Intracellular microRNA Research Articles

Dispersion-to-localization of catalytic hairpin assembly for sensitive sensing and imaging microRNAs in living cells from whole blood

Localized DNA circuits have shown good performance regarding reaction rate and sensitivity for sensing intracellular microRNAs (miRNAs). However, these methods reported recently require large kinds of DNA strands and suffer from low signal-to-background (S/B) ratio, which hinder their clinical application. To circumvent these issues, we herein developed a novel strategy for sensitive sensing and imaging miRNAs in living cells based on dispersion-to-localization of catalytic hairpin assembly (DL-CHA). This strategy consists of only three classes of DNA strands (two hairpins and a linker strand), which largely reduces sequence design complexity. Additionally, owing to the unique engineering of the substrate transformation from dispersion to localization, the DL-CHA exhibits not only minimal background leakage but also intensive signal amplification, thus significantly improving the S/B ratio. In particular, the simple sensing method is capable of imaging miRNAs in cells from clinical blood samples for the diagnosis of breast cancer. Therefore, this work provides a powerful tool for intracellular molecules detection and gives a much broader design space for constructing high-performance DNA circuits.

Multiple amplified microRNAs monitoring in living cells based on fluorescence quenching of Mo2B and hybridization chain reaction

Imaging intracellular microRNAs (miRNAs) demonstrated an essential role in exposing their biological and pathological functions. However, the detection of sequence-specific miRNAs in living cells remains a key challenge. Herein, a facile amplified multiple intracellular miRNAs imaging platform was constructed based on Mo2B nanosheets (NSs) fluorescence (FL) quenching and hybridization chain reaction (HCR). The Mo2B NSs demonstrated strong interaction with the hairpin probes (HPs), ssDNA loop, and excellent multiple FL dyes quenching performance, achieving ultralow background signal. After transfection, the HPs recognized specific targets miRNAs, the corresponding HCR was triggered to produce tremendous DNA-miRNA duplex helixes, which dissociated from the surface of the Mo2B NSs to produce strong FL for miRNAs detection. It realized to image multiple miRNAs biomarkers in different cells to discriminate cancer cells from normal cells owing to the excellent sensitivity, and the regulated expression change of miRNAs in cancer cells was also successfully monitored. The facile and versatile Mo2B-based FL quenching platform open an avenue to profile miRNAs expression pattern in living cells, and has great applications in miRNAs based biological and biomedical research.

Sustainable and cascaded catalytic hairpin assembly for amplified sensing of microRNA biomarkers in living cells

The sensing of intracellular microRNAs (miRNAs) is of significance for early-stage disease diagnosis and therapeutic monitoring. DNA is an interesting building material that can be programed into assemblies with rigid and branched structures, especially suitable for imaging intracellular biomolecules or therapeutic drug delivery. Here, by introducing the palindromic sequences into the programmable DNA hairpins, we describe an endogenous target-responsive three-way branched and palindrome-assisted catalytic hairpin assembly (3W-pCHA) approach for imaging miRNA-155 of living tumor cells with high sensitivity. The miRNA-155 triggers autonomous assembly of the fluorescently quenched signal hairpin and two hairpin dimers formed via hybridization of their respective palindromic sequences to yield branched DNA junctions, which carry the unopened hairpins and thus provide addressable substrates for continuous assembly formation of DNA nanostructures. During the formation of the DNA nanostructures, the miRNA-155 is cyclically reused and many signal probes are unfolded to show highly intensified fluorescence for detecting miRNA-155 down to 6.9 pM in vitro with high selectivity. More importantly, these probes can be transfected into live cancer cells to initiate the assembly process triggered by intracellular miRNA-155, which provides a new way for imaging highly under-expressed miRNAs in cells. Besides, this approach can also be employed to differentiate miRNA-155 expression variations in different cells, indicating its promising potentials for early-stage disease diagnosis and biological studies in cells.

