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Construction of an Autonomous Nonlinear Hybridization Chain Reaction for Extracellular Vesicles-Associated microRNAs Discrimination Qiong Wu, Hong Wang, Keke Gong, Jinhua Shang, Xiaoqing Liu, and Fuan Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02181 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Analytical Chemistry

Construction of an Autonomous Nonlinear Hybridization Chain Reaction for Extracellular Vesicles-Associated microRNAs Discrimination Qiong Wu, Hong Wang, Keke Gong, Jinhua Shang, Xiaoqing Liu, Fuan Wang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China ABSTRACT: Extracellular vesicles (EVs) have emerged as promising tumor biomarkers for early cancer diagnosis as primary tumorsecreted EVs carry characteristic molecular information of parent cells. It is thus desirable to realize the efficient discrimination of the signatured EVs-associated miRNAs with low expression and subtle variation. Here, we introduce an autonomous nonlinear enzyme-free signal amplification paradigm for EVs discrimination through a highly sensitive and selective detection of their inherent miRNAs in situ. Our proposed amplifier consists of a modularized DNAzyme-amplified two-staged cascaded hybridization chain reaction (CHCR-DNAzyme) circuit, where the analyte-generated output of the preceding hybridization chain reaction (HCR1) stage serves as input to motivate the following hybridization chain reaction (HCR2) stage and the concomitant assembly of numerous DNAzyme biocatalysts. By incorporating a flexibly configurable sensing module, this modular CHCR-DNAzyme circuit can further extend to “plug-and-play” sensing mode that enables the miRNA assay with high specificity. The sophisticated design and the detecting performance of our CHCR-DNAzyme scheme were systematically investigated in vitro. The optimized CHCR-DNAzyme system was further applied for distinguishing EVs derived from different cells through the amplified detection of a putative miRNA biomarker in EVs. This compact CHCR-DNAzyme amplifier provides a universal and facile toolbox for highly efficient identification of multiple miRNAs-involved EVs and thus holds great potential for early cancer diagnosis.

EVs are membrane-enclosed vesicles of endocytic origin and are demonstrated to carry various signaling molecules (nucleic acids and proteins) for reflecting their parent cells and tissues.1,2 Significantly, EVs have emerged as mediators for shuttling molecular information in pathophysiological processes, such as pre-metastatic niche formation and cancer progression.3-6 Many pathological conditions, especially cancer-involved diseases, lead to higher production of vesicles inherited from their parent cells.7,8 Consequently, tumorderived EVs possess unique protein and miRNA profiles which could be used as prominent clues for tracking their original cells.2,9 Therefore, the tumor-derived circulating EVs have been recognized as promising noninvasive biomarkers for early cancer diagnosis.10,11 Currently, the identification and quantification of specific EVs are realized for achieving early cancer diagnosis through their exterior membrane proteins.12-14 These strategies have been developed for precisely predicting their parental tumor cells. Different tumor-secreted EVs present quilt-like tapestry of membrane protein markers.15,16 Yet these EVs share some common protein patterns with subtle variations. In addition, the translation of these proteins into distinguishable signals also represents a challenge due to the time-consuming target screen/enrichment, the lack of representative cancerous EVs biomarkers and the limited signal gain (immunoassay)17, especially in early cancer stage. Meanwhile, miRNAs are deeply involved in cancerassociated genome regulations and are considered as significantly important diagnostic and prognostic biomarkers for distinguishing different oncological diseases.18-22 Similarly, the entrapped miRNAs in circulating EVs (EVs-miRNAs) are demonstrated to be encoded with similar expression patterns of tumor cells.2,23 These EVs-encapsulated miRNAs are protected

