Duplex-specific Nuclease Research Articles

A sensitive RNA chaperone assay using induced RNA annealing by duplex specific nuclease for amplification

The hybridization of two complementary RNAs in single cells depends on their complementary sequences and secondary structures, and is usual inefficient at the low concentrations. The bacterial RNA chaperone Hfq increases the rate of base pairing hybridization of mRNA, and stabilizes sRNA-mRNA duplexes. However, The RNA chaperone Hfq accelerates the RNA annealing between two complementary pair RNAs with a still unknown mechanism. So the sensitivity assay of Hfq induced RNA annealing is very important. By using a 2-OMe-RNA modified molecular beacon as a reporter, which can be specificity cleavage by DSN, we observed the amplification reaction kinetics (κrea) is 0.16 s−1. Our results showed that the Hfq hexamer directly induced the RNA annealing, and DSN aided the ultra-sensitivity assay reaction with 0.18 fM Hfq/RNA1/MB1.

Short-probe-based duplex-specific nuclease signal amplification strategy enables imaging of endogenous microRNAs in living cells with ultrahigh specificity

Specific nucleic acids amplification at a constant and mild temperature is important for imaging assay of endogenous microRNAs (miRNAs) in living cells. Duplex-specific nuclease (DSN) is attractive in one-step isothermal assay of miRNA; however, its intrinsic limitations of low amplification specificity and high reaction temperature greatly restrict the application scope. Herein, we present a short-probe-based DSN signal amplification (spDSNSA) strategy enabling analysis of miRNAs at body temperature with significantly high specificity. From systematic investigation of amplification reaction on different types of DNA probes, we revealed that the annealing rate between probe and target miRNA greatly affects the dynamics of amplification process. By simply shortening the length of DNA probe, the spDSNSA remarkably improved specificity without loss of amplification efficiency at 37 °C. As a proof-of-concept, let-7a was sensitively detected by spDSNSA with a limit of detection down to 30 p.M., and a specificity 102 ‒ 104 folds higher than those of traditional DSNSA methods. The analysis of the let-7a in the lysates of A549 human lung cancer cells and BEAS-2B human lung normal bronchial epithelial cells exhibited well agreement with rt-qPCR method. Furthermore, the endogenous let-7a in A549 and BEAS-2B living cells was clearly imaged without damaging the original morphology of cells. The method provide a facile idea for extension of DNS related signal amplification strategies in the application in living cells and POCTs, and would pose a great impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.

Modified beacon probe assisted dual signal amplification for visual detection of microRNA

In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity.

Stimuli-Responsive DNA Microcapsules for SERS Sensing of Trace MicroRNA.

In this work, one stimuli-responsive DNA microcapsule was designed to combine duplex-specific nuclease (DSN) amplification strategy and surface-enhanced Raman spectroscopy (SERS) technology for sensitive detection of microRNA 155 (miRNA 155). First, toluidine blue (TB) as Raman dye and CaCO3 as core templates co-precipitated to form TB@CaCO3 composite. Then, DNA networks were layer by layer constructed with oligonucleotide layers cross-linked by linker ssDNA L to lock TB@CaCO3 inside. In the presence of ethylenediaminetetraacetic acid, the core CaCO3 would be dissolved to form TB-loading DNA microcapsule. With target miRNA 155-induced DSN signal amplification, a large amount of simulative target ssDNA D was obtained, which can completely complement with the linker L on the DNA networks, destroying the microcapsule to release TB and obtain a strong Raman signal. So by this smart design, a SERS platform was fabricated on the basis of the stimuli-responsive DNA microcapsule to detect miRNA 155 from 1 fM to 10 nM with a detection limit of 0.67 fM. In the present study, the programmable property and rapid response speed of DNA microcapsule, which helped in fabrication of a new potential biosensing technology for miRNA detection that is anticipated to be applied for clinical diagnosis.

Cascade Amplification-Mediated In Situ Hot-Spot Assembly for MicroRNA Detection and Molecular Logic Gate Operations.

MicroRNAs (miRNAs) play important roles in many biological processes and are associated with various diseases, especially cancers. Combination of technological developments such as nanomaterials, functional enzyme-mediated reactions, and DNA nanotechnology holds great potential for high-performance detection of miRNAs in molecular diagnostic systems. In this work, we have fabricated a cascade signal amplification platform through integrating duplex-specific nuclease (DSN)-assisted target recycling with catalytic hairpin assembly (CHA) reaction for the detection of microRNA-141 (miR-141). The target recycling process driven by DSN results in highly amplified translation of target miRNA to single-stranded connector DNA fragments. The CHA reaction is further initiated by connector DNAs using hairpin-modified gold nanoparticles (HP-AuNPs) as the sensing unit, leading to the formation of AuNP network architecture on the electrode for electrochemical and photoelectrochemical detection of miR-141 in signal-on and signal-off modes, respectively. The developed electrochemical biosensor exhibits a detection limit down to 25.1 aM miR-141 (60 copies in 4 μL sample) and excellent selectivity to discriminate a single base-mismatched sequence and other miRNAs. This assay is also applied to the determination of miR-141 in total RNAs extracted from human breast cancer cells (MDA-MB-231), confirming the applicability of this method for absolute quantification of specific miRNAs in real-world samples. Furthermore, two-input AND and INHIBIT (INH) logic gates are constructed to detect miRNAs. In particular, the AND gate achieves cell-specific gate activation based on expression profiles of miR-141 and microRNA-21 (miR-21). Therefore, our proposed cascade amplification platform has great potential applications in miRNA-related clinical diagnostics and biochemical research.

Ultrasensitive detection of miRNA-155 using multi-walled carbon nanotube-gold nanocomposites as a novel fluorescence quenching platform

Multi-walled carbon nanotube-gold nanocomposites (MWCNT/AuNCs) were prepared and used as a novel fluorescence quenching platform for ultrasensitive detection of miRNA-155. In the designed system, fluorophore labeled DNA probe was introduced, and the fluorescence signal of them were effectively quenched due to the adsorption of them on MWCNT/AuNCs. Upon the addition of target miRNA-155, DNA-RNA heteroduplexes were formed and desorbed from MWCNT/AuNCs to yield a strong fluorescence signal. In the presence of duplex-specific nuclease (DSN), DNA-RNA heteroduplexes became a substrate for the enzymatic hydrolysis of DNA strands and triggered cyclic signal amplification. Under optimal conditions, MWCNT/AuNCs displayed 93.2% of the quenching efficiency to the fluorophore labeled DNA probe, paving the way for ultrasensitive miRNA-155 detection with a detection limit down to 33.4 fM. Meantime, the present strategy showed excellent selectivity for miRNA-155 detection against other miRNAs including miRNA-21, miRNA-141, and mutated miRNA-155 strands. Moreover, satisfactory results were obtained for miRNA-155 detection in human serum samples, illustrating that the proposed sensing platform is applicable in disease diagnosis.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

MoS2 Nanoprobe for MicroRNA Quantification Based on Duplex-Specific Nuclease Signal Amplification.

MicroRNAs (miRNAs) play significant regulatory roles in physiologic and pathologic processes and are considered as important biomarkers for disease diagnostics and therapeutics. Simple, fast, sensitive, and selective detection of miRNAs, however, is challenged by their short length, low abundance, susceptibility to degradation, and homogenous sequence. Here, we report a novel design of nanoprobes for highly sensitive and selective detection of miRNAs based on MoS2-loaded molecular beacons (MBs) and duplex-specific nuclease (DSN)-mediated signal amplification (DSNMSA). We show that MoS2 nanosheets not only exhibit high affinity toward MBs but also act as an efficient quencher for absorbed MBs. The strong fluorescence-quenching ability of MoS2 in combination with cyclic DSNMSA contributes to the superior sensitivity of our method, with a limit of detection 4 orders of magnitude lower than that of traditional hybridization methods. Moreover, the nanoprobes also show high selectivity for discriminating homogenous miRNA sequences with one-base differences because of the discrimination ability of MBs and DSN. Furthermore, we demonstrate that the MoS2-loaded MB nanoprobes can be utilized for multiplexed detection of miRNAs. Given its high sensitivity and specificity, as well as the multiplexed function; this novel method as an effective tool shows a great promise for simultaneous quantitative analysis of multiple miRNAs in biomedical research and clinical diagnosis.

