Detection Of Target miRNA Research Articles

Split Probe-Induced Protein Translational Amplification for Nucleic Acid Detection.

Nucleic acid detection is important in a wide range of applications, including disease diagnosis, genetic testing, biotechnological research, environmental monitoring, and forensic science. However, the application of nucleic acid detection in various fields is hindered by the lack of sensitive, accurate, and inexpensive methods. This study introduces a simple approach to enhance the sensitivity for the accurate detection of nucleic acids. Our approach combined a split-probe strategy with in vitro translational amplification of reporter protein for signal generation to detect nucleic acids with high sensitivity and selectivity. This approach enables target-mediated translational amplification of reporter proteins by linking split probes in the presence of a target microRNA (miRNA). In particular, the fluorescence split-probe sensor adopts a reporter protein with various fluorescence wavelength regions, enabling the simultaneous detection of multiple target miRNAs. Moreover, luminescence detection by merely altering the reporter protein sequence can substantially enhance the sensitivity of detection of target miRNAs. Using this system, we analyzed and quantified target miRNAs in the total RNA extracted from cell lines and cell-derived extracellular vesicles with high specificity and accuracy. This split-probe sensor has potential as a powerful tool for the simple, sensitive, and specific detection of various target nucleic acids.

Chain extension-mediated colorimetric and sensitive detection of osteosarcoma-related miRNA based on CHA-cooperated self-priming

The accurate and dynamic monitoring of osteosarcoma, one of the most prevalent cancers, has a substantial impact on the quality of life of numerous individuals. This study proposes a sample, sensitive, and portable approach to miRNA detection that integrates a catalytic hairpin assembly (CHA) circuit with self-priming mediated chain extension. This method employs CHA as a protein enzyme-free isothermal amplification technique to amplify miRNA signals and induce self-priming through a specially designed hairpin probe. The pyrophosphate sensing probe (pp probe) was employed, which enables dual high-efficiency and stable colorimetric signaling, in contrast to traditional colorimetric modes. This novel system for the colorimetric detection of target miRNA is operated with high sensitivity (LOD of 3.5 fM) and selectivity, with the entire detection procedure requiring approximately 60 min. Additionally, this system could detect miRNA from constructed serum samples with high stability and recovery. This system is unique, extremely simple, and rapid, and it is designed to detect osteosarcoma-related miRNA through a highly sensitive color change. It is likely to be useful in applications that necessitate point-of-care detection of osteosarcoma.

Automated System for Attomolar-Level Detection of MiRNA as a Biomarker for Influenza A Virus.

We have developed an automated sensing system for the repeated detection of a specific microRNA (miRNA) of the influenza A (H1N1) virus. In this work, magnetic particles functionalized with DNAs, target miRNAs, and alkaline phosphate (ALP) enzymes formed sandwich structures. These particles were trapped on nickel (Ni) patterns of our sensor chip by an external magnetic field. Then, additional electrical signals from electrochemical markers generated by ALP enzymes were measured using the sensor, enabling the highly sensitive detection of target miRNA. The magnetic particles used on the sensor were easily removed by applying the opposite direction of external magnetic fields, which allowed us to repeat sensing measurements. As a proof of concept, we demonstrated the detection of miRNA-1254, one of the biomarkers for the H1N1 virus, with a high sensitivity down to 1 aM in real time. Moreover, our sensor could selectively detect the target from other miRNA samples. Importantly, our sensor chip showed reliable electrical signals even after six repeated miRNA sensing measurements. Furthermore, we achieved technical advances to utilize our sensor platform as part of an automated sensing system. In this regard, our reusable sensing platform could be utilized for versatile applications in the field of miRNA detection and basic research.