Intelligent assembly of Y-shaped DNA nanostructures for intracellular microRNA imaging

Highly sensitive and specific imaging of low-level microRNAs (miRNAs) in cytoplasm is vital for early diagnosis of cancers. In this work, we have developed the amplification strategies for miRNA-155 detection based on the combination the nicked rolling circle amplification (N-RCA) and catalyzed hairpin assembly (CHA). In this system, the target miRNA-155 acts as a polymerase primer to activate N-RCA to produce nicked fragment1 (NF1) and NF2. NF1 acted as new primer could further initiate a new N-RCA reaction over and over. Then, the NF2s could serve as triggers to induce the CHA reaction, and the Y-shaped DNA nanostructure (Y-SDN) was formed. Thus, an amplified fluorescence signal was obtained based on the multiple amplification. Under the optimized experimental conditions, a high sensitivity with a detection limit as low as 1.8 pM at 3σ miRNA-155 and excellent specificity in buffer condition have been achieved by applying this method. Meanwhile, the proposed method enables the application in miRNA-155 detection in human serum. Moreover, we have shown that the method performs well for the intracellular miRNA-155 imaging in cellular environments. Therefore, the present strategy was expected to apply into the clinical disease diagnosis effectively.

Ratiometric FRET Encoded Hierarchical ZrMOF @ Au Cluster for Ultrasensitive Quantifying MicroRNA In Vivo.

Here, Zirconium metal-organic frameworks @ gold (ZrMOF @ Au) cluster architectures have been fabricated and then functionalized with two fluorescent dyes (Quasar [QS] and Cyanine5.5 [Cy5.5]) through deoxyribonucleic acid hybridization, to form a fluorescence resonance energy transfer (FRET) encoded ZrMOF @ Au-QS/Cy5.5 complex. In the presence of the target intracellular microRNA (miR)-21, the fluorescence of Cy5.5 at 705nm (F705 ) decreases and the fluorescence of QS at 665nm (F665 ) increases when Cy5.5 is released from the surface of ZrMOF @ Au-QS/Cy5.5. The change in the fluorescence ratio (F705 /F665 ) shows an outstanding linear range of 0.006-67.9 amol/ngRNA , and the limit of detection is 4.51 zmol/ngRNA in living cells. The high ratio loading of nucleic acid on surface of ZrMOF @ Au cluster and two fluorescence encoded signal enables better sensitivity and reliability. Zeptomolar sensitivity and good linearity against target affords distinct imaging-based monitoring of the cancer marker miR-21 both in living cells and in vivo. At the same time, the architecture displays remarkable photothermal conversion efficiency (53.7%) and gives rise to outstanding therapy ability in vivo. This strategy offers new avenues for the intelligent quantification of miRNAs for simultaneous diagnoses and treatments of early-stage cancers.

Exosomal let-7e, miR-21-5p, miR-145, miR-146a and miR-155 in Predicting Antidepressants Response in Patients with Major Depressive Disorder.

The intracellular microRNAs that negatively regulate Toll-like receptor 4 signaling pathways in peripheral blood mononuclear cells are associated with major depressive disorder (MDD). However, that the distribution of these microRNAs in exosomes could be a biomarker of central nervous system diseases is just beginning to be explored. In the present study, we isolated serum exosomes from patients with MDD and healthy controls to explore the levels of exosomal microRNAs, including let-7e, miR-21-5p, miR-223, miR-145, miR-146a, and miR-155. We also investigated the changes of these exosomal microRNAs after antidepressant treatment and their association with clinical changes in scores on the Hamilton Depression Rating Scale. An ANCOVA adjusted by age, sex, BMI, and smoking showed higher expression levels of miR-146a (p = 0.006) in patients with MDD compared to controls. Patients who achieved remission showed significantly lower let-7e, miR-21-5p, miR-145, miR-146a, and miR-155 levels before treatment and increased levels after antidepressant treatment compared with the non-remission group. Through receiver operating characteristic (ROC) analysis, let-7e, miR-145, and miR-146a showed acceptable discrimination between the remission and non-remission groups, whereas miR-21-5p and miR-155 showed poor discrimination. These findings demonstrate that exosomal microRNAs may play essential roles in predicting antidepressants response.