An electrochemical biosensor for microRNA-196a detection based on cyclic enzymatic signal amplification and template-free DNA extension reaction with the adsorption of methylene blue

A simple and sensitive electrochemical biosensor was developed for microRNA-196a detection, which is of important diagnostic significance for pancreatic cancer. It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction. In the presence of microRNA-196a, duplex-specific nuclease (DSN) catalyzed the digestion of the 3′-PO4 terminated capture probe (CP), resulting in the target recycling amplification. Meanwhile, the 3′-OH terminal of CP was exposed. Then, template-free DNA extension reaction was triggered by terminal deoxynucleotidyl transferase (TdT), producing amounts of single-stranded DNA (ssDNA). After ssDNA absorbed numerous methylene blue (MB), an ultrasensitive electrochemical readout was obtained. Based on this dual amplification mechanism, the proposed biosensor exhibited a high sensitivity for detection of microRNA-196a down to 15 aM with a linear range from 0.05 fM to 50 pM. This biosensor displayed high specificity, which could discriminate target microRNAs from one base mismatched microRNAs. It also showed good reproducibility and stability. Furthermore, it was successfully applied to the determination of microRNA-196a in plasma samples. In conclusion, with the excellent analytical performance, this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer.

Label-free and sensitive microRNA detection based on a target recycling amplification-integrated superlong poly(thymine)-hosted copper nanoparticle strategy

Poly(thymine)-hosted copper nanoparticles (poly T-CuNPs) have emerged as a promising label-free fluorophore for bioanalysis, but its application in RNA-related studies is still rarely explored. Herein, by utilizing duplex-specific nuclease (DSN) as a convertor to integrate target recycling mechanism into terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly T-CuNPs platform, a specific and sensitive method for microRNA detection has been developed. In this strategy, a 3′-phosphorylated DNA probe can hybridize with target RNA and then be cut by DSN to produce 3′-hydroxylated fragments, which can be further tailed by TdT with superlong poly T for fluorescent CuNPs synthesis. As proof of concept, an analysis of let-7d was achieved with a good linear correlation between 20 and 1000 pM (R2 = 0.9965) and a detection limit of 20 pM. Moreover, both homologous and heterologous microRNAs were also effectively discriminated. This strategy might pave a brand-new way for designing label-free and sensitive microRNA assays.

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform.

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

Triple Signal Amplification Strategy for Ultrasensitive Determination of miRNA Based on Duplex Specific Nuclease and Bridge DNA-Gold Nanoparticles.

Abnormal expression of miRNAs always occurs in solid tumors. Thus, it is critical to sensitively and selectively detect such biomarkers for the diagnosis and prognosis of diseases. Here, we report a biosensing scheme for the determination of miRNA with triple signal amplification based on target-triggered cyclic duplex specific nuclease digestion and bridge DNA-gold nanoparticles. Electrochemical signals are recorded to present initial levels of miRNA. This method is ultrasensitive with a wide linear range of 10-17 to 10-11 M. The limit of detection is down to 6.8 aM. Moreover, the overexpression of miR-21 is confirmed in lung cancer patients by the proposed method, which is in good accordance with qRT-PCR results. In addition, the developed biosensor does not need a reverse transcription process or any thermal cycling processes. Its performance satisfies the requirement for convenient, rapid, sensitive, and specific early diagnosis of cancers. Therefore, it may have great potential utility in the near future.

On-Particle Rolling Circle Amplification-Based Core-Satellite Magnetic Superstructures for MicroRNA Detection.

Benefiting from the specially tailored properties of the building blocks as well as of the scaffolds, DNA-assembled core-satellite superstructures have gained increasing interest in drug delivery, imaging, and biosensing. The load of satellites plays a vital role in core-satellite superstructures, and it determines the signal intensity in response to a biological/physical stimulation/actuation. Herein, for the first time, we utilize on-particle rolling circle amplification (RCA) to prepare rapidly responsive core-satellite magnetic superstructures with a high load of magnetic nanoparticle (MNP) satellites. Combined with duplex-specific nuclease-assisted target recycling, the proposed magnetic superstructures hold great promise in sensitive and rapid microRNA detection. The long single-stranded DNA produced by RCA serving as the scaffold of the core-satellite superstructure can be hydrolyzed by duplex-specific nuclease in the presence of target microRNA, resulting in a release of MNPs that can be quantified in an optomagnetic sensor. The proposed biosensor has a simple mix-separate-measure strategy. For let-7b detection, the proposed biosensor offers a wide linear detection range of approximately 5 orders of magnitude with a detection sensitivity of 1 fM. Moreover, it has the capability to discriminate single-nucleotide mismatches and to detect let-7b in cell extracts and serum, thus showing considerable potential for clinical applications.