MiRNA Is Involved in the Pathogenesis of Multiple Diseases by Targeting Osteoprotegerin

As a member of the tumor necrosis factor receptor family, osteoprotegerin (OPG) is highly expressed in adults in the lung, heart, kidney, liver, spleen, thymus, prostate, ovary, small intestines, thyroid gland, lymph nodes, trachea, adrenal gland, the testis, and bone marrow. Together with the receptor activator of nuclear factor-κB (RANK) and the receptor activator of nuclear factor-κB ligand (RANKL), it forms the RANK/RANKL/OPG pathway, which plays an important role in the molecular mechanism of the development of various diseases. MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs performing regulatory functions in eukaryotes, with a size of about 20-25 nucleotides. miRNA genes are transcribed into primary transcripts by RNA polymerase, bind to RNA-induced silencing complexes, identify target mRNAs through complementary base pairing, with a single miRNA being capable of targeting hundreds of mRNAs, and influence the expression of many genes through pathways involved in functional interactions. In recent years, a large number of studies have been done to explore the mechanism of action of miRNA in diseases through miRNA isolation, miRNA quantification, miRNA spectrum analysis, miRNA target detection, in vitro and in vivo regulation of miRNA levels, and other technologies. It was found that miRNA can play a key role in the pathogenesis of osteoporosis, rheumatoid arthritis, and other diseases by targeting OPG. The purpose of this review is to explore the interaction between miRNA and OPG in various diseases, and to propose new ideas for studying the mechanism of action of OPG in diseases.

A bioinspired heterostructured nanochannel based on dendrimer-gold network and aluminum oxide arrays for sensitive detection of miRNA

BackgroundMicroRNAs, as oncogenes or tumor suppressors, enable to up or down-regulate gene expression during tumorigenesis. The detection of miRNAs with high sensitivity is crucial for the early diagnosis of cancer. Inspired by biological ion channels, artificial nanochannels are considered as an excellent biosensing platform with relatively high sensitivity and stability. The current nanochannel biosensors are mainly based on homogeneous membranes, and their monotonous structure and functionality limit its further development. Therefore, it is necessary to develop a heterostructured nanochannel with high ionic current rectification to achieve highly sensitive miRNA detection. ResultsIn this work, an asymmetric heterostructured nanochannel constructed from dendrimer-gold nanoparticles network and anodic aluminum oxide are designed through an interfacial super-assembly method, which can regulate ion transport and achieve sensitive detection of target miRNA. The symmetry breaking is demonstrated to endow the heterostructured nanochannels with an outstanding ionic current rectification performance. Arising from the change of surface charges in the nanochannels triggered by DNA cascade signal amplification in solution, the proposed heterogeneous nanochannels exhibits excellent DNA-regulated ionic current response. Relying on the nucleic acid's hybridization and configuration transformation, the target miRNA-122 associated with liver cancer can be indirectly quantified with a detection limit of 1 fM and a wide dynamic range from 1 fM to 10 pM. The correlation fitting coefficient R2 of the calibration curve can reach to 0.996. The experimental results show that the method has a good recovery rate (98%–105 %) in synthetic samples. SignificanceThis study reveals how the surface charge density of nanochannels regulate the ionic current response in the heterostructured nanochannels. The designed heterogeneous nanochannels not only possess high ionic current rectification property, but also enable to induce superior transport performance by the variation of surface chemistry. The proposed biosensor is promising for applications in early diagnosis of cancers, life science research, and single-entity electrochemical detection.

Selective miR-21 detection technology based on photocrosslinkable artificial nucleic acid-modified magnetic particles and hybridization chain reaction

Recently, microRNA (miRNA) detection in blood has attracted attention as a new early detection technology for cancer. The extraction of target miRNA is a necessary preliminary step for detection; however, currently, most extraction methods extract all RNA from the blood, which limits the detection selectivity. Therefore, a method for the selective extraction and detection of target miRNA from blood is very important. In this study, we utilized photocrosslinkable artificial nucleic acids and the hybridization chain reaction (HCR) in an attempt to improve upon the current standard method RT-qPCR, which is hampered by problems with primer design and enzymatic amplification. By introducing photocrosslinkable artificial nucleic acids to oligonucleotide probes modified with magnetic particles with a sequence complementary to that of the target miRNA and irradiating them with light, covalent bonds were formed between the target miRNA and the oligonucleotide probes. These tight covalent bonds enabled the capture of miRNA in blood, and intensive washing ensured that only the target miRNA were extracted. After extraction, two types of DNA (H1 and H2) modified with fluorescent dyes were added and the fluorescence signals were amplified by the HCR in the presence of the target miRNA bound to the photocrosslinkable artificial nucleic acids, allowing for isothermal and enzyme-free miRNA detection. The novel method is suitable for selective miRNA detection in real blood samples. Because the reaction proceeds isothermally and no specialized equipment is used for washing, this detection technology is simple and selective and suitable for application to point-of-care technology using microfluidic devices.