from degradation by ribonuclease or suboptimal storage,24 making the EVs-miRNAs promising biomarkers for predicting parental tumors. Therefore, the identification of endogenous EVs-miRNAs may provide an alternative non-invasive approach for achieving early cancer diagnosis. Considering that the early-staged circulating EVs are associated with low and subtle miRNAs variations, numerous signal amplification strategies have thus proposed to sensitively detect miRNA, which also favors the efficient EVs discrimination.19-25 Conventional quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) has been initially introduced for EVs-miRNA quantification, yet challenges still exist for the following reasons: the cancer cell-derived EVs represent only a small fraction of total cell-secreted EVs. Then all EVs are lysed and mixed to extract total RNAs for analysis regardless of their origins, leading to the dilution of the dysregulated EVsmiRNAs. Therefore, it is highly desirable to implement the facile isothermal amplification system for directly detecting the inherent analytes inside EVs. The isothermal amplification strategies attract more attentions than the conventional polymerase chain reaction (PCR)26,27 due to their superior efficiency, moderate speed and ease of use (constant temperature environment). Indeed, various enzyme-involved isothermal amplification strategies, including rolling circle amplification (RCA),28 polymerization/nicking replication29,30 and exonuclease-mediated regeneration31 of analyte, have been developed for amplified biosensing. DNAzyme is further integrated into these enzymes-based amplification systems.32,33 DNAzyme is a single-stranded catalytic nucleic acid that can mimic the functions of proteinenzymes,34 such as horse radish peroxidase (HRP)35 or endonucleases.36 The flexibility in encoding recognition

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functions into DNAzyme sequences, the intrisic cyclic biotransformations and their reduced non-specific absorptions, turn the DNAzyme into an ideal candidate for developing amplified sensing platforms.37 For example, the isothermal rolling circle polymerization of the hemin/G-quadruplex DNAzyme,38 the autonomous replication/nicking generation of DNAzymes,39 and the exonuclease III-stimulated regeneration of analyte and successive production of DNAzymes40 have been identified as ultrasensitive biosensing strategies. Albeit substantial progress is accomplished, these enzymatic strategies have the environmentally sensitive limitations that might otherwise prohibit protein enzymes. Besides enzymes and DNAzymes, hybridization-based chain reaction (HCR) represents a new important nonenzymatic tool for realizing the isothermal autonomous amplification.41-44 HCR involves a self-sustained hybridization through which two metastable DNA hairpins react with each other to generate long dsDNA polymers upon their exposure to analyte.45,46 This isothermal amplification system is further realized by encoding RNA-cleaving or hemin/G-quadruplex DNAzyme.36,47 Recently, nonlinear HCR has been exploited for the assembly of branched DNA nanostructures to achieve hypersensitive bioassay.48,49 A tandem HCR is integrated for acquiring branched DNA nanostructures by taking advantage of flexible circuit design and reduced unpredictable signal leakages.50-52 Importantly, the optimized HCR circuit could serve as compact and universal module for realizing multiple miRNAs analysis. Considering the aforementioned complex interior constituent of EVs, the integration of nonlinear HCR system with DNAzyme transduction represents an ideal candidate for specifically and reliably detecting EVs-miRNAs with early cancer diagnosis purpose. Herein, we propose a facile and modular cascaded HCRDNAzyme (CHCR-DNAzyme) amplifier for differentiating different EVs based on the detection of inherent miRNA biomarker. Here miR-21 is selected as a model biomarker, which is enriched in OSCC cell (Cal 27)-secreted EVs as compared with that of normal cells (HaCat, HIOEC). The optimized enzyme-free CHCR-DNAzyme circuit is systematically investigated in vitro and then is transfected into EVs to investigate the in situ miRNA assay. The amplifier is initiated by the endogenous miRNA and generates branched Mg2+-dependent DNAzyme nanowires, which recognize and cleave the substrate to achieve an amplified readout. The isothermal autonomous DNA amplifier integrates three successive cascaded amplicon stages: the preceding HCR1, the following HCR2 and the DNAzyme amplifier unit. Initiator triggers the autonomous cross-opening of HCR1 reactants, generating HCR1 dsDNA nanowire backbone. The tandem rudiment triggers of HCR1 nanowires act as intermediate transducers for the following HCR2 that activate and generate numerous DNAzyme branched nanowires, achieving an amplified fluorescence readout. The CHCR-DNAzyme system could be used as a universal sensing platform for analyzing more other targets without significant optimization, which is anticipated to realize multiple EVs-miRNAs discrimination and early cancer diagnosis. EXPERIMENTAL SECTION Materials. 4-(2-hydroxyethyl) piperazine-1 ethanesulfonic acid sodium salt (HEPES), (3-aminopropyl)trimethoxysilane (APTES), magnesium chloride and sodium chloride were purchased from Sigma-Aldrich. All DNA primers were