A method to directly assay circRNA in real samples.

A method to directly assay circular RNA (circRNA) is proposed in this work by utilizing the 'microRNA (miRNA) sponge' nature of circRNA and by taking the advantage of duplex-specific nuclease. Moreover, miRNA absorption site-mediated hairpin DNA unfolding and nuclease-assisted target recycling have also been designed in this method to amplify the signal readout, thus sensitive assay of circRNA can be achieved in real samples.

Ratiometric electrochemical assay for sensitive detecting microRNA based on dual-amplification mechanism of duplex-specific nuclease and hybridization chain reaction

We propose a ratiometric electrochemical assay for detecting microRNA (miRNA) on the basis of dual-amplification mechanism by using distinguishable electrochemical signals from thionine (Thi) and ferrocene (Fc). The thiol-modified and ferrocene-labeled hairpin capture probes (CP) are first immobilized on an Au electrode via Au-S reaction. The target miRNA hybridizes with CP and unfolding the hairpin structure of CP to form miRNA-DNA duplexes. Then, kamchatka crab duplex specific nuclease (DSN) specifically cleaves the DNA in miRNA-DNA duplexes, leading to the release of miRNA and another cleaves cycle, meanwhile, numerous Fc leaves away from the electrode surface and leads to the signal-off of Fc. The residual fragment on electrode surface acts as a HCR primer to form dsDNA polymers through in situ HCR with the presence of the primer and two probes (HDNA and HDNA’), resulting in the capture of numerous DNA/Au NPs/Thi and the signal-on of Thi. The dual-amplification mechanism significantly amplifies the decrease of Fc signal and the increase of Thi signal for ratiometric readout (IThi/IFc), thus providing a sensitive method for the selective detection of miR-141 with a detection limit down to 11aM. The dual-signal ratiometric outputs have an intrinsic self-calibration to the effects from system, which is promising to be applied in biosensing and clinical diagnosis.

Facile detection of microRNA based on phosphorescence resonance energy transfer and duplex-specific nuclease-assisted signal amplification

MicroRNAs (miRNAs) play an important role in many biological processes, and its level in plasma and other biological fluids is closely related to many diseases. In this work, a selective room-temperature phosphorescence (RTP) detection method for miRNA was developed based on a duplex-specific nuclease (DSN) -assisted signal amplification strategy and phosphorescence resonance energy transfer (PRET) between poly-diallyldimethylammonium chloride-modified quantum dots (QDs@PDDA) and 6-carboxy-X-rhodamine-modified miRNA sequences complementary oligonucleotide (ROX-ssDNA). The positively charged QDs@PDDA could adsorb negatively charged ROX-ssDNA by electrostatic interaction, whereas the RTP signal of QDs@PDDA could be efficiently quenched by ROX-ssDNA via PRET. In the presence of microRNA-21 (miR-21) and DSN, miR-21 hybridized with ROX-ssDNA initially to form a DNA-RNA heteroduplex as the substrate of DSN, then ssDNA in DNA-RNA heteroduplex would be cleaved into small fragments by DSN and liberate miR-21 to hybridize with another ROX-ssDNA. Eventually, due to weak interaction between ROX-ssDNA fragments and QDs@PDDA, PRET efficiency continually decreased whereas the RTP signal was significantly amplified. By employing the strategy above, quantitative detection of miR-21 in the range of 0.25–40 nM with a detection limit of 0.16 nM was realized, showing excellent performance with simplicity, good selectivity and the ability to be a promising method for miRNA detection.

Quantitative detection of exosomal microRNA extracted from human blood based on surface-enhanced Raman scattering

Since the nature of the exosomal lipid bilayer can allow miRNAs to be protected from degradation by cellular RNAses in body fluids, exosomal microRNA (miRNA) has become an ideal source of non-invasive biomarkers for the early diagnosis and prognosis. In this paper, a new surface-enhanced Raman scattering (SERS) analysis strategy combining stable SERS reporter element and duplex-specific nuclease (DSN)-assisted signal amplification for quantitative detection of exosomal miRNA extracted from human blood is proposed. Firstly, we prepared SERS signal reporter of Au@R6G@AgAu nanoparticles (R6G attachment on the gold nanoparticles, then encapsulated in AgAu alloy shell nanoparticles named as ARANPs) with an inter small nanogap to generate stable SERS signal. Then, ARANPs and separating substrate of silicon microbead (SiMB) were then covalently attached to the 3′- and 5′- end of capture probe (CP) targeting exosomal miRNA. Upon target miRNA binding, DNA in heteroduplexes could be specifically cleaved by DSN and resulted in the release of ARANPs from the surface of SiMB. Meanwhile, target miRNA remained intact and subsequently involved in the next round of target-recycling amplification. The combination of stable SERS intensity and signal amplification significantly improved the sensitivity of the sensing systems, resulting in detection limits of 5 fM. More importantly, this method also could be used for the detection of exosomal miRNAs extracted from the blood collected from patients of recurrence in non-small-cell lung cancer (NSCLC), with a detection of 5.0μL of sample volume, which has potential for point-of-care testing (POCT) in clinical analysis.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3’ end modified with biotin) for a target miRNA of miR-21 and capture sequence (5’ end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

Nanopore-Based Electrochemiluminescence for Detection of MicroRNAs via Duplex-Specific Nuclease-Assisted Target Recycling.

In this study, we proposed a nanopore-based electrochemiluminescence (ECL) sensor combined with duplex-specific nuclease (DSN)-assisted target recycling amplification to detect microRNAs. Because of the synergetic effect of electrostatic repulsion and volume exclusion of gold nanoparticle-labeled DNA capture (DNA-Au NPs) to the negatively charged luminol anion probe, the DNA-Au NP-modified anodized aluminum oxide (AAO) nanopore electrode exhibited high ECL decline in comparison with the bare AAO electrode. Upon the introduction of DSN and target microRNA, the specific DNA-RNA binding and enzyme cleaving could trigger the detachment of capture DNA from the membrane surface, resulting in uncapping of AAO and an increased ECL signal. For comparison, positively charged Ru(bpy)32+ was used as the ECL probe instead of luminol. Because the electrostatic attraction effect between DNA and Ru(bpy)32+ is partially offset by the volume exclusion effect of Au NPs, the AAO electrode modified with only DNA capture is more suitable for the Ru(bpy)32+ case. In our experiment, the case of negatively charged luminol combined with the synergetic effect of electrostatic repulsion and volume exclusion of DNA-Au NPs provides a quantitative readout proportional to the target microRNA concentration in the range of 1.0 fM to 1.0 nM, with a lower detection limit of 1 fM.

Target-initiated labeling for the dual-amplified detection of multiple microRNAs

Herein we exploited a novel target-initiated labeling strategy for the multiplex detection of microRNAs (miRNAs) by coupling duplex-specific nuclease (DSN) with terminal deoxynucleotidyl transferase (TdT). In the presence of target miRNA, the immobilized and 3′-blocked capture probes hybridized with target and thus the formed DNA-RNA hybrid was recognized by DSN. DSN mediated the digestion of 3′-phosphated capture probes (CPs) in the hybrids and synchronously target was released and recycled for another round of hybridization and cleavage. The cleaved CP fragments with a free 3′-OH were then elongated and labeled with multiple biotin-dUTP nucleotides by TdT. Fluorescence reporter streptavidin-phycoerythin was finally added to react with the immobilized biotins and render fluorescence signals. This dual-amplification labeling strategy was successfully demonstrated to sensitively detect multiple miRNAs, taking advantage of DSN-mediated target recycling and TdT-catalyzed multiple signal modification with analysis by a commercial Luminex xMAP array platform. Our experimental results showed the simultaneous quantitative measurement of three sequence-specific miRNAs at concentrations from 1 pM to 2.5 nM. Attempts were also made to directly detect miRNAs in total RNA extracted from cancer cells. The dual-amplification labeling strategy reported here shows a great potential for the development of a method for the multiplexed, sensitive, selective, and simple analysis of multiple miRNAs in tissues or cells for biomedical research and clinical early diagnosis.