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Oriented tetrahedron-mediated protection of catalytic DNA mo-lecularscale detector against in vivo degradation for intracellular miRNA detection Congcong Li, Chang Xue, Jue Wang, Mengxue Luo, Zhifa Shen, and Zai-Sheng Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00860 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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Analytical Chemistry

Oriented tetrahedron-mediated protection of catalytic DNA molecular-scale detector against in vivo degradation for intracellular miRNA detection Congcong Li,a Chang Xue,a Jue Wang,b Mengxue Luo,a Zhifa Shenb and Zai-Sheng Wua* aCancer

Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, National & Local Joint Biomedical Engineering Research Center on Photodynamic Technologies, Fujian Engineering Research Center for Drug and Diagnoses-Treat of Photodynamic Therapy, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350002, China bKey

Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China *Corresponding

Author: Email: [email protected] (Z.S. Wu)

ABSTRACT: We report a tetrahedron-based DNAzyme probe (Tetra-ES) for intracellular miRNA detection. Two DNA tetrahedrons (Tetra) were arranged at the different positions of the enzyme (E)/substrate (S) complex in a unique direction. A Na+-dependent DNAzme was designed to be initially locked to inhibit the activity of the DNAzyme. Fluorescence imaging and gel electrophoresis analyses demonstrated that the silenced DNAzyme could be specifically initiated by intracellular target miRNA. The activated DNAzyme repeatedly cleaved the substrates, allowing the controllable signal transduction and amplification effect. The combination of spatially controlled arrangement of DNA tetrahedrons with the stimuli-responsive behavior of the locked DNAzyme improved cell permeability and desirable nuclease resistance. Tetra-ES detector exhibited at least 10 times higher detection sensitivity (LOD of 16 pM) than that of the non-amplification molecular beacon counterpart and was capable of discriminating miRNA target from the corresponding family members. The expression level of target miRNA inside the cells of interest as well as different miRNAs inside the same type of cell lines were reliably screened utilizing the Tetra-ES detector. As an intracellular probe, Tetra-ES may provide valuable insight into developing a homogeneous DNA nanostructure-based controllable signal transduction strategy suitable for detection of miRNA and potential application to cancer diagnosis, prognosis and therapeutics.

INTRODUCTION MicroRNAs (miRNAs) are an emerging type of short (1824 nucleotides), endogenous, single-stranded non-protein coding RNA that play a significant role in regulating the expression of target genes in a post-transcriptional manner.1-4 Benign tumors, malignant tumors and corresponding normal tissues often have different miRNA expression profiles. Several key miRNAs could be used as the criterion for determining whether tissues have become cancerous. According to the literature,5-7 abnormal expression of miRNA-21 is often detected in various tumor specimens and cell lines, including breast cancer, prostate cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, colorectal cancer, glioma, cervical cancer and lung cancer. Hence, miRNA-21 is a recognized oncogenic miRNA.

The accurate identification and quantitative analysis of miRNAs are of great significance in the diagnosis and treatment of tumors, in understanding the mechanism of their biofunctions and in the development of related gene drugs. However, the small size, sequence homology, and low abundance of miRNAs pose a formidable challenge for their reliable detection. To screen the expression levels of miRNAs, some signaling methods have been developed. For example, northern blotting8,9 is the conventional method for miRNA detection on the basis of hybridization and blotting with complementary radioactive or fluorescent probes. However, this method is sample-consuming, labor-intensive, insensitive, and can introduce environmental contamination. Another method known as miRNA microarray analysis involves using a high-throughput analytic platform capable of detecting cancer-specific expression of hundreds of miRNAs in many samples.10-13 However, this method suffers from