Ratiometric Fluorescence Imaging of Intracellular MicroRNA with NIR-Assisted Signal Amplification by a Ru-SiO2@Polydopamine Nanoplatform.

Accurate and sensitive fluorescence imaging of intracellular miRNA is essential for understanding the mechanism underlying some physiological and pathological events, as well as the prevention and diagnosis of diseases. Herein, a highly sensitive ratiometric fluorescent nanoprobe for intracellular miRNA imaging was fabricated by integrating a Ru-SiO2@polydopamine (Ru-SiO2@PDA) nanoplatform with a near-infrared light (NIR)-assisted DNA strand displacement signal amplification strategy. The Ru-SiO2@PDA spheres have excellent biosafety, high photothermal effect, and unique photophysical properties that can both emit a stable red fluorescence and well quench the fluorophores getting closer to them. So, when the fuel DNA and carboxyfluorescein (FAM)-labeled signal DNA are co-assembled on their outer surfaces, the FAM's green fluorescence is quenched, and a low ratiometric signal is obtained. However, in the presence of miRNA, the target displaces the signal DNA from the capture DNA, releasing the signal DNA far away from the Ru-SiO2@PDA. Then, the green fluorescence recovers and leads to an enhanced Igreen/Ired value. Under NIR light irradiation, the Ru-SiO2@PDA increases the local temperature around the probe and triggers the release of fuel DNA, which thus recycles the target miRNA and effectively amplifies the ratiometric signal. Using A549 cells as a model, the nanoprobe realizes the highly sensitive ratiometric fluorescence imaging of miRNA let-7a, as well as its in vivo up- and down-regulation expressions. It provides a facile tool for highly sensitive and accurate intracellular miRNA detection through one-step incubation and may pave a new avenue for single-cell analysis.

DNA Logic Circuits for Multiple Tumor Cells Identification Using Intracellular MicroRNA Molecular Bispecific Recognition.

The aberrant expression level of intracellular microRNAs (miRNAs) holds great promise for differentiating cell types at the molecular level. However, cell subtype discrimination based on a single miRNA molecular level is not sufficient and reliable. Herein, multiple identifiable and reporting modules are integrated into a single DNA circuit for multiple cancer cell subtypes identification based on miRNAs bispecific recognition. The DNA three-dimensional (3D) logic gate nano-hexahedron framework extends three recognition modules and three reporting modules to form three "AND" logic gates. Each Boolean operator "AND" returns an "ON" signal in the presence of bispecific miRNAs, simultaneously enabling three types of cell subtype identification. It not only enables the discrimination of cancer cells A549 and MCF-7 from normal cells NHDF but also successfully distinguishes different types of cancer cells. The bispecific intracellular miRNA controllable DNA circuit, with low signal-to-noise ratio, easily extends to various cell type discrimination by adjusting the miRNA species, provides huge opportunities for accurately differentiating multiple cell types at the molecular level.

Near-Infrared Light Controllable DNA Walker Driven by Endogenous Adenosine Triphosphate for in Situ Spatiotemporal Imaging of Intracellular MicroRNA.

