neill, Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21, Nat. Immunol. 11 (2010) 141e147. [33] C. Suzuki, R.G. Garces, K.A. Edmonds, S. Hiller, S.G. Hyberts, A. Marintchev, G. Wagner, PDCD4 inhibits translation initiation by binding to elF4A using both its MA3 domains, P. Natl. Acad. Sci. USA. 105 (2008) 3274e3279.