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poor sensitivity, long hybridization time and the requirement for an extremely precise instrument which can restrict its wide application. Hence, efficient amplification strategies are often required to develop highly sensitive assay systems for miRNA detection. For example, the rolling circle amplification (RCA) technology was explored for the amplification detection of mRNAs due to its advantages of simple operation, high sensitivity, and high specificity.14 However, tradeoffs including the time-consuming amplification process, difficultly in synthesis of templates and expensive enzymes currently impede its further applications. Although quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered a gold standard with a high sensitivity and good universality for the detection of different miRNAs,15-18 its widespread use still remains a challenge due to the complex design of primers, need for expensive equipment and false positives for miRNA detection.19,20 Moreover, several problems such as surface modification, inhomogeneous signal transduction, harsh reaction conditions, the involvement of one or more enzymes and/or transfection reagents can make these conventional strategies unsuitable for the signaling of intracellular biomolecules especially inapplicable for in vivo applications. With the discovery of an increasing number of miRNAs in different organisms and the rapid progress in the understanding of their functions in many biological processes, there is an urgent need to develop convenient, biocompatible and reliable techniques for miRNA assessment in vivo. Direct imaging of biomolecules within living cells would be able to reveal the expression level of cancer-related miRNAs and can offer valuable information on cancer diagnosis, staging, progression, prognosis and response to drug treatment in real time,21,22 Unfortunately, the image contrast of these strategies (e.g., FISH, fluorescence in situ hybridization) is easily limited by a high background fluorescence originating from the nonspecifically bound so-called “always-on” probes. However, activatable DNA probes would provide new opportunities for the construction of competent platforms capable of sensing the expression level of low-abundance miRNA molecules inside cells.23 DNAzymes are a class of DNA-based biocatalysts that can catalyze a variety of biological and chemical transformations in the presence of specific metal ion cofactors. In this way, DNAzymes were used to develop DNAzymatic sensors for a variety of metal ions in aqueous solution via modification of these molecules with different functional agents such as fluorophores and thiol.24,25 Detection of the target species can cause the biosensor to increase its fluorescence intensity by more than 10-fold with picomolar to low nanomolar sensitivity and response time on the order of several min. Besides sensing in environmental samples, DNAzymes as a molecular tool also show considerable promise for the design of signaling probes for endogenous metal ions and biomolecules in living cells.24,26-29 While these advances are encouraging, some significant unaddressed issues hamper the further utility of DNAzymes for the

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intracellular imaging of biomolecules. These issues include poor cellular uptake of the DNAzyme probe due to the electrostatic repulsion between the probe and cell membrane, and the natural susceptibility of the probe to nuclease-mediated degradation in a physiological environment. To overcome these deficiencies, transfection agents, inorganic nanomaterials and/or chemical modification are often used to offer protective moieties as required. However, the modification can cause significant toxicity to cells and organisms, increase the assay cost and complicate the probe synthesis. It is thus necessary to develop activatable DNAzyme-based probes via assembling multifunctional DNA nanoarchitecture, where non-nucleic acid materials are completely circumvented. As such, besides good biocompatibility, these newly-developed probes can actively enter cells, achieve intracellular signal activation and show a self-protection against enzymatic degradation in cellular environments. Herein, a biostable Tetra-ES detector is developed for intracellular miRNA detection via the assembly of two DNA tetrahedrons (named Tetra) onto a lockedDNAzyme (called E)/hairpin-type substrate strand (abbreviated as S) complex (named ES) and orienting them at different directions to allow ES to acquire the adequate resistance against in vivo degradation. The tetrahedral DNA nanostructure has a great significance in the operation of Tetra-ES detector within living cell owing to its low toxicity, high resistance against degradation in biological media and the desirable capability to enter cells in the absence of transfection reagents.30-34 Specifically, E hybrid is designed on the basis of a Na+-dependent DNAzyme as Na+ abundantly exists inside living cells35 and the DNAzyme is silenced by a locking strand. S duplex are made up of two parts: 5'-fluorophore (FAM)labeled strand with a hairpin structure (Sub-f) and 3'quencher (Dabcyl)-attached partly complementary strand (Se-dab). Sub-f contains a single adenosine ribonucleotide (rA) in the loop segment as the scissile cleavage site, while Se-dab is capable of hybridizing with Sub-f, thus bringing FAM and Dabcyl in close proximity to each other. The ES complex are assembled to have two sticky ends with the same base sequence that are employed to arrange two DNA tetrahedrons at different positions because each tetrahedron has a complementary singlestranded overhang. In the absence of target miRNA, the FAM fluorescence of Sub-f is quenched by Dabcyl of Sedab due to fluorescence resonance energy transfer (FRET), and no obvious fluorescence change is detected even if Tetra-ES detector is incubated in a detrimental biological environment. This is because threedimensional DNA tetrahedrons endow the Tetra-ES assembly with excellent biostability. However, once the Tetra-ES detector is internalized into living cells of interest, the target miRNA can peel off the locking strand through a hybridization reaction and activate the DNAzyme, forming the active secondary structure in the catalytic cores. Therefore, the substrate strand is irreversibly cleaved at the scissile site, and the small


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cleaved FAM-labeled segment moves easily away from the residual Dabcyl-attached DNA nano-architecture. As a result, the fluorophore is completely separated from the quencher, leading to a sharp increase in the fluorescent intensity. Monitoring the fluorescence change of chemically-modified DNA molecules enables the realtime observation of intracellular interaction between Tetra-ES detectors and miRNAs, allowing us to precisely signal the expression level of target miRNAs within living cells. Characterization of the Tetra-ES detector shows desirable analytical capabilities such as a low detection limit, high detection specificity and rapid response rate.

EXPERIMENTAL SECTION Chemicals and Materials. The oligonucleotides were self-designed and provided by Sangon Biotech Co., Ltd. (Shanghai, China). Their base sequences are shown in Table S1. All stock solutions of oligonucleotides were prepared via dissolving in 1×TAMg buffer (40 mM Tris, 7.6 mM MgCl2.6H2O, pH 7.4). The low molecular weight ladders, DNAase I, PrimeScript RT reagent Kit with gDNA Eraser and TaKaRa Ex Taq HS in TB Green™Premix Ex Taq™ Kit were obtained from New England Biolabs (Beijing, China). The 17 β-Estradiol (E2) was obtained from Sigma-Aldrich (USA). Hoechst 33342, Gel loading dye (6 × ) and Trizol reagent were provided by Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). All aqueous solutions were prepared by ultrapure water (≥18 MΩ cm resistivity, Milli-Q, Millipore). All other chemical reagents were of analytical grade. Preparation of Tetra-ES detector. Tetrahedral DNA nanostructures were prepared according to the previous method with slight modification.36,37 Four designed strands (Sa to Sd, 1 µL, 10 µM each) were mixed at a molar ratio of 1:1:1:1 in 1×TAMg buffer (pH 7.4). The resulting solution was heated to 90 °C for 5 min and then slowly cooled to room temperature. Afterwards, the linker strand (1 µL, 10 µM) was incubated with tetrahedral DNA nanostructure at an equimolar ratio at 37 °C for 2 h, forming the tetrahedron-linker assembly. The final concentration of tetrahedron-linker assembly was 0.4 µM. For the preparation of locked-DNAzyme, the Locking-4 was mixed with DNAzyme at a molar ratio of 1:1, followed by diluting with 1×TAMg buffer (pH 7.4). The resulting mixture was heated at 90 °C for 2 min and then cooled to room temperature gradually, in which the DNAzyme/locking-4 duplex concentration was 0.4 µM. Subsequently, the locked-DNAzyme was incubated with an equal volume of the above-mentioned DNA tetrahedron-linker at 37 °C for 2 h to form 0.2 µM DNA tetrahedron-DNAzyme (Tetra-E). Substrate hybrid was the duplex of Sub-f and Se-dab at a molar ratio of 1:1 (0.4 µM). The mixture of Sub-f and Sedab was heated at 90 °C for 2 min and then cooled to

room temperature gradually. Afterwards, the substrate hybrid solution was incubated with an equal volume of the above-mentioned DNA tetrahedron at 37 °C for 2 h, forming 0.2 µM DNA tetrahedron-substrate complex (Tetra-S). Finally, Tetra-E was incubated with the Tetra-S at 37 °C for 2 h by a ratio of 1:1, obtaining the expected 0.1 µM Tetra-ES detector. Cell Culture. HeLa cells, MCF-7 cells, and L02 cells were obtained from Chinese Academy of Sciences (Shanghai). These cells were cultured in DMEM complete medium via incubating in a temperature-controlled incubator at 37 °C. Cells were plated on glass dishes and allowed to grow to 70-90% confluence before use for the confocal fluorescence imaging experiments. Confocal Fluorescence Imaging. All cell lines were plated on glass slides for 24 h. For the comparative evaluation of the capability between Tetra-ES and ES to exert the living cell imaging, HeLa cells freshly prepared were separately incubated with Tetra-ES and ES at the final concentration of 50 nM for 4 h. After washing by PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH=7.4), the cells were incubated with 1 μM Hoechst dye for 10 min and then washed by PBS again. Afterwards, the cells were fixed with 4% paraformaldehyde for 10 min. Finally, the cells were washed with PBS before florescence imaging. HeLa cells and L02 cells were employed for the evaluation of the expression level of miRNA-21 in different cells. The two types of cells were incubated with Cy5labeled Tetra-ES at the final concentration of 50 nM for 4 h and then washed with PBS. The nuclear staining and cell fixing were performed according to the same procedure as that described above before exerting the fluorescence imaging.