As a powerful signal amplification tool, the DNA walker has been widely applied to detect rare microRNA (miRNA) in vivo. Despite the significant advances, a near-infrared (NIR) light controllable DNA walker for signal amplification powered by an endogenous initiator has not been realized, which is crucial for spatiotemporal imaging of miRNA in living cells with high sensitivity. Herein, we constructed a NIR-photoactivatable DNA walker system, which was powered by endogenous adenosine triphosphate (ATP) for in situ miRNA imaging with spatial and temporal resolution. The system was very stable with an extremely low fluorescent background for the bioimaging in living cells. We employed upconversion nanoparticles (UCNPs) as the carriers of the DNA probe and transducers of converting NIR to UV light. Coupled with the DNA walker fueled by intracellular ATP, a smart system based on the NIR light initiated DNA walker was successfully developed for precise spatiotemporal control in living cells. Triggered by NIR light, the DNA walker could autonomously and progressively travel along the track with the assistance of intracellular ATP. The system has been successfully applied for in situ miRNA imaging in different cell lines with highly spatial and temporal resolution. This strategy can expand NIR photocontrol the DNA walker for precise imaging in a biological system.

MicroRNA and diabetic retinopathy—biomarkers and novel therapeutics

Diabetic retinopathy (DR) accounts for ~80% of legal blindness in persons aged 20–74 years and is associated with enormous social and health burdens. Current therapies are invasive, non-curative, and in-effective in 15–25% of DR patients. This review outlines the potential utility of microRNAs (miRNAs) as biomarkers and potential therapy for diabetic retinopathy. miRNAs are small noncoding forms of RNA that may play a role in the pathogenesis of DR by altering the level of expression of genes via single nucleotide polymorphism and regulatory loops. A majority of miRNAs are intracellular and specific intracellular microRNAs have been associated with cellular changes associated with DR. Some microRNAs are extracellular and called circulatory microRNAs. Circulatory miRNAs have been found to be differentially expressed in serum and bodily fluid in patients with diabetes mellitus (DM) with and without retinopathy. Some miRNAs have been associated with the severity of DR, and future studies may reveal whether circulatory miRNAs could serve as novel reliable biomarkers to detect or predict retinopathy progression. Therapeutic strategies can be developed utilizing the natural miRNA/long noncoding RNA (lncRNA) regulatory loops. miRNAs and lncRNAs are two major families of the non-protein-coding transcripts. They are regulatory molecules for fundamental cellular processes via a variety of mechanisms, and their expression and function are tightly regulated. The recent evidence indicates a cross-talk between miRNAs and lncRNAs. Therefore, dysregulation of miRNAs and lncRNAs is critical to human disease pathogenesis, such as diabetic retinopathy. miRNAs are long-distance communicators and reprogramming agents, and they embody an entirely novel paradigm in cellular and tissue signaling and interaction. By targeting specific miRNAs, whole pathways implicated in the pathogenesis of DR may potentially be altered. Understanding the endogenous roles of miRNAs in the pathogenesis of diabetic retinopathy could lead to novel diagnostic and therapeutic approaches to managing this frequently blinding retinal condition.

Stimuli-Responsive Autonomous-Motion Molecular Machine for Sensitive Simultaneous Fluorescence Imaging of Intracellular MicroRNAs.

DNAzymes with enzymatic activity identified from random DNA pools by in vitro selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.

3D DNA Scaffold-Assisted Dual Intramolecular Amplifications for Multiplexed and Sensitive MicroRNA Imaging in Living Cells.

The simultaneous live-cell imaging of multiple intracellular and disease-related microRNAs (miRNAs) with low abundances is highly important to enhance specificity and accuracy for disease diagnosis. On the basis of the improved cell internalization and accelerated reaction kinetics, we develop a three-dimensional (3D) DNA nanoprobe that integrates intramolecular DNAzyme (intra-Dz) and catalytic hairpin assembly (intra-CHA) amplifications to simultaneously monitor multiple miRNAs in living cells. The sensing components are loaded on a DNA scaffold via the sticky-end hybridization of the DNA sequences to increase the local concentrations of the signal probes. The miRNA-21 and miRNA-155 target sequences can trigger intra-Dz and -CHA amplifications on the nanoprobes to show significantly amplified and distinct fluorescence at different wavelengths for simultaneously monitoring low levels of miRNAs. Real-time fluorescence microscopy reveals that such a 3D DNA nanoprobe design with the intra-Dz and -CHA amplifications can accelerate the reaction rate compared to that of the conventional free Dz and CHA because of the increased local concentrations of the sensing components. Importantly, the 3D DNA nanoprobe has desirable stability and biocompatibility and can be readily delivered into living cells to achieve multiplexed and highly sensitive sensing of intracellular miRNA-155 and miRNA-21 sequences. With the demonstration of its intracellular application, the developed 3D DNA nanoprobe thus holds promising potential for biological studies and accurate disease diagnosis.