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synthesized and HPLC-purified by Sangon Biotech Co., Ltd. (Shanghai, China). The ribonucleobase (rA)-containing substrate was purchased from TaKaRa Bio. Inc. (Dalian, China). Table S1 depicts the sequences of these oligonucleotides. Double-distilled ultrapure water was obtained from a Millipore Milli-Q water purification system (Merck Millipore, France), and was used in all experiments. Construction of CHCR-DNAzyme Amplifier. All experiments were prepared in HEPES (10 mM, pH 7.2) with 1 M NaCl and 50 mM MgCl2. Each hairpin (4 μM) was heated at 95 °C for 5 min, and then was rapidly cool down to 25 °C for 2 h at least. For amplified detection of target DNA, different concentrations of which were introduced into the present CHCR-DNAzyme system (0.2 μM of each hairpin). For the analysis of miR-21, except for the concentration of “helper” hairpin H7 was 50 nM, the others were the same. After the substrate (0.5 μM) was added to the mixture, fluorescence measurements were conducted at a spectrophotometer with λex=490 nm and λem=520 nm for FAM immediately at 25 °C. The fluorescence spectra and absorption spectra were acquired by Cary elipse spectrophotometer and Cary 100 UV-vis spectrophotometer (Agilent Technologies, U.S.A), respectively. Native Polyacrylamide Gel Electrophoresis (PAGE). Each of these DNA mixtures (0.2 μM of each hairpin, 50 nM of initiator I) were reacted for 5 h. Then 10 µL of each sample was mixed loading buffer and transferred into the 9% gel for running at 120 V (3 h). After staining, the gel was imaged by using the Fluorchem FC3 System (Proteinsimple, U.S.A). Atomic Force Microscopy (AFM) Imaging. Mica was modified with APTES to possess positively charged surface. The DNA sample was deposited onto the modified mica for 15 min, following by gently washing with ultrapure water. AFM imaging was then conducted by Multimode 8 Atomic Force Microscope (Bruker Inc., U.S.A). EVs Isolation for CHCR-DNAzyme Execution. Cal 27 with overexpressed miR-21 was chosen as model cell. HIOEC and HaCat were chosen as control cells. All cells were plated in culture flask and grown to 80-90% confluence. Then these cells were subjected to 48 h of serum-deprived medium incubation, the supernatants of the cells were collected and centrifuged at 2000 g for 20 min to discard the cell debris and apoptotic bodies in the precipitate. Next, the supernatant was collected for centrifuging at 120,000 g for 60 min and the EVs were acquired in the pellets. The obtained EVs were re-suspended in sterile PBS and further centrifuged at 120,000 g for 60 min, and the purified EVs were dispersed in sterile PBS and stored in -80 °C. The concentration of EVs were quantified by BCA assays. For CHCR-DNAzyme loading, MVs (10 μg) and CHCRDNAzyme mixture were endured an electroporation at 250 V and 350 μF, after 20 min of standing, the excess DNAs were eliminated by centrifugation. The CHCR-DNAzyme-loaded EVs were dispersed and reacted in opti-MEM medium. RESULTS AND DISCUSSION The principle of our nonlinear autonomous HCR system is illustrated in Scheme 1. The isothermal CHCR-DNAzyme system consists of two successive HCR circuits, HCR1 and HCR2, and a DNAzyme amplifier unit. Both HCRs can only be initiated by their corresponding triggers to form the energetically favored nicked dsDNA structures analogous to alternating copolymers. In order to integrate the CHCRDNAzyme circuit, the product of upstream HCR1 should be


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able to trigger downstream HCR2, which means that the HCR2 trigger should be carefully engineered and encoded into the HCR1 reactants. In addition, the reconstituted DNAzyme subunits should be introduced into the HCR2 reactants, of which the active DNAzyme can only be generated with the concomitant activation of the CHCR-DNAzyme circuit. The hierarchical reaction acceleration features of CHCR-DNAzyme circuit lead to a progressively sequential signal amplification analogue to the avalanche photodiode detector (APD) electronic devices.