Identifying Genetic Signatures from Single-Cell RNA Sequencing Data by Matrix Imputation and Reduced Set Gene Clustering

In this current era, the identification of both known and novel cell types, the representation of cells, predicting cell fates, classifying various tumor types, and studying heterogeneity in various cells are the key areas of interest in the analysis of single-cell RNA sequencing (scRNA-seq) data. Due to the nature of the data, cluster identification in single-cell sequencing data with high dimensions presents several difficulties. In this paper, we introduce a new framework that combines various strategies such as imputed matrix, minimum redundancy maximum relevance (MRMR) feature selection, and shrinkage clustering to discover gene signatures from scRNA-seq data. Firstly, we conducted the pre-filtering of the “drop-out” value in the data focusing solely on imputing the identified “drop-out” values. Next, we applied the MRMR feature selection method to the imputed data and obtained the top 100 features based on the MRMR feature selection optimization scores for further downstream analysis. Thereafter, we employed shrinkage clustering on the selected feature matrix to identify the cell clusters using a global optimization approach. Finally, we applied the Limma-Voom R tool employing voom normalization and an empirical Bayes test to detect differentially expressed features with a false discovery rate (FDR) < 0.001. In addition, we performed the KEGG pathway and gene ontology enrichment analysis of the identified biomarkers using David 6.8 software. Furthermore, we conducted miRNA target detection for the top gene markers and performed miRNA target gene interaction network analysis using the Cytoscape online tool. Subsequently, we compared our detected 100 markers with our previously detected top 100 cluster-specified markers ranked by FDR of the latest published article and discovered three common markers; namely, Cyp2b10, Mt1, Alpi, along with 97 novel markers. In addition, the Gene Set Enrichment Analysis (GSEA) of both marker sets also yields similar outcomes. Apart from this, we performed another comparative study with another published method, demonstrating that our model detects more significant markers than that model. To assess the efficiency of our framework, we apply it to another dataset and identify 20 strongly significant up-regulated markers. Additionally, we perform a comparative study of different imputation methods and include an ablation study to prove that every key phase of our framework is essential and strongly recommended. In summary, our proposed integrated framework efficiently discovers differentially expressed stronger gene signatures as well as up-regulated markers in single-cell RNA sequencing data.

Detection of transcriptome-wide microRNA–target interactions in single cells with agoTRIBE

MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.

Nanoribbon Biosensor-Based Detection of microRNA Markers of Prostate Cancer.

Prostate cancer (PC) is one of the major causes of death among elderly men. PC is often diagnosed later in progression due to asymptomatic early stages. Early detection of PC is thus crucial for effective PC treatment. The aim of this study is the simultaneous highly sensitive detection of a palette of PC-associated microRNAs (miRNAs) in human plasma samples. With this aim, a nanoribbon biosensor system based on "silicon-on-insulator" structures (SOI-NR biosensor) has been employed. In order to provide biospecific detection of the target miRNAs, the surface of individual nanoribbons has been sensitized with DNA oligonucleotide probes (oDNA probes) complementary to the target miRNAs. The lowest concentration of nucleic acids, detectable with our biosensor, has been found to be 1.1 × 10-17 M. The successful detection of target miRNAs, isolated from real plasma samples of PC patients, has also been demonstrated. We believe that the development of highly sensitive nanotechnology-based biosensors for the detection of PC markers is a step towards personalized medicine.

AIEgens-integrated structural color barcodes for binary screening of microRNAs

Highly sensitive microRNAs (miRNA) imaging is of great value in the early diagnosis of tumors. Here, we present a novel aggregation-induced emission luminogens (AIEgens) integrated structural color barcode for binary screening of miRNAs. The barcode particles consist of periodically ordered silica nanoparticles encoded cores, and tetraphenylethylene carboxylate derivatives TPE-(COOH)4 integrated inverse opal polyacrylamide (PAAm) hydrogel shells. As the miRNA probes are decorated in the adjacent TPE-(COOH)4 molecules, the skeletal networks of the hydrogel as well as the TPE-(COOH)4 molecules would be closer with the presence of target miRNA due to the formation of a double helix. This phenomenon could not only result in fluorescence change of the TPE-(COOH)4 molecules, but also contribute to structural color variation of shell hydrogel. These two optical signals could be employed as binary channels for more accurate detection of target miRNAs. During this process, the solid cores of the barcodes could keep stable structural colors, thus providing foundation codes for multiplexed detection of miRNAs. Based on these features, we have demonstrated that the AIEgens-integrated structural color barcodes could realize the multiplexed screening of miRNAs in a microfluidics device for in vitro diagnosis, which implies the potential prospects in biomedical fields.

