G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets.

MicroRNAs (miRNAs) play indispensable roles in post-transcriptional gene regulation. The identification of target mRNAs is essential for dissecting the recognition basis, dynamics, and regulatory mechanism of miRNA-mRNA interactions. However, the lack of an unbiased method for detecting weak miRNA-mRNA interactions remains a long-standing obstacle for miRNA research. Here, we develop and provide proof-of-concept evidence demonstrating a chemical G-clamp-enhanced photo-cross-linking strategy for covalent capture of intracellular miRNA targets in different cell lines. This approach relies on an aryl-diazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe containing an alkynyl group that improves the thermal stability of miRNA-target mRNA duplex molecules and can rapidly cross-link with the complementary strand upon UV 365 nm activation, enhancing the transient capture of mRNA targets. After validating the accuracy and binding properties of ARAGON-based miRNA probes through the successful enrichment for the known targets of miR-106a, miR-21, and miR-101, we then extend ARAGON's application to screen for previously unknown targets of different miRNAs in various cell lines. Ultimately, results in this study uncover GAB1 as a target of miR-101 in H1299 lung cancer cells and show that miR-101 silencing of GAB1 can promote apoptosis in H1299 cells, suggesting an oncogenic mechanism of GAB1. This study thus provides a powerful and versatile tool for enhanced screening of global miRNA targets in cells to facilitate investigations of miRNA functions in fundamental cellular processes and disease pathogenesis.

Multiply Guaranteed Catalytic DNA Circuit for Cancer-Cell-Selective Imaging of miRNA and Robust Evaluation of Drug Resistance.

Catalytic DNA circuits are desirable for sensitive bioimaging in living cells; yet, it remains a challenge to monitor these intricate signal communications because of the uncontrolled circuitry leakage and insufficient cell selectivity. Herein, a simple yet powerful DNA-repairing enzyme (APE1) activation strategy is introduced to achieve the site-specific exposure of a catalytic DNA circuit for realizing the selectively amplified imaging of intracellular microRNA and robust evaluation of the APE1-involved drug resistance. Specifically, the circuitry reactants are firmly blocked by the enzyme recognition/cleavage site to prevent undesirable off-site circuitry leakage. The caged DNA circuit has no target-sensing activity until its circuitry components are activated via the enzyme-mediated structural reconstitution and finally transduces the amplified fluorescence signal within the miRNA stimulation. The designed DNA circuit demonstrates an enhanced signal-to-background ratio of miRNA assay as compared with the conventional DNA circuit and enables the cancer-cell-selective imaging of miRNA. In addition, it shows robust sensing performance in visualizing the APE1-mediated chemoresistance in living cells, which is anticipated to achieve in-depth clinical diagnosis and chemotherapy research.

DNA Nanospheres Assisted Spatial Confinement Signal Amplification for MicroRNA Imaging in Live Cancer Cells.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

Rolling circle amplification assisted CRISPR/Cas12a dual-cleavage photoelectrochemical biosensor for highly sensitive detection of miRNA-21.

BACKGROUND: MicroRNA-21 has been determined to be the only microRNA overexpressed in 11 types of solid tumors, making it an excellent candidate as a biomarker for disease diagnosis and therapy. Photoelectrochemical (PEC) biosensors have been widely used for quantification of microRNA-21. However, most PEC biosensing processes still suffer from some problems, such as the difficulty of avoiding the influence of interferents in complex matrices and the false-positive signals. There is a pressing need for establishing a sensitive and stable PEC method to detect microRNA-21. RESULTS: Herein, a nicking endonuclease-mediated rolling circle amplification (RCA)-assisted CRISPR/Cas12a PEC biosensor was fabricated for ultrasensitive detection of microRNA-21. The p-p type heterojunction PbS QDs/Co3O4 polyhedra were prepared as the quencher, thus the initial PEC signal attained the "off" state. Furthermore, the target was specifically identified and amplified by the RCA process. Then, its product single-stranded DNA S1 activated the cis- and trans-cleavage abilities of CRISPR/Cas12a, leading to almost all of the PbS QDs/Co3O4 polyhedra to leave the electrode surface, the p-n semiconductor quenching effect to be disrupted, and the signal achieving the "super-on" state. This pattern of PEC signal changed from "off" to "on" eliminated the interference of false-positive signals. The proposed PEC biosensor presented a satisfactory linear relationship ranging from 1 fM to 10 nM with a detection limit of 0.76 fM (3 Sb/N). SIGNIFICANCE AND NOVELTY: With innovatively synthesized PbS QDs/Co3O4 polyhedra as the effective quencher for PEC signal, the CRISPR/Cas12a dual-cleavage PEC biosensor possessed excellent selectivity, stability and repeatability. Furthermore, the detection of various miRNAs can be realized by changing the relevant base sequences in the constructed PEC biosensor. It also provides a powerful strategy for early clinical diagnosis and biomedical research.

miPEPPred-FRL: A Novel Method for Predicting Plant MiRNA-Encoded Peptides Using Adaptive Feature Representation Learning.