A One-Two-Three Multifunctional System for Enhanced Imaging and Detection of Intracellular MicroRNA and Chemogene Therapy.

Simultaneous imaging, diagnosis, and therapy can offer an effective strategy for cancer treatment. However, the complex probe design, poor drug release efficiency, and multidrug resistance remain tremendous challenges to cancer treatment. Here, a novel one-two-three system is built for enhanced imaging and detection of miRNA-21 (miR-21) overexpressed in cancer cell and chemogene therapy. The system consists of dual-mode DNA robot nanoprobes assembled by two types of hairpin DNAs and three-way branch DNAs modified on gold nanoparticles, with intercalating anticancer drugs (doxorubicin), into DNA duplex GC base pairs. In the system, via intracellular ATP-accelerated cyclic reaction triggered by miR-21, fluorescence and SERS signals were alternated with DNA structure switch, and the precise SERS detection of miRNA and fluorescence imaging oriented "on-demand" release of two types of anticancer drugs (anti-miR-21 and Dox) are achieved. Thus, "one-two-three" means one kind of miR-21-triggered endogenous substance accelerated cyclic reaction, two modes of signal switch, and three functions including enhanced imaging, detection, and comprehensive treatment. The one-two-three system has some notable merits. First, ATP as an endogenous substance promotes DNA structure switching and accelerates the cyclic reaction. Second, the treatment with a dual-mode signal switch is more reliable and accurate and can provide more abundant information than a single-mode treatment platform. Thus, the imaging and detection of intracellular miRNA and effective comprehensive therapy are realized. In vivo results indicate that the system can provide new insights and strategies for diagnosis and therapy.

Autonomously driving multiplexed hierarchical hybridization chain reaction of a DNA cobweb sensor for monitoring intracellular microRNA

Here, we present an autonomously driving multiplexed hierarchical hybridization chain reaction (MH-HCR) of a DNA cobweb sensor for monitoring intracellular microRNAs. The amplifier of the sensor consists of an enzyme-free amplification, two-step and multiple round hybridization chain reaction (HCR) circuit, where the target miRNA-triggered output of the first HCR stage (HCR1) serves as input to generate the following HCR stage (HCR2) and then produces n cycles (HCR1-HCR2) amplifying processes to result in a DNA cobweb sensor. By introducing a pair of fluorescence resonance energy transfer (FRET) probes, this MH-HCR-based DNA cobweb sensor enables selective and sensitive detection of miRNAs down to 1 fM. In situ imaging of miRNAs in several living cells was successfully achieved. Compared with other nonlinear HCRs, the initiation sequence is enclosed in the stable multiple capture probe, avoiding interference reaction of the capture probe with a hairpin containing DNA fluorescent agent to generate false signals. This MH-HCR amplifier provides a general and robust method for the highly sensitive and selective detection of multiple miRNAs and has the potential for early cancer diagnosis.

Many Birds, One Stone: A Smart Nanodevice for Ratiometric Dual-Spectrum Assay of Intracellular MicroRNA and Multimodal Synergetic Cancer Therapy