Rapid and ultrasensitive miRNA detection by combining endonuclease reactions in a rolling circle amplification (RCA)-based hairpin DNA fluorescent assay.

MicroRNA (miRNA) sensing strategies employing rolling circle amplification (RCA) coupled with the hairpin DNA (HD) probe-mediated FRET assay have shown promise, but achieving rapid, sensitive, and specific detection of target miRNA remains a challenge in clinical diagnostics. Herein, we incorporate PstI endonuclease cleavage (PEC) into a conventional RCA-based HD probe FRET assay to develop an effective and feasible method. Long single-stranded RCA products are synthesized from miRNA-21 loaded on a circular dumbbell DNA, and the resultant RCA products self-assemble to generate long HD structures with double-stranded stem regions that are specifically recognized and cleaved by PstI endonucleases when incubated with PstI enzymes. This releases large amounts of short single-stranded DNA fragments that hybridize and open to the complementary loop-stem regions of HD probes labeled with FAM at one end and BHQ-1 at the other, resulting in a reduction in FRET efficiency. This assay achieves a 39.7 aM detection limit for target miRNA-21, approximately 37-fold higher than that of the conventional assay (1.5 fM). Moreover, quantitative detection is possible in a wide range from 1 aM to 1pM within 90min with high sequence specificity. We demonstrate the assay with the detection of target miRNA-21 in total RNA extracted from MCF-7 cancer cells.

Self-Priming DNA Polymerization-Propelled Stochastic Walkers on Magnetic Microbeads for Amplified Detection of miRNA.

Sensitive detection of miRNA targets in complex biological samples possesses great value in biopsy analysis and disease diagnosis but is still challenging because of low abundance and nonspecific interferences. In this work, self-primer DNA polymerization-propelled stochastic walkers (SWs) were proposed to detect miRNA-24 by combining magnetic microbeads (MMBs) and flow cytometry. The MMBs not only provide a three-dimensional interface for DNA walkers but also facilitate the enrichment and isolation of RNA targets from complex biological samples such as serum. The SWs can be initiated to walk through the entire surface of MMBs and transduce RNA walking into amplified fluorescence signals, with the detection limit of miRNA-24 at 0.95 pM. Moreover, this strategy integrating with flow cytometry was demonstrated to have good specificity with other homologous miRNAs. This platform offers promising applications in RNA biosensing and biomedical diagnostics.

Catalyst-Accelerated Circular Cascaded DNA Circuits: Simpler Design, Faster Speed, Higher Gain.

DNA cascaded circuits have great potential in detecting low abundance molecules in complex biological environment due to their powerful signal amplification capability and nonenzymatic feature. However, the problem of the cascaded circuits is that the design is relatively complex and the kinetics is slow. Herein, a new design paradigm called catalyst-accelerated circular cascaded circuits is proposed, where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration. This new design is very simple and only requires three hairpin probes. Meanwhile, the results of a series of studies demonstrate that the reaction speed can be accelerated and the sensitivity can be also improved. Moreover, endogenous mRNA can also be used as a catalyst to drive the circuits to amplify the detection of target miRNA in live cells and in mice. These catalyst-accelerated circular cascaded circuits can substantially expand the toolbox for intracellular low abundance molecular detection.

Palindrome-Embedded Hairpin Structure and Its Target-Catalyzed Padlock Cyclization for Label-Free MicroRNA-Initiated Rolling Circle Amplification.