Scheme 1. Schematic illustration of the isothermal DNAzymeamplified two-staged cascaded hybridization chain reaction (CHCR-DNAzyme) circuit. Complementary domains are marked with an asterisk (e.g., domain a* complements with domain a).

All hairpin reactants, comprising the CHCR-DNAzyme circuit, are metastable without the corresponding initiator. The upstream HCR1 system consists of hairpins H1 and H2. The inactive trigger subunits of downstream HCR2, segments d and e, are grafted into 5'- and 3'-ends of H2, respectively. Upon the introduction of initiator I, H1 and H2 autonomously cross-open and successively bring the separated end-grafted domains d and e into close proximity, resulting in the assembly of long dsDNA nanowires that consist of tandem adjacent regions d and e (trigger T of HCR2) (part A of Scheme 1, Figure S1). Thus, each I can propagate a cross-hybridization of H1 and H2 in upstream HCR1, forming a linear dsDNA backbone nanowire to initiate downstream HCR2 branching circuit. The downstream HCR2 unit consists of hairpins H3, H4, H5 and H6. The Mg2+-dependent DNAzyme is split into two catalytically inactive DNAzyme subunits, I and II, which are grafted into the 3'-end of H3 and 5'-end of H5, respectively. Upon the generation of I-assembled HCR1 nanowires, the newly exposed trigger T hybridizes with the accessible sticky end e* of H3 and motivates an autonomous cross-opening of HCR2 reactants. This leads to the unlocking and integrating of two DNAzyme subunit-functionalized hairpins, and to the generation of concatenated and repeated catalytically active DNAzyme units (details see Figure S2). The CHCR-DNAzyme circuit thus contributes to the construction of branched DNA nanostructures consisting of one dsDNA copolymeric HCR1 backbone and tandem HCR2 DNAzyme nanowires branches (part B of Scheme 1, Figure S3). The binding of quencher/fluorophore (BHQ/FAM)-labeled substrate to the newly assembled DNAzyme results in the recovery of FAM fluorescence due to the DNAzyme-catalyzed cleavage of

substrate (Scheme 1, part C). The inherent catalytic ability of DNAzyme leads to an amplified fluorescence readout. In brief, the upstream HCR1-motivated successive cross-opening of H1 and H2 provides numerous accessible stages for downstream HCR2-triggered cyclic sequential hybridizations, and eventually the DNAzyme transductions. Through the nonlinear CHCR-DNAzyme pathway, the synergistic effect between HCR1, HCR2 and DNAzyme amplicons progressively accelerates the generation of an amplified readout. The nonlinear CHCR-DNAzyme circuit is kinetically impeded in a metastable state without target, due to the closed formation of hairpins (Figure S4). It should be noted that the thermal stability of hairpin reactants is crucial to avoid spontaneous and false cross opening without initiator. The CHCR-DNAzyme system should not be activated until the introduction of a specific analyte. In order to fully eliminate the false signal transduction, part of trigger subunit d was caged into the stem region of H2. This can be confirmed by native gel electrophoresis, Figure 1A. Each stage of the CHCR circuit, upstream HCR1 and downstream HCR2, was first validated separately to verify the execution of CHCR as designed. Scarcely no side product can be observed, implying that no internal signal leakage occurs for each CHCR component. As expected, several new bands of dsDNA polymers were obtained with a concomitant bands dissipation of the primary hairpins mixtures for the activated HCR1 or HCR2. Furthermore, nearly no new band appeared for hairpin mixtures without initiator while many new bands with high-molecular-weight dsDNA emerged for CHCR-DNAzyme-motivated system, demonstrating an efficient consumption of the hairpins components and an utmost generation of the dsDNA products. These results clearly reveal that the upstream HCR1, downstream HCR2 or CHCR-DNAzyme circuits can only be activated by their corresponding initiators to produce distinct dsDNA structures with high efficiency. The formation of supramolecular dsDNA products is evident as revealed by gel electrophoresis, then the morphological information of the resulting DNA nanostructures is probed by AFM. The analyteassembled HCR1 dsDNA nanowires provided numerous triggers T acting as anchoring rudiment sites for HCR2generated DNAzyme concatamer nanobranches. As expected, micrometer-long branched DNA structures are observed for the initiated CHCR-DNAzyme amplifier (Figure 1B), confirming that the system proceeded as anticipated. The height of DNA nanochains corresponds to ~2 nm (Figure 1B inset), a characteristic height of dsDNA. Moreover, the partial nanochains bundling might originate from the crossinteractions between DNAzyme subunits. Figure 1C shows the AFM image of non-triggered CHCR system. Only tiny spots of hairpin monomers were observed without any assembled products, demonstrating that no crosstalk occurred. In addition, a large amount of long linear dsDNA structures were detected for the initiator-triggered HCR1 system (as a positive control, Figure 1D), validating the feasibility of our proposed system as expected. Albeit gel electrophoresis and AFM characterizations have confirmed the construction of high-molecular-weight branched dsDNA nanostructures, the effect of DNAzyme is still unclear for the CHCR-DNAzyme system. The DNAzyme-mediated cyclically hydrolytic cleavage of the substrate could, in principle, further strengthens the CHCR-DNAzyme amplifier. Here the ribonucleobase-containing DNAzyme substrate is modified at its 5'- and 3'-ends with a fluorophore/quencher