RESULTS AND DISCUSSION Assembly and Working Principle of Tetra-ES Detector. DNAzyme/substrate hybrid probes have a great potential for intracellular analysis and living cell imaging. Such applications include, for example, metal ion sensing26,27,29 and miRNA detection within cells.28 However, the susceptibility of DNA strands to enzymatic degradation in vivo, especially the design of an internal RNA base required at the cleavage site of substrate strand, makes the DNAzyme/substrate hybrid probe vulnerable to endogenous-nuclease activity. Moreover, the distribution of metal ion cofactors is uncontrollable and they essentially exist in both extra- and intracellular environments. As a result, nuclease-induced the degradation and metal-catalyzed cleavage occur to varying degrees before a DNAzyme/substrate probe can reach its cellular destination. This can cause an increase in background fluorescence and can completely change the functional conformation of the DNAzyme which inevitably compromises the sensing performance and defeats the purpose of target-mediated signal



transduction. Additionally, the difficulty in the internalization of negatively charged DNAs into living cells also severely hampers the application of DNAzyme/substrate probe in cellular analysis.38,39 To promote the cellular entry and protect DNA strands from enzymatic degradation, various materials such as cationic transfection agents, liposomes and modified viruses were often involved in the exploration of oligonucleotide-based drug delivery or therapeutic systems. However, these materials suffer from several drawbacks, such as cytotoxicity, an inability to be degraded in living organism and nonspecific immunogenic responses.40 To address these challenges, a locking strand was designed to silence the DNAzyme. This strand is able to be peeled off by hybridization with the intracellular miRNA of interest so that the DNAzyme could remain in a catalytically inactive state during the cell internalization until it is specifically activated. Two tetrahedral DNA nanostructures were arranged at the different positions of the DNAzyme/substrate hybrid to facilitate the cellular internalization of probes and offer enhanced resistance against degradation in intracellular milieu. Substantial internalization of DNA tetrahedra into living cells was observed even without the help of transfection reagents and the uptaken tetrahedral nanodevices maintained their structural integrity in cells for a long period of time.30,33,34 Moreover, the DNA Tetrahedron positioned at the end of oligonucleotide probes can efficiently protect probes from enzymatic degradation.30,32 The locked DNAzyme (E)/substrate (S) hybrid probe was called ES detector, the tetrahedral DNA nanostructure was abbreviated as Tetra, and the Tetra-protected DNAzyme/substrate nano-architecture was named TetraES detector. Scheme S1 illustrates the hybridizations among nucleic acid sequences responsible for the formation of ES detector and the Tetra/linker. For the construction of the Tetra-ES detector, we prepared separately the Tetra-E and Tetra-S in advance and then mixed them at equal molar ratio as illustrated in Scheme 1A. The stepwise assembly of Tetra-E and Tetra-S is schematically depicted in Scheme S2. Two sticky ends designed in the loop part and at the stem terminal of the ES detector (seen in Scheme S1A) are responsible for the oriented anchoring of two DNA tetrahedra onto the different positions, and two single-stranded spacers are separately inserted between tetrahedra and ES detector so that the DNA tetrahedra could swing and adequately protect the ES detector from nuclease degradation. Scheme 1B depicts the cell uptake of Tetra-ES detector and its intracellular operation. In order to monitor the fluorescence change of the Tetra-ES detector, we labeled the hairpin-type substrate strand and its partly complementary strand (Se) with carboxyfluorescein (FAM) and Dabcyl, respectively. For the intact Tetra-ES detector, the fluorescence of FAM is quenched by Dabcyl because they are brought by a DNA duplex structure into close proximity. With the aid of DNA tetrahedra, the Tetra-ES detector is easily internalized into cells and

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preserves its structural integrity until touching the target fuel. When the intracellular target miRNA peels off the locking strand via hybridization, the released DNAzyme strand folds into a catalytically active conformation and then binds to and cleaves the substrate strand with the help of an intracellular Na+ cofactor. Because of the decrease in the binding affinity, the cleaved fluorescentlymodified fragment moves away from the Dabcyl-attached fragment, resulting in the separation of the fluorophorequencher pair. Thus, the fluorescence increase is observed owing to the restoration of quenched FAM emission originating from the loss of the short distance-dependent FRET effect. Moreover, due to its higher binding affinity, the activated DNAzyme can compete for the next substrate strand with the inactive DNAzyme to initiate another cycle of hybridization, cleavage and turnover. This leads to the generation of an amplification signal, thus allowing for the sensitive analysis of intracellular miRNA of interest.

Scheme 1. Schematic illustration of oriented tetrahedronmediated catalytic DNA molecular-scale probe for intracellular miRNA detection. (A) The assembly of Tetra-ES detector. (B) Cellular internalization of Tetra-ES detector and intracellular miRNA detection mechanism.

Feasibility for Target MiRNA-21 Detection. The Tetra-ES detector assembly was confirmed by gel electrophoresis analysis, and the experimental results are shown in Figure S1. The feasibility for target detection is explored in the following test. Since miRNA-21 plays an essential role in the occurrence and development of common human cancers, this miRNA was often chosen as the target model.41 Taking the higher chemical stability of DNA over RNA into account, the DNA mimic of miRNA21 was instead used in the preliminary experiments, while miRNA-21 was used in the detection experiments and intracellular fluorescence imaging. We first performed nPAGE analysis to validate the target miRNA-based displacement reaction, and the results are shown in


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Figure 1A. Lanes 1, 2 and 3 depict the target band, locking strand band and DNAzyme band, respectively. Besides the new bands in Lane 4 or Lane 5, both target band and locking strand band disappear in Lane 4, and the locking strand band and DNAzyme band are not observed in Lane 5. This demonstrates the formation of a target/locking hybrid and DNAzyme/locking hybrid. In Lane 6, the brightness of DNAzyme/locking band decreases, and two bands, target/locking hybrid and DNAzyme are clearly detected, indicating that, in the presence of target sequence, the DNAzyme can be released from the silenced conformation via hybridizing locking strand with the target species.

Figure 1. Signaling capability of Tetra-ES detector at the molecular scale. (A) 12% native PAGE characterization of the assembly and initiation of locked DNAzyme, where Sybr green I was used to stain DNAs because they were not fluorescently labeled. Lane M: DNA ladder; Lane 1: DNA target; Lane 2: Locking-4; Lane 3: DNAzyme; Lane 4: Locking-4 + DNA target; lane 5: Locking-4 + DNAzyme; Lane 6: Locking-4 + DNAzyme + DNA target. (B) 6% native PAGE analysis of the cleavage activity of ES detector, where the fluorescence emission is from FAM covalently attached to DNA substrate stand without additional staining. Lane M: DNA ladder; Lane 1: Sub-f; Lane 2: Locking-4 + DNAzyme + Sub-f + Se-dab; Lane 3: Locking-4 + DNAzyme + Sub-f + Sedab + DNA target; Lane 4: Locking-4 + DNAzyme + Sub-f + Se-dab + Na+; Lane 5: Locking-4 + DNAzyme + Sub-f + Sedab + DNA target + Na+. (C) and (D) are the measured data from the real-time monitoring of the changes in fluorescence emission intensity of 50 nM ES detector and 50 nM Tetra-ES detector, respectively, upon addition of 5 nM target species. For the Blank, 1×TAMg buffer was used instead of target solution.

Further supporting data are shown in Figure 1B. The Sub-f band is clearly visible in Lane l. The difference in the band position between Lane 2 (ES detector) and Lane 3 (ES detector activated by target) demonstrates that the target species can peel off the locking strand via hybridization reaction, making the DNAzyme fold into its catalytically active conformation. In this case, once a metal ion cofactor is introduced, the DNAzyme cleaves the substrate stand, generating a short fluorescently

modified fragment. This can cause a substantial fluorescence increase due to the spatial separation of the fluorophore FAM and quencher Dabcyl. As a result, a very bright band with low MW is observed in Lane 5. In contrast, in the absence of the target strand, the Na+ cofactor cannot make the ES detector change its position (and fluorescence) as shown in Lane 4. These data demonstrate that the designed ES detector is initially silenced, but the target species can cause the ES detector to switch its structure from the inactive to active conformation. Figure 1C shows the fluorescence change of the ES detector induced by target species through real-time fluorescence measurements. The target sample can cause the fluorescence intensity of ES to continue to increase (first rapidly, then gradually and finally slowly) compared to the Blank. The fluorescence intensity almost reaches a plateau at 10 min. Similarly, the real-time fluorescence monitoring of Tetra-ES detector in the presence of target strand is shown in Figure 1D. The dramatic difference between target sample and Blank is observed, demonstrating that the assembly of the two DNA tetrahedra onto the loop and stem terminal of ES detector does not compromise its capability to signal target species, and thus the as-constructed Tetra-ES detector can be used to detect the target species. The cleavage activity was also explored under the given conditions. As shown in Figure S2, the kinetic curve can be fitted to the first-order reaction with a single-turnover cleavage rate constant (kobs) of 0.1 min−1. This cleavage rate coincides well with the literature value,26 indicating a substantially faster cleavage than other DNAzymes and naturally occurring ribozymes.26 To validate that the activated DNAzyme can be recycled, Tetra-ES was mixed with target miRNA at a molar ratio of 1:1 (the final concentration, 5 nM) and the fluorescence change was monitored in real time. After 30 min, an additional Tetra-ES (the final concentration, 10 nM) or an equal volume of TAMg buffer was added, followed immediately by the real-time fluorescence monitoring for another 30 min. The measured data, accompanied by more details on assay experiments, are shown in Figures S3A and S3B. The fluorescence intensity of Tetra-ES in a signal plateau is capable of continuing to increase by a factor of 1.9 upon introduction of additional Tetra-ES (Figure S3A), while introduction of additional TAMg buffer cannot induce the fluorescence increase (Figure S3B). These results demonstrate that the activated DNAzyme in the initial Tetra-ES can be recycled to initiate the operation of other locked Tetra-ES detectors. To further offer the evidence for the target-induced amplification signal, we monitored the fluorescence change of sensing systems at different concentrations of Tetra-ES detector upon addition of the same concentration of target. As shown in Figure S3C, when Tetra-ES concentration increases from 0.5 nM to 2 nM and finally to 10 nM, the florescence signal increases from 18 a.u. to 46 a.u., and 130 a.u., respectively. Thus, the 10 nM Tetra-ES system offers a