Highly sensitive detection of microRNAs (miRNAs) is of great significance in early diagnosis of cancers. Here, we develop a palindrome-embedded hairpin structure and its target-catalyzed padlock cyclization for rolling circle amplification, named PHP-RCA for simplicity, which can be applied in label-free ultrasensitive detection of miRNA. PHP-RCA is a facile system that consists of only an oligonucleotide probe with a palindrome-embedded hairpin structure (PHP). The two ends of PHP were extended as overhangs and designed with the complementary sequences of the target. Hence, the phosphorylated PHP can be cyclized by T4 DNA ligase in the presence of the target that serves as the ligation template. This ligation has formed a palindrome-embedded dumbbell-shaped probe (PDP) that allows phi29 polymerase to perform a typical target-primed RCA on PDP by taking miRNA as a primer, resulting in the production of a lengthy tandem repeat. Benefits from the palindromic sequences and hairpin-shaped structure in padlock double-stranded structures can be infinitely produced during the RCA reaction and provide numerous binding sites for SYBR Green I, a double-stranded dye, achieving a sharp response signal for label-free target detection. We have demonstrated that the proposed system exhibits a good linear range from 0.1 fM to 5 nM with a low detection limit of 0.1 fM, and the non-target miRNA can be clearly distinguished. The advantages of high efficiency, label-free signaling, and the use of only one oligonucleotide component make the PHP-RCA suitable for ultrasensitive, economic, and convenient detection of target miRNAs. This simple and powerful system is expected to provide a promising platform for tumor diagnosis, prognosis, and therapy.

Partial collapse of DNA tetrahedron for miRNA assay with duplex-specific nuclease-assisted amplification.

We establish a facile electrochemical approach for detecting miRNA. Programmable DNA tetrahedron is designed using thiol groups for electrode modification, the amino group for the localization of electrochemical species and a hairpin structure that responds to target miRNA. In addition, duplex-specific nuclease-assisted amplification helps improve the sensitivity of this biosensor. The target-initiated partial collapse of the DNA tetrahedron event integrates recognition, electrode immobilization, signal recruitment and amplification. By measuring the sharp silver stripping peak, the highly sensitive detection of miRNA is achieved, which also performs satisfactorily challenging biological samples. This method is featured with simple operation, high sensitivity and practical utility, exhibiting great application potential in clinical diagnosis.

Quantitative SERS detection of multiple breast cancer miRNAs based on duplex specific nuclease-mediated signal amplification.

Simultaneous and ultrasensitive detection of multiple microRNA (miRNA) biomarkers is an essential precondition for early cancer diagnosis and treatment. Here we developed a sandwich surface-enhanced Raman scattering (SERS) sensor based on Au@Ag core-shell nanorods combined with duplex specific nuclease-mediated signal amplification (DSNSA) for quantitative detection of multiple breast cancer miRNA biomarkers. The DSNSA strategy enables quantitative detection of target miRNA through rehybridizing the capture probe DNA-SERSnanotag conjugates to trigger signal amplification. The Au@Ag core-shell nanorods coated with an Ag shell exhibit excellent SERS performance, implying that molecules can be concentrated by the Ag shell at the hot spots. By monitoring the Raman signal attenuation of hot spots in the presence of target miRNAs, three breast cancer associated miRNAs (miR-21, miR-155, and let 7b) were simultaneously determined using the sandwich SERS sensor, and their detection limits (LODs) were 0.05 fM, 0.063 fM and 0.037 fM, respectively. These results indicated that our sandwich SERS sensor combined with the DSNSA strategy holds remarkable promise for multiplex detection of cancer biomarkers and contributes to early diagnosis of cancer.

A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans.

Circulating microRNAs (miRNAs) have become increasingly popular biomarker candidates in various diseases. However, heparin-based anticoagulants might affect the detection of target miRNAs in blood samples during quantitative polymerase chain reaction (qPCR)-based analysis of miRNAs involving RNA extraction, cDNA synthesis and the polymerase catalyzed reaction. Because low-molecular-weight heparins (LMWH) are widely used in routine healthcare, we aimed to investigate whether a prophylactic dose of the LMWH tinzaparin influences qPCR-based quantification of circulating miRNAs. A total of 30 subjects were included: 16 fracture patients with tinzaparin treatment and 14 non-fracture controls without anticoagulation therapy. To control for the effect of tinzaparin on miRNA analysis an identical concentration of synthetic miRNAs was added to plasma, isolated RNA and prepared complementary DNA (cDNA) from all samples in both groups. No significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin-treated patients with untreated controls. Among the tinzaparin-treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and one to six hours after a subcutaneous injection of tinzaparin 4500IU. No significant effect was observed for any of the investigated miRNAs. A prophylactic dose of 4500IU tinzaparin does not seem to affect cDNA synthesis or qRT-PCR-based quantification of circulating miRNAs.