(F/Q) pair, respectively, resulting in the effective quenching of fluorophore (FAM). The CHCR-assembled DNAzyme nanochains transduce the system through the cyclic cleavage of substrate. Figure 2A shows the time-dependent fluorescence changes of the CHCR-DNAzyme system in the absence and presence of an initiator. The mixture shows no obvious fluorescence change (curve a, Figure 2A), indicating these hairpins remain in a closed structure with negligible signal leakage (spontaneous CHCR or/and DNAzyme reactions). However, upon the introduction of initiator I, a dramatically increased fluorescence was observed with prolonged reaction time and started to retard after ca. 5 h (curve b, Figure 2A). In addition, an exponential fluorescence growth was observed during the earlier stage of the CHCR-DNAzyme system, which was consistent with the nonlinear CHCR amplification and the cyclic DNAzyme reaction. The optimized incubation time was chosen as 5 h to acquire fluorescence spectra (Figure 2B), considering an adequate balance between effective signal gain and minimal background.

Figure 1. (A) Native gel electrophoresis of CHCR-DNAzyme and the corresponding HCR1 and HCR2 systems. (B) AFM characterization and cross-section analysis of the CHCRassembled dsDNA products. (C) AFM image of the non-triggered CHCR-DNAzyme system. (D) AFM characterization of HCR1assembled products.

It should be noted that the CHCR-DNAzyme system was optimized through caging part of the trigger subunit d into the stem region of hairpin H2 (Figure S5). In addition, the subunits of DNAzyme were facilely integrated into the end of respective hairpins without further optimization (Figure S6). Control experiments were carried out to further expatiate the whole working principle. The effect of HCR1 was first evaluated by subtracting hairpin H1 or H2, the key HCR1 constitute, from the system. The downstream HCR2 and DNAzyme cannot be activated without upstream HCR1 execution, thus no fluorescence generation is expected. Indeed, the H1- or H2expelled CHCR-DNAzyme system shows no fluorescence change even with initiator I (Figures S7A and S7B). Furthermore, more control experiments were implemented to elaborate the effect of downstream HCR2 by removing hairpin H4 or H6, the core component of HCR2, out of the CHCR system. No fluorescence change is observed for analyte-

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triggered H4-substracting CHCR system (Figure S7C), which is reasonable since the DNAzyme subunits could not be activated without H4 concatamer. Both HCRs and DNAzyme assembly are interrupted for H4-excluded HCR2 circuit, leading to the blockage of CHCR even the upstream HCR1 circuit proceeds smoothly. Moreover, the H6-excluded CHCR system represents a characteristic traditional HCR-DNAzyme circuit where the H6-expelled HCR2 can only transduce the upstream HCR1 circuit without amplification. As anticipated, a moderate fluorescence change was observed for H6-expelled CHCRDNAzyme circuit with initiator (Figure S7D). Unsurprisingly, no fluorescence change was observed for DNAzyme-mutated CHCR-DNAzyme circuit where the DNAzyme subunits were replaced with polyT sequences (Figures S7E and S7F), indicating that the amplified readout was indeed originated from the DNAzyme catalysis. All control experiments indicate that the CHCR-DNAzyme circuit leads to the cyclic crosshybridizations and to the multiple DNAzymes assembly, resulting in an effective fluorescence signal.

Figure 2. (A) Time-dependent fluorescence changes and (B) Fluorescence spectra of the CHCR-DNAzyme system in the absence (a) and presence (b) of analyte (100 nM). (C) Fluorescence spectra of the CHCR-DNAzyme system upon the introduction of different concentrations of I: 0, 1×10-12, 5×10-12, 1×10-11, 5× 10-11, 1×10-10, 5×10-10, 1×10-9, 5×10-9, 1×10-8, 5×10-8 and 1 ×10-7 M. Inset is the resulting calibration curve. (D) Fluorescence spectra of the CHCR-DNAzyme system in the presence of complementary target (a), one-base mutant (b), two-base mutant (c), three-base mutant (d) and no analyte (e). Inset is the fluorescence intensity ratio at λem=520 nm. Error bars were acquired by 3 parallel experiments.

These results convinced us to utilize the present CHCRDNAzyme circuit for amplified detection of initiator. Thus the CHCR-DNAzyme scheme was carried out for analyzing analyte I (Figure 2C, details see Figure S8). The fluorescence spectra intensified with increasing concentration of DNA analyte, which was consistent with the enhanced assembly of tandem DNAzyme arrays and the efficient generation of fluorescence. The derived calibration curve was acquired with a detection limit corresponding to 0.4 pM based on the 3σ/S method (σ is the standard deviation of the background signal


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while S refers to the slope of fluorescence intensity versus analyte concentration). The dramatic signal amplification capacity is attributed to the synergistic effect of HCR1, HCR2 and DNAzyme amplifiers that go through avalanche-mimicking hierarchical accelerating reaction scheme. The continuous and cyclic hybridization chain-migrations lead to the growth of a two-staged branched DNA structure and the assembly of tandem DNAzyme nanowires. The high performance of the present nonlinear HCR sensing platform is not only reflected by its improved sensitivity but also by its high selectivity (Figures 2D and S9). Target I generates a much higher fluorescence readout (curve a) than its single-base mutant I1 (curve b). Moreover, the fluorescence spectra of the two-base mutant I2 (curve c) and three-base mutant I3 (curve d) mutants show almost identical intensities to the background signal (curve e), indicating the high selectivity of the present CHCR-DNAzyme system.

Figure 3. (A) Schematic illustration of the general CHCRDNAzyme circuit for miR-21 assay by introducing a foreign helper H7. (B) Fluorescence spectra of the updated CHCR-DNAzyme system by introducing different concentrations of miR-21: 0 (a), 1 ×10-12 (b), 5×10-12 (c), 1×10-11 (d), 5×10-11 (e), 1×10-10 (f), 5× 10-10 (g), 1×10-9 (h), 5×10-9 (i), 1×10-8 (j), 5×10-8 (k) and 1×107 M (l). (C) The corresponding calibration curve of (B). (D) Specificity of the CHCR-DNAzyme system upon exposure to different analytes: (a) no target, (b) miR-31, (c) let-7a, (d) miR-205 and (e) miR-21. Inset is the fluorescence ratios of different RNAs. (E) Stability of the CHCR-DNAzyme system in different serums: (a’) buffer only, (b’) 5% serum only, (c’) 10% serum only, (a) 50 nM miR-21 in buffer, (b) 50 nM miR-21 in 5% serum, (c) 50 nM miR-21 in 10% serum. Inset is the fluorescence ratios in different serums. F0 represents the background signal of the system.

The CHCR-DNAzyme system can be extended as a universal sensing platform for amplified detection of other biologically important analyte, e.g., microRNA-21 (miR-21), without further optimization. The varied miR-21 expression is supposed to correlate closely with various anticancer treatments, making miR-21 an important therapeutic target. As shown in Figure 3A, this miRNA sensing platform was constructed by introducing a sensing module composed of a foreign “helper” hairpin H7 into the CHCR-DNAzyme circuit. The specific recognition of hairpin H7 by miR-21 leads to the release of I analogue sequence for initiating the assembly of branched DNAzyme nanowires and the ultimate fluorescence transduction. Figure S10 shows the time-dependent fluorescence changes of the updated miR-21-sensing platform with and without target RNA. Negligible fluorescence change was observed for the extended CHCR-DNAzyme system without miR-21, revealing the high stability of the sensing module. In the presence of miR-21, the fluorescence intensified with time and eventually leveled off after ~5 h. These results definitely confirmed the successful CHCR-generated tandem DNAzyme nanowires for facilitating signal amplification. Figure 3B depicts the representative fluorescence spectra corresponding to different concentrations of miR-21 after 5 h. The fluorescence intensity increases obviously with elevated concentration of miR-21. The present RNA-sensing platform is capable of sensitively detecting miR-21 with a detection limit of 0.3 pM (Figure 3C). Such a detection limit is comparable with most of the isothermal enzyme-free miRNA detection methods (Table S2). Thus the CHCR-DNAzyme amplicon could be used as a universal amplification module for detecting lower amount of analyte with the assistance of a sensing module through a simple ‘plug-and-play’ format. As a robust and versatile sensing platform, the specificity is another important parameter that needs to bring into considerations. We then challenged the updated CHCRDNAzyme system for detecting several interfering nucleic acids: miR-31, let-7a miRNA and miR-205. It is obviously that the fluorescence intensities generated by the different interfering nucleic acids are almost close to the background signal of the system (Figure 3D), indicating the failure of the interfering triggers-motivated CHCR-DNAzyme process. These results definitely demonstrate the excellent selectivity of our proposed system which can easily discriminate the target microRNA from other interfering sequences. The potential applications of the CHCR-DNAzyme circuit relate to its practical utility under biological environment, e.g., serum samples. Then miR-21 was analyzed by the present updated CHCR-DNAzyme sensing system in diluted human serum. Figure 3E shows the fluorescence spectra for analyzing 50 nM miR-21 in 5% and 10% human serum samples (details see Figure S11). Clearly, the target RNA could be analyzed without obvious interference in diluted serum samples, implying an acceptable accuracy of the present CHCR-DNAzyme circuit in complex biological fluids. The effectiveness of our CHCR-DNAzyme system was further extended to evaluate the varied miRNA expressions in different cells-secreted EVs. Cal 27, a human oral squamous cell carcinoma (OSCC) cell line with overexpressed miR-21, was chosen as model cells. HIOEC and HaCat, which are normal human immortalized oral epithelial cell and immortal human keratinocyte line, were chosen as control cells. We anticipated that their generated EVs might also encode with a similar miR-21 expression.



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Figure 4. Detection of miR-21 in Cal 27 cancer cells-derived EVs. (A) CLSM characterization and (B) the corresponding Pearson’s correlation coefficient (PCC) of Cal 27 EVs after transfecting the miR-21-targeting CHCR-DNAzyme system for different time-intervals. (C) Flow cytometric analysis and (D) the corresponding fluorescence intensity of Cal 27 EVs probed by CHCR-DNAzyme system with different reaction durations. Ft represents the fluorescence intensity of the system at indicated time point. All scale bars correspond to 20 μm.

Here the cancerous Cal 27 cells-derived EVs (nominating as Cal 27 EVs) and the non-cancerous HIOEC/HaCat cells-derived EVs (nominating as HIOEC or HaCat EVs) were introduced as model systems for studying the real time applicability of our miR-21-targeting CHCR-DNAzyme platform. After the updated CHCR-DNAzyme reactants were transfected into EVs by electroporation, the EVs-containing miR-21 could initiate the autonomous generation of DNAzymes for cleaving the FAM/BHQ-labelled substrates in EVs, generating an amplified green fluorescence. Meanwhile, these EVs were labelled with red fluorescent membrane dyes (DiI) for optical EVs colocalization, through which the operation of our CHCRDNAzyme circuit was demonstrated inside EVs, not outside of EVs. Confocal laser scanning microscopy (CLSM) characterization showed a negligible green fluorescence at the beginning of each reaction, demonstrating a robust feature of CHCR-DNAzyme system in EVs. A bright green FAM fluorescence was gradually revealed with an increasing reaction time from 0 to 4 h in Cal 27 EVs since the readout signal coincided perfectly with the red fluorescent EVs membrane (Figures 4A and 4B). However, a much weaker green fluorescence was observed for HIOEC and HaCat EVs even after 4 h, which was consistent with their much lower Pearson’s correlation coefficient (PCC) (Figures S12 and S13). The CHCR-DNAzyme-amplified fluorescence transduction of these different EVs was furtherly evaluated and quantified by flow cytometry. The fluorescence of Cal 27 EVs intensified with prolonged reaction time and generated an almost 4.5-fold higher fluorescence after 4 h (Figures 4C and 4D) while the HIOEC EVs and HaCat EVs produced only 1.06and 1.25-fold of fluorescence (Figures S12 and S13), respectively. Clearly, the CHCR-DNAzyme system was effectively triggered by Cal 27 EVs, implying a higher enrollment of miR-21 in Cal 27 EVs. The feasibility of our CHCR-DNAzyme-mediated EVs discrimination was proved by

qRT-PCR analysis (Figure S14), which revealed a higher miR21 expression in Cal 27 EVs than in HIOEC and HaCat EVs. Obviously, the performance of our CHCR-DNAzyme system is comparable with conventional qRT-PCR method. Notably, the CHCR-DNAzyme system could accurately discriminate different EVs through a sensitive and real-time miRNA assay. Importantly, the modular CHCR-DNAzyme circuit could be easily extended to detect multiple EVs-miRNAs biomarkers by simply redesigning the auxiliary sensing module, and thus holds great promise in realizing EVs-miRNAs-mediated early cancer diagnosis. CONCLUSIONS In conclusion, the facilely designed CHCR-DNAzyme biocircuit was proposed as an autonomous nonlinear signal amplification paradigm for highly sensitive and selective miRNA detection and the ultimate EVs differentiation. Target triggers the successive cross-opening of HCR1 reactants to generate dsDNA copolymers consisting of numerous HCR2 initiators. These assembled intermediate initiators stimulate the subsequent assembly of tandem DNAzyme nanowires that catalyze the generation of a significantly amplified fluorescence readout. The whole CHCR-DNAzyme working principle was systematically investigated with high signal gain yet low background. The multiple guaranteed recognitions and synergistic signal amplifications of our CHCR-DNAzyme system enabled the accurate and sensitive detection of miR-21 down to 0.3 pM. In addition, the modular CHCR-DNAzyme platform was successfully utilized for differentiating cellsecreted EVs through monitoring a given miRNA biomarker in situ. Comparing to conventional EVs-targeted detection methods, our CHCR-DNAzyme system shows more advantages including high signal gain and the flexible design. It is of great importance since the EVs-miRNAs are lowly and subtly varied expressed from cells of different types or stages, especially at the earlier stage of disease development. This


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‘plug-and-play’ sensing scheme provides a versatile approach for identifying miRNAs patterns of EVs, thus holds great potential in early cancer diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed schematic illustration of HCR1 and HCR2 circuits, PAGE and AFM characterizations, sequence optimization, CLSM, FC and qRT-PCR analysis of miR-21, and more control experiments.

AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed. E-mail: [email protected].

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors sincerely acknowledge the financial support from Natural Science Foundation of China (21503151 and 81602610), National Basic Research Program of China (973 Program, 2015CB932601) and Fundamental Research Funds for the Central Universities (No. 2042018kf0210).

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