7-fold increase in fluorescence signal compared with 0.5 nM Tetra-ES even if the target concentration does not change. The corresponding background fluorescence is very low as shown in Figure S3D. Furthermore, the signal amplification efficiency would be further improved by increasing the concentration of Tetra-ES detector. For example, as shown in the left panel of Figure S5A where the Tetra-ES concentration is 50 nM, 0.5 nM target species can make the florescence increase to 240 a.u., indicating 13-fold signal amplification. Along this line, we further evaluate the signal amplification efficiency of 50 nM Tetra-ES upon the higher concentration of target species. The florescence intensity of 50 nM Tetra-ES detector induced by 5 nM DNA targets was 447 a.u. (Figure S5A), while the florescence intensity of 5 nM Tetra-ES detector in the presence of 5 nM DNA targets (at an equal molar ratio) was 65 a.u. (Figure S3A). These results demonstrate that 50 nM Tetra-ES detector offers a 7-fold amplification of fluorescence signal for 5 nM target species. Thus, the amplification efficiency of 50 nM TetraES detector for the low concentration of target species is higher than that for the high target concentration. In vitro detection of MiRNA-21. Following the feasibility of Tetra-ES detector for target miRNA detection and the optimization of detection conditions (Figure S4), its capability to quantify target species in vitro was explored before employing to screen the intracellular miRNAs. The fluorescence intensity of the Tetra-ES detector in the presence of the varied concentrations of target miRNA was detected, and the relationship between fluorescence change and target concentration is presented in Figure S5A. One can see that the fluorescence intensity increases with rising target concentration (ranging from 0 ~ 100 nM), and the target species can be quantified in the concentration range of 50 pM to 50 nM. The regression equation could be expressed as F = 161.4 LogC + 325.9, where F indicates the fluorescence intensity and C is the target concentration. The correlation coefficient of the calibration curve is 0.9914. At the higher concentration of target, for example, 100 nM, the response-point is beyond the quantitative concentration range. The longer reaction time cannot lead to a considerable increase in the fluorescence intensity as shown in Figure S6. On the basis of the Blank plus 3σ (3 time the standard deviation) method, the limit of detection (LOD) was found to be 16 pM, representing more than 10-fold improvement over the corresponding non-amplification molecular beacon (Figure S7). Such an assay capability is much higher than other impressive DNAzyme cleavage-based sensing methods for the detection of intracellular miRNA such as the DNAzymebased nanomachine42 and walker.43 Detection specificity of target miRNA. One of the important prerequisites for potential applications in genetic diagnosis is that the developed signaling probe must be able to discriminate the complementary target from mismatched nontargets with a desirable selectivity. In order to validate the detection specificity of Tetra-ES,

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we first designed several spurious non-target sequences containing mismatched bases as the controls and the corresponding signals were separately recorded. Compared with the sample in the absence of target species, there is the remarkable fluorescence increase for the target (Figure S5B). The fluorescence intensity induced by Mismatch-1 target is about 30% of that by fully matched target, indicating a single-base discrimination capability superior to that of the literature method.44 Moreover, no substantial fluorescence change is observed for each of the other four mismatched targets, indicating desirable sequence recognition capability. Besides the preliminary validation of Tetra-ES to identify the DNA mimic of the miRNA target from mutant DNA strands, it is necessary to investigate its detection specificity towards miRNA-21 because of the coexistence of different miRNAs and even different members with the high sequence similarity in miRNA family in target cells. In order to prove the sequence specificity towards miRNA-21, the fluorescence intensity of the Tetra-ES detector in the presence of related miRNA family members were explored under identical conditions. According to previous literature,28,45 miR-141, miR-429, miR-200b, let-7d and miR-21 belong to the same miRNA family, the miR-200 family. Thus, the four DNA counterparts of the non-target miRNAs were employed as the controls of miR-21. As shown in Figure S5C, compared with the strong fluorescence intensity of miR-21D (about 440 a.u.), the florescence intensities corresponding to miR-141D, miR-200bD, miR-429D, let-7dD and Blank are 43, 40, 30, 29 and 15 a.u., respectively. If the fluorescence signal induced by miR-21D is defined as 100%, each DNA mimic of nontarget miRNAs is not more than 10%, demonstrating that miR-21D can specifically induce Tetra-ES detector to emit the fluorescence signal. We also measured the fluorescence response of Tetra-ES upon miR-200 family members (not DNA mimics). As shown in Figure S5D, the results are in accordance with Figure S5C. The Tetra-ES detector can discriminate the miRNA-21 target from the other member of the miR-200 family.

Figure 2. Confocal fluorescence imaging of HeLa cells with Tetra-ES or ES detectors. Scale bar is 20 μm. The details on fluorescence imaging experiments are seen in the section of “Confocal Fluorescence Imaging” of “EXPERIMENTAL SECTION”. The quantitative fluorescence analysis is shown in Figure S11.

Additionally, the relationship between the fluorescence signal of Tetra-ES detector and the metal ion nature was investigated by monitoring the target-induced


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fluorescence change in the presence of different metal ions (data shown in Figure S8). It is well known that the metal ion cofactor is vital for the catalytic activity of DNAzyme probes, and thus the dependence of its signaling capability on metal ions indicates their distinct functional mechanisms to some extent.46,47 The experimental results represented in Figure S8 are consistent with literature reports,26,35 suggesting that the operational mechanism of adapted DNAzyme in the Tetra-ES detector is similar to that of the original Na+dependent DNAzyme and its intact properties are not changed by the introduction of miRNA recognition element and DNA tetrahedra. Intracellular fluorescence detection of MiRNA-21. It has been known that the weak cell penetration of nucleic acid always limits the intracellular application of DNA nanomaterials.48 However, in addition to being stable against the nonspecific enzymatic degradation which reduces false-positive signals, the DNA tetrahedron exhibits a self-delivery ability.30,38 Thus, we introduced DNA tetrahedral structures to eliminate the false-positive signal and enable the entry of ES detector into the cells easily. As shown in Figure S9, the degradation resistance of Tetra-ES detector has been effectively improved and its false-positive signal is negligible. In contrast, as shown in Figure S10, Lip-ES is very susceptible to enzymatic degradation and thus is not adopted although Lip-3000 is one of the most effective commercially available transfection agents. Since HeLa cells and MCF-7 cells include the high expression of miRNA-21 and L02 cells have an extremely low expression of miRNA-21,49,50 the fluorescence imaging of these cell lines was explored as target cells or control cells. At first, the difference in the capability to execute intracellular fluorescence imaging between ES and Tetra-ES was evaluated to prove the cell permeability of DNA tetrahedron. We incubated HeLa cells separately with Tetra-ES and ES for 4 h, followed by the fluorescence imaging experiments. As shown in Figure 2, the fluorescence intensity caused by Tetra-ES is much higher than that of ES. Because the fluorescence imaging analyses were conducted using the same cell lines under identical conditions, these experimental results demonstrate that DNA tetrahedra with selfdelivery ability promote the cell entry of the Tetra-ES detector.

Figure 3. The capability of Tetra-ES detector to execute confocal fluorescence imaging within living cells. (A) Fluorescence images of HeLa cells and L02 cells after incubation with Tetra-ES detector capable of recognizing miR-21 for 4 h. Scale bar is 20 μm. (B) Intracellular fluorescence detection of miRNA-21 within MCF-7 cells treated and untreated with estrogen (E2). Scale bar is 20 μm.

Next, we performed the miRNA fluorescence detection within HeLa cells and L02 cells. To monitor the cell entry of Tetra-ES, Sa-Cy5 was used instead of Sa for Tetra-ES preparation, besides employing the FAM fluorescence to observe the cleavage reaction of Sub-f by activated DNAzyme. The confocal fluorescence images of HeLa cells and L02 cells conducted under identical conditions are shown in Figure 3A. The fluorescence brightness of green FAM of HeLa cells is significantly higher than that of L02 cells, but there is no substantial difference in the red fluorescence intensity (Cy5). These experimental results illustrate that the Tetra-ES detector not only was able to enter HeLa cells but also was almost equally internalized into L02 cells. Moreover, the different expression levels of miRNA-21 in the two types of cells can be efficiently screened. The quantitative analysis of cleavage process induced by intracellular miRNA-21 using Image 2J software can be seen in Figure S12A. The expression levels of miRNA in the same cells may be at various stages in the initiation and progression of human cancer. Thus, to assess the ability of Tetra-ES to screen the intracellular miRNA at different expression levels, E2 was used to downregulate the miRNA-21 expression of MCF-7 cells according to the literature method.51 Then, E2-treated MCF-7 cells and untreated MCF-7 cells were separately incubated with Cy5-labeled Tetra-ES for 4 h under identical conditions, followed by performing the confocal fluorescence imaging. As shown in Figure 3B, the Cy5 fluorescence intensity for untreated MCF-7 cells is almost the same as the image of E2-treated MCF-7 cells, illustrating almost an equal amount of TetraES detectors entering the two groups of cells. However, the fluorescence intensity of FAM for untreated MCF-7 cells is higher than that detected for E2-treated MCF-7



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cells. The results indicate that more locked DNAzymes were activated in untreated MCF-7 cells and cleaved more FAM-labeled substrate strands, which is consistent with a fact that the expression of miRNA-21 triggers was inhibited in E2-treated MCF-7 cells. The quantitative analysis of the substrate cleavage in the two groups of cells is described in Figure S12B. These measured results from the confocal fluorescence imaging experiments were also confirmed by qRT-PCR assay (Figure S13, Table S2). Native MCF-7 cells do have a higher miR-21 expression level than E2-treated MCF-7 cells, which is in good accordance with previous reports.52,53 The universality of Tetra-ES detector is verified by extending it for the detection of microRNA-31 and microRNA-221 according to the same procedure. The details on the target assay are shown in Figure S14 and Figure S15.

nucleic acid-based nanostructures to play an essential role in molecular biological studies, early diagnosis and management of human cancers.

It is worth mentioning that, besides a high abundance Na+ in living cells, another reason for choosing Na+specific DNAzyme is that it exhibits a higher signaling capability than Mg2+-specific DNAzyme. The measured data are seen in Figure S16.

Notes

CONCLUSIONS In summary, we have succeeded in devising a tetrahedral nanostructure-based DNAzyme molecular probe (TetraES) on the basis of a Na+-dependent DNAzyme. DNA tetrahedra with enzymatic stability and self-delivery for targeting cells are arranged onto the DNAzyme and substrate strand, respectively, endowing the subsequent ES detector with the remarkably improved resistance to degradation by nucleases and active cell permeability. The sodium ion abundantly existing in biological systems is used as the cofactor for the DNAzyme that is efficiently silenced until encountering the intracellular target miRNA. The as-developed DNAzyme detector can easily enter cells without the aid of any exogenous transfection agent. In the presence of target miRNAs, the silenced DNAzyme can be specifically activated in a predictable manner and folds into the catalytically active conformation, followed by the circular cleavage of the substrate strands, each of which contains an internal scissile position of ribonucleotide adenosine (rA). This leads to a significantly amplified fluorescence signal because the fluorophores are released from the quenchers in a one-to-many manner different from the conventional molecular beacon (MB). Thus, the detection ability is improved by at least 10 times compared with the MB counterpart. Due to the desirable assay specificity and high sensitivity, the Tetra-ES is able to be used to discriminate target miRNA from the corresponding family members and accurately sense the expression level of target miRNA within living cells. Furthermore, the universality is validated by extending the newly-proposed signaling scheme to the detection of the other two miRNAs. Together, these experimental results demonstrate that the Tetra-ES detector is a promising platform for the detection of specific miRNA inside target living cells, providing a unique opportunity for functional

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Corresponding Author: Email: [email protected]

ORCID Congcong Li: 0000-0001-8126-6211 The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Pascal Chartrand for discussion and manuscript review. This work is supported by the National Natural Science Foundation of China (NSFC) (grant NO: 21775024) and Key Project of Natural Science Foundation of Fujian Province (Grant NO: 2019J02005).

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