MicroRNAs (miRNAs) are an essential type of small molecule RNAs that play significant regulatory roles in organisms. Recent studies have demonstrated that small open reading frames (sORFs) harbored in primary miRNAs (pri-miRNAs) can encode small peptides, known as miPEPs. Plant miPEPs can increase the abundance and activity of cognate miRNAs by promoting the transcription of their corresponding pri-miRNAs, thereby modulating plant traits. Biological experiments are the most effective way to accurately identify miPEPs; however, they are time-consuming and expensive. Hence, an efficient computational method for the identification of miPEPs on a large scale is highly desirable. Up to now, there have been no specialized computational tools for identifying miPEPs. In this work, a novel predictor named miPEPPred-FRL based on an adaptive feature representation learning framework that consists of the feature transformation module and the cascade architecture has been proposed. The feature transformation module integrating a newly designed feature selection method and classifier selection rule is developed to convert sequence-based features into primary class and probabilistic features, which are then fed into the improved cascade architecture to obtain more stable and discriminative augmented features. Finally, the augmented features are utilized to construct the final predictor. Cross-validation experiments illustrate that the novel feature selection method and classifier selection rule contribute to boosting the feature representation ability of the framework. Furthermore, the high accuracy of miPEPPred-FRL on independent testing data suggests that it is a trustworthy and valuable tool for the identification of miPEPs.

A micro-nano interface integrated SERS-based microfluidic sensor for miRNA detection using DNAzyme walker amplification.

BACKGROUND: Precise and specific miRNA detection plays a vital role in exploring development mechanisms of cancer disease, thereby it can significantly improve relevant prevention and treatment strategies. RESULTS: In this work, a surface-enhanced Raman spectroscopy (SERS)-based microfluidic chip has been devised with a microcone array SERS substrate (MCASS) for the miR-141 detection. This substrate excels in unique SERS activity and large surface area for DNA oligonucleotide modification. As the presence of miR-141, the DNAzyme walker induced cleavage reaction took place on the finely designed and prepared dual DNA conjugated SERS nanoprobes. The SERS nanoprobes can anchor on MCASS by the DNA hybridization that achieved an impressive detection limit in the femtomolar level. SIGNIFICANCE: With this integrated SERS-based microfluidic chip, we provided a miRNA detection strategy using DNAzyme walker amplification technology. It is believed that this strategy could be a powerful tool for miRNA detection and related cancer screening test.

Nano-matrixes propped self-enhanced electrochemiluminescence biosensor for microRNA detection.

MicroRNAs (miRNA) are the potential biomarker for breast cancer, a biosensor for detecting miRNA-21 was successfully prepared by covalently linking carbohydrazide (CON4H6) and tris (4,4 '- dicarboxylic acid-2,2' - bipyridyl) ruthenium dichloride (Ru (dcbpy)32+) as a self-enhanced emitter (Ru-CON4H6). The biosensor was prepared by coating the electrode with mesoporous silica encapsulated Ru-CON4H6 as luminophores (RMSNs) to covalently link a couple of DNA strands (Q1-H2). The RMSNs coated electrode exhibited strong ECL emission due to the intramolecular electron transfer between the electrochemically oxidized Ru (dcbpy)32+ and co-reactant CON4H6. In the presence of target miRNA-21 and an assistant hairpin H1, H2 could be released from the surface through a strand displacement reaction (SDR), and the reserved Q1 could form G-quadruplex upon the addition of K+. The formed G-quadruplex then interacted with Q2-Fc in the presence of Mg2+ to form a DNA complex on the biosensor surface, which quenched the nano-matrixes propped self-enhanced ECL emission through the electron exchange between Fc and electrode or oxidized ECL intermediates. Under optimal conditions, the ECL decrease showed a correlation with target concentration, leading to a biosensing method for sensitive detection of miRNA-21. The proposed ECL method demonstrated a detectable concentration range from 0.1 fM to 1 nM along with a detection limit of 0.03 fM, good accuracy, and acceptable reproducibility, showing that the self-enhanced ECL biosensing strategy supported by nano-matrix provided a new way for the ultrasensitive detection of miRNA, and promoted the development of breast cancer diagnosis.