The development of a theragnostic platform integrating precise diagnosis and effective treatment is significant but still extremely challenging. Herein, an integrated smart nanodevice composed of Au@Cu2-xS@polydopamine nanoparticles (ACSPs) and fuel DNA-conjugated tetrahedral DNA nanostructures (fTDNs) was constructed, in which the ACSP nanoprobe played multiple key roles in antitumor therapy as well as in situ monitoring of microRNAs (miRNAs) in cancer cells. Regarding the analysis, the ACSP probe contained two optical properties: excellent surface-enhanced Raman scattering (SERS) enhancement and high fluorescence (FL) quenching performance. Employing the ACSPs as the high-efficiency detection substrate combined with the fTDN-assisted DNA walking nanomachines as the superior amplification strategy, a SERS-FL dual-spectrum biosensor was constructed, which achieved an ultralow background signal and excellent sensitivity with detection limits of 0.11 pM and 4.95 aM by FL and SERS, respectively. Moreover, the rapid FL imaging and precise SERS quantitative detection for miRNA in cancer cells were also achieved by dual-signal ratio strategy, improving the accuracy of diagnosis. Regarding the therapeutic application, due to the high reactive oxygen species generation ability and excellent photothermal conversion efficiency, the ACSPs can also act as an all-in-one nanoagent for multimodal collaborative tumor therapy. Significantly, both in vivo and in vitro experiments confirmed its high biological safety and strong anticancer effect, indicating its promising theragnostic applications.

Modulations of obesity-related microRNAs after exercise intervention: a systematic review and bioinformatics analysis.

Obesity is one of the prevalent health-threatening conditions; however, it is preventable by lifestyle interventions such as exercise. The molecular mechanisms underlying physiological adaptation to physical activity are not fully understood. It has been documented that both intracellular and extracellular (circulating) microRNAs (miRNAs) are involved in both obesogenic and exercise adaptation mechanisms. We aimed to conduct a systematic review of publications that examined the effect of exercise on the expression of miRNAs in individuals with obesity. In addition, bioinformatics analysis was performed on most repetitive miRNAs. PubMed, Scopus, and Google Scholar were searched with relevant keywords. We only included studies that utilized exercise as a modality for the health management of human subjects with obesity to evaluate the changes in expression of obesity-related miRNAs. Through checking of 211 retrieved articles, we reached 12 eligible studies. Some studies reported a statistically significant correlation between the change of miRNAs and clinical parameters such as body mass index and fasting glucose. In silico analysis of most repetitive miRNAs i.e. miR-126, miR-21, miR-146a, miR-221, and miR-223 resulted in the molecular signaling pathways that potentially involve in cellular adaption to exercise in people with obesity. miRNAs partake in health-related benefits of physical activity on obesity-associated cellular and molecular phenomena. However, our understanding of the exact mechanism is still in its infancy. Consistently, the clinicians waiting for the result of more integrated experiments to develop a miRNAs panel as a predictive biomarker of exercise in patients with obesity.

Fluorescence on-off-on with small and charge-tunable nanoparticles enables highly sensitive intracellular microRNA imaging in living cells

Nanomaterial-based on-off-on fluorescence sensing strategies are significant particularly in intracellular nucleic acids imaging assay. There still remains challenge to rationally balance fluorescence quenching efficiency and recovery dynamics. We assume that the performance of on-off-on fluorescence sensing strategy can be fundamentally improved on small zero-dimensional (0D) nanomaterial with precisely modulated surface charge. For a proof-of-concept demonstration, silicon nanoparticle (SiNP) with ~4 nm was synthesized and used as the quencher model, of which the surface charge density was modulated by modification of triphenylphosphonium (TPP). The influence of particle size, surface charge and charge density of the nanomaterials on sensing performance was systematically investigated. The strategy showed a low limit of detection (LOD) as 26 pM for target model miR-494, which is one of the lowest in nanomaterial-based on-off-on sensing platforms. And the LOD is even comparable to amplification-based methods in a greatly shortened assay time (2.5 h). The miR-494 expresses in cancerous and normal living cells of human cervical carcinoma (HeLa), human lung carcinoma (A549), human breast cancer (MCF-7), and normal human mammary epithelial (MCF-10A) cells were imaged and localized with significantly improved sensitivity and specificity. These excellent performances insure it a promising candidate as convenient and non-enzymatic sensing platform for miRNA-associated disease detection and early diagnosis.