Target-triggered hybridization chain reaction for ultrasensitive dual-signal miRNA detection

A signal amplification sensing system with target-triggered DNA cascade reaction combined with dual-signal readout technology was designed for ultrasensitive analysis of miRNA. The highly conductive metal organic frameworks (MOFs) derivative, N-doped carbon dodecahedron (N-PCD) was deposited with gold nanoparticles as the electrode substrate, which could assist the electron transfer between the molecular probe and the electrode surface, and could remarkably enhance electrochemical response. Tetrahedral DNA nanostructure (T4-DNA) with high structural stability and mechanical stiffness was designed to improve the loading capacity and binding efficiency of the target, thus increasing the sensitivity of the system. The non-enzymatic amplification method based on the DNA cascade reaction allows the electrochemical responses from dual signal DNA probes labeled with ferrocene (Fc) and methylene blue (MB), respectively in turn to improve the reliability of detection. Under optimal conditions, the sensor has a linear range of 5–1.0 × 104 fM, and the limit of detection is as low as 1.92 fM and 3.74 fM for Fc and MB labeled probe, respectively. This strategy raises the promising application for the rapid detection of miRNA targets with low abundance in complex biological systems.

A two-photon metal-organic framework nanoprobe with catalytic hairpin assembly for amplified MicroRNA imaging in living cells and tissues

Monitoring miRNAs can provide critical information for the generation and progress of various cancers. However, the existing DNA amplification strategies-based fluorescent nanoprobes for intracellular miRNAs imaging are often confronted with problems such as shallow light-penetration depth, enzymatic degradation, and low DNA loading efficiency. Herein, we developed a new two-photon nanoprobe (TP-CHA-MOFs) by using MOF as the nanocarrier and hairpins as a two-photon fluorescence signal amplifier for intracellular miRNA imaging. After cell penetration, intracellular phosphate could coordinate with Zr atoms in the MOFs, leading to the release of hairpins from the surfaces of TP-CHA-MOFs. The intracellular miRNA could trigger the formation of the H1-H2 duplex, which led to a turn-on TP fluorescence signal, realizing amplified detection of target miRNA. TP-CHA-MOFs nanoprobe showed high loading capacity of nucleic acid molecules, high biostability toward enzyme, and good biocompatibility. Because of the employment of TP fluorophore, TP-CHA-MOFs achieved sensitive imaging of intracellular miRNA and differentiated tumor cells with different miRNA levels. Moreover, sensitive imaging of miRNA in deep tumor tissues was realized with a penetration depth of 160 µm. The TP-CHA-MOFs nanoprobe is highly promising for future application in medical diagnostics.

Biologically stable threose nucleic acid-based probes for real-time microRNA detection and imaging in living cells

We successfully fabricated threose nucleic acid (TNA)-based probes for real-time monitoring of target miRNA levels in cells. Our TNA probe is comprised of a fluorophore-labeled TNA reporter strand by partially hybridizing to a quencher-labeled TNA that is designed to be antisense to a target RNA transcript; this results in effective quenching of its fluorescence. In the presence of RNA targets, the antisense capture sequence of the TNA binds to targeted transcripts to form longer, thermodynamic stable duplexes. This binding event displaces the reporter strand from the quencher resulting in a discrete “turning-on” of the fluorescence. Our TNA probe is highly specific and selective toward target miRNA and is able to distinguish one to two base mismatches in the target RNA. Compared with DNA probes, our TNA probes exhibited favorable nuclease stability, thermal stability, and exceptional storage ability for long-term cellular studies. Our TNA probes are efficiently taken up by cells with negligible cytotoxicity for dynamic detection of target miRNAs and can also differentiate the distinct target miRNA expression levels in different cell lines. This work illuminates for using TNA as a building component to construct a biocompatible probe for miRNA detection that offers alternative molecular reagents for miRNA-related diagnostics.