Stimuli-responsive probes for amplification-based imaging of miRNAs in living cells.

MicroRNAs (miRNAs) have emerged as important biomarkers in biomedicine and bioimaging due to their roles in various physiological and pathological processes. Real-time and in situ monitoring of dynamic fluctuation of miRNAs in living cells is crucial for understanding these processes. However, current miRNA imaging probes still have some limitations, including the lack of effective amplification methods for low abundance miRNAs bioanalysis and uncontrollable activation, leading to background signals and potential false-positive results. Therefore, researchers have been integrating activatable devices with miRNA amplification techniques to design stimuli-responsive nanoprobes for "on-demand" and precise imaging of miRNAs in living cells. In this review, we summarize recent advances of stimuli-responsive probes for the amplification-based imaging of miRNAs in living cells and discuss the future challenges and opportunities in this field, aiming to provide valuable insights for accurate disease diagnosis and monitoring.

Near-Infrared-Driven Nanorocket for Rapid and Ultrasensitive Detection of MicroRNA.

Herein, by introducing gold nanostars (AuNSs) as fuel core, a near-infrared-driven nanorocket (NIDNR) with pretty fast walking was exploited for ultrasensitive miRNA detection. Compared with traditional nanomaterials-comprised nanomachines (NMs), the NIDNR possesses much better kinetic and thermodynamic performance owing to the extra photothermal driving force from localized surface plasmon (LSP). Impressively, the whole reaction time of NIDNR down to 15 min was realized, which is almost more than 8 times beyond those of conventional DNA-based NMs. This way, the inherent obstacle of traditional NMs, including long reaction time and low efficiency, could be easily addressed. As a proof of concept, the NIDNR was successfully applied to develop an electrochemical biosensing platform for rapid and sensitive detection of miRNA with an LOD down to 2.95 aM and achieved the real-time assay of real biological samples from human hepatocellular carcinoma cells (MHCC97L) and HeLa, thus providing an innovative insight to design more versatile DNA nanomachines for ultimate application in biosensing platform construction and clinical sample detection.

Three-Dimensional CHA-HCR System Using DNA Nanospheres for Sensitive and Rapid Imaging of miRNA in Live Cells and Tissues.

Isothermal, enzyme-free amplification techniques, such as the hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), have gained increasing attention for miRNA analysis. However, current methodological challenges, including slow kinetics, low amplification efficiency, difficulties in efficient cellular internalization of DNA probes, and concerns regarding the intracellular stability of nucleic acids, need to be addressed. To this end, we propose a novel strategy for sensitive miRNA detection based on a three-dimensional (3D) CHA-HCR system. This system comprises two DNA nanospheres, named DS-13 and DS-24, which are functionalized with CHA and HCR hairpins. Target miR-21 initiates CHA between the two nanospheres, thereby activating downstream HCR and bringing cyanine 3 (Cy3) and cyanine 5 (Cy5) into proximity. The 3D CHA-HCR process leads to the formation of large DNA aggregates and the generation of fluorescence resonance energy transfer signals. In this strategy, the employment of a cascaded reaction and spatial confinement effect improve sensitivity and kinetics, while the use of DNA nanocarriers facilitates cellular delivery and protects nucleic acid probes. The experimental results in vitro, in living cells, and in clinical tissue samples demonstrated the desirable sensing performance. Collectively, this approach holds promise as a valuable tool for cancer diagnosis and biomedical research.

Entropy-driven DNA circuit with two-stage strand displacement for elegant and robust detection of miRNA let-7a.

MicroRNAs (miRNAs) research in cancer diagnosis is expanding, on account of miRNAs were demonstrated to be key indicator of gene expression and hopeful candidates for biomarkers. In this study, a stable miRNA-let-7a fluorescent biosensor was successfully designed based on an exonuclease Ⅲ-assisted two-stage strand displacement reaction (SDR). First, an entropy-driven SDR containing a three-chain structure of the substrate is used in our designed biosensor, leading to reduce the reversibility of the target recycling process in each step. The target acts on the first stage to start the entropy-driven SDR, which generates the trigger used to stimulate the exonuclease Ⅲ-assisted SDR in the second stage. At the same time, we design a SDR one-step amplification strategy as a comparison. Expectly, this developed two-stage strand displacement system has a low detection limit of 25.0 pM as well as a broad detection range of 4 orders of magnitude, making it more sensitive than the SDR one-step sensor, whose detection limit is 0.8 nM. In addition, this sensor has high specificity across members of the miRNA family. Therefore, we can take advantage of this biosensor to promote miRNA research in cancer diagnosis sensing systems.

Artificial miRNAs derived from miR-181 family members have potential in cancer therapy due to an altered spectrum of target mRNAs.

miRNAs are a class of noncoding RNAs with gene regulation properties, and they function as key factors in cell homeostasis. The interaction of miRNAs with their target mRNAs is largely considered to rely on sequence complementarity; however, some evidence indicates that mature miRNAs can adopt diverse conformations with implications for their function. Using the oncogenic miR-181 family as a study model, we suggest that a potential relationship between the primary sequence and secondary structure of miRNAs may have an impact on the number and spectrum of targeted cellular transcripts. We further emphasize that specific alterations in miR-181 primary sequences might impose certain constraints on target gene selection compared with the wild-type sequences, leading to the targeting of new transcripts with upregulated function in cancer.

Comparative Biochemical Studies of Disease-Associated Human Dicer Mutations on Processing of a Pre-microRNA and snoRNA.

Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer's catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitro biochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.

Programming inactive RNA-binding small molecules into bioactive degraders.

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

TNFα, IL-6, miR-103a-3p, miR-423-5p, miR-23a-3p, miR-15a-5p and miR-223-3p in the crevicular fluid of periodontopathic patients correlate with each other and at different stages of the disease.

Periodontitis is one of the main frequent intraoral diseases. Pathogenesis triggers are the immune responses with pro-inflammatory cytokines production and non-coding RNAs expression. The purpose of the present study was to evaluate the involvement of selected miRNAs in various stages of periodontitis and their relationship with the levels of inflammatory mediators in gingival crevicular fluid (GCF). For this study, 36 subjects (21 with periodontal disease, 15 healthy controls) were selected with an age mean of 59.1 ± 3.7 years. Clinical parameters included plaque index, gingival index, sulcus bleeding index, pocket depth, and clinical attachment level. The GCF samples were taken using capillary paper. The levels of miRNAs in GCF were estimated using a Real-Time PCR and TNFα and IL-6 levels were assessed by enzyme-linked immunosorbent assay (ELISA). The results indicated that the miRNA-103a-3p, miRNA-23a-3p, miRNA-15a-5p, and miRNA-223-3p were significantly upregulated with respect to healthy controls. Significant differences were observed for miRNA-23a-3p, miRNA-103a-3p and miRNA-423-5p levels in accord with the disease stages. Inflammatory mediators evaluated in GCF correlate well with the clinical parameters and the severity of the periodontal disease. miRNAs can represent biomarkers of disease stage and can be investigated as a possible therapeutic target, as well as levels of TNFα and IL-6 may drive the disease progression by acting as prognostic markers.

Chemiluminescence Sensor for miRNA-21 Detection Based on CRISPR-Cas12a and Cation Exchange Reaction.

Herein, a chemiluminescence (CL) biosensor based on CRISPR-Cas12a and cation exchange reaction was constructed to detect the biomarker microRNA-21 (miRNA-21). The rolling circle amplification (RCA) reaction was introduced to convert each target RNA strand into a long single-stranded DNA with repeated sequences, which acted as triggers to initiate the transcleavage activity of CRISPR-Cas12a. The activated Cas12a could cleave the biotinylated linker DNA of CuS nanoparticles (NPs) to inhibit the binding of CuS NPs to streptavidin immobilized on the surface of the microplate, which strongly reduced the generation of Cu2+ from a cation exchange between CuS NPs and AgNO3, and thus efficiently suppressed the CL of Cu2+-luminol-H2O2 system, giving a "signal off" biosensor. With the multiple amplification, the detection limit of the developed sensor for miRNA-21 reached 16 aM. In addition, this biosensor is not only suitable for a professional chemiluminescence instrument but also for a smartphone used as a detection tool for the purpose of portable and low-cost assay. This method could be used to specifically detect quite a low level of miRNA-21 in human serum samples and various cancer cells, indicating its potential in ultrasensitive molecular diagnostics.

An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.

Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.