Pyrene-Modified Guanine Cluster Probes Forming DNA/RNA Hybrid Three-Way Junctions for Imaging of Intracellular MicroRNAs.

MicroRNAs (miRNAs) regulate gene expression in cells; high levels of expression are associated with various cancers. In this paper, we describe PyA-modified nucleic acid probes that can detect intracellular miRNAs by forming DNA/RNA hybrid three-way junction structures containing a fluorescent scaffold-a so-called G-cluster. This G-cluster featured two mismatched strands, four guanine residues, and one fluorescent adenine residue having a pyrene moiety covalently connected at the 8-position through an acetylene linker. The scaffold underwent a dramatic shift in its emission wavelength when two mismatched strands formed a duplex, similar to the behavior of an adenine pentad system (A-cluster). We applied the G-cluster scaffold in a three-way junction system to probe for miRNAs; its red-shifted fluorescence intensity and stability were greater than those reported previously for A-cluster three-way junction probes. Furthermore, confocal microscopy of cancer cell lines revealed bright fluorescence emissions in response to the miRNAs in the cells. Thus, this system can be applied intracellularly as a potential fluorescent probe for the detection of various biologically important nucleic acids.

NIR Light-Propelled Janus-Based Nanoplatform for Cytosolic-Fueled microRNA Imaging.

Various nanoplatforms have been developed to visualize intracellular microRNAs (miRNAs) because of their clinical significance in tumor progression and diagnosis. However, the diffusion-limited motion of the nanoplatforms penalizes the miRNA imaging efficiency in cells. Herein, we fabricated a near-infrared (NIR) light-propelled Janus-based nanoplatform to advance the imaging response. The Janus nanomotor covered with an Au half-shell was loaded by the endocytosis adjuvant of the MnO2 nanosheet for delivering a miRNA-responsive hQN (hairpin DNA quadrangular nanostructure) probe with a catalyzed hairpin assembly (CHA). Once the nanoplatform entered into cells, the MnO2 nanosheet was degraded to Mn2+ by endogenous fuels (such as glutathione) to release the hQN probe. The NIR light irradiation of the nanoplatform generated a heat gradient and thus propelled motion of the nanoplatform. This process accelerated the intracellular reaction of the hQN probe with miRNAs to trigger the cascade CHA amplification with an enhanced fluorescence readout.

Encoding DNA Frameworks for Amplified Multiplexed Imaging of Intracellular microRNAs.

Real-time imaging of multiple low-abundance microRNAs (miRNAs) simultaneously in living cells with high sensitivity is of vital importance for accurate cancer clinical diagnosis and prognosis studies. Maintaining stability of nanoprobes resistant to enzyme degradation and enabling effective signal amplification is highly needed for in vivo imaging studies. Herein, a rationally designed one-pot assembled multicolor tetrahedral DNA frameworks (TDFs) by encoding multicomponent nucleic acid enzymes (MNAzymes) was developed for signal-amplified multiple miRNAs imaging in living cells with high sensitivity and selectivity. TDFs could enter cells via self-delivery with good biocompatibility and stability. Two kinds of MNAzymes specific for miRNA-21 and miRNA-155 with fluorescein labeling were encoded in the structure of TDFs respectively through one-step thermal annealing. In the intracellular environment, the TDFs could be specifically bound with its specific miRNA target and form an active DNAzyme structure. The cleavage of the active site would trigger the release of target miRNA and circular fluorescence signal amplification, which enabled accurate diagnosis on miRNA identifications of different cell lines with high sensitivity. Meanwhile, with the specific AS1411 aptamer targeting for nucleolin overexpressed on the surface of the carcinoma cells, this well-designed TDFs nanoprobe exhibited good discrimination between cancer cells and normal cells. The strategy provides an efficient tool for understanding the biological function of miRNAs in cancer pathogenesis and therapeutic applications.