Collapse of DNA Tetrahedron Nanostructure for “Off–On” Fluorescence Detection of DNA Methyltransferase Activity

As a potential detection technique, highly rigid and versatile functionality of DNA tetrahedron nanostructures is often used in biosensing systems. In this work, a novel multifunctional nanostructure has been developed as an “off–on” fluorescent probe for detection of target methyltransferase by integrating the elements of DNA tetrahedron, target recognition, and dual-labeled reporter. This sensing system is initially in an “OFF” state owing to the close proximity of fluorophores and quenchers. After the substrate is recognized by target methyltransferase, the DNA tetrahedron can be methylated to produce methylated DNA sites. These sites can be recognized and cut by the restriction endonuclease <i>Dpn</i>I to bring about the collapse of the DNA tetrahedron, which leads to the separation of the dual-labeled reporters from the quenchers, and thus the recovery of fluorescence signal to produce an “ON” state. The proposed DNA tetrahedron-based sensing method can detect Dam methyltransferase in the range of 0.1–90 U mL^–1 with a detection limit of 0.045 U mL^–1 and shows good specificity and reproducibility for detection of Dam methyltransferase in a real sample. It has been successfully applied for screening various methylation inhibitors. Thus, this work possesses a promising prospect for detection of DNA methyltransfrase in the field of clinical diagnostics

Quantitative Proteomic Analysis To Identify Differentially Expressed Proteins in Myocardium of Epilepsy Using iTRAQ Coupled with Nano-LC–MS/MS

Epilepsy is a difficult-to-manage neurological disease that can result in organ damage, such as cardiac injury, that contributes to sudden unexpected death in epilepsy (SUDEP). Although recurrent seizure-induced cardiac dysregulation has been reported, the underlying molecular mechanisms are unclear. We established an epileptic model with Sprague–Dawley rats by applying isobaric tags for a relative and absolute quantification (iTRAQ)-based proteomics approach to identify differentially expressed proteins in myocardial tissue. A total of seven proteins in the acute epilepsy group and 60 proteins in the chronic epilepsy group were identified as differentially expressed. Bioinformatics analysis suggested that the pathogenesis of cardiac injury in acute and chronic epilepsy may be due to different molecular mechanisms. Three proteins, a receptor for activated protein kinase C1 (RACK1), aldehyde dehydrogenase 6 family member A1 (ALDH6A1), and glycerol uptake/transporter 1 (Hhatl), were identified as playing crucial roles in cardiac injury during epilepsy and were successfully confirmed by Western blot and immunohistochemistry analysis. Our study not only provides a deeper understanding of the pathophysiological mechanisms of myocardial damage in epilepsy, but also suggests some potential novel therapeutic targets for preventing cardiac injury and reducing the incidence of sudden death due to heart failure

Catalytic Hairpin Assembly Actuated DNA Nanotweezer for Logic Gate Building and Sensitive Enzyme-Free Biosensing of MicroRNAs

A target-switched DNA nanotweezer is designed for AND logic gate operation and enzyme-free detection of microRNAs (miRNAs) by catalytic hairpin assembly (CHA) and proximity-dependent DNAzyme formation. The double crossover motif-based nanotweezer consists of an arched structure as the set strand for target inputs and two split G-rich DNAs at the termini of two arms for signal output. Upon a CHA, a small amount of binary target inputs can switch numerous open nanotweezers to a closed state, which leads to the formation of proximity-dependent DNAzyme in the presence of hemin to produce a highly sensitive biosensing system. The binary target inputs can be used for successful building of AND logic gate, which is validated by polyacrylamide gel electrophoresis, surface plasmon resonance and the biosensing signal. The developed biosensing system shows a linear response of the output chemiluminescence signal to input binary miRNAs with a detection limit of 30 fM. It can be used for miRNAs analysis in complex sample matrix. This system provides a simple and reusable platform for logic gate operation and enzyme-free, highly sensitive, and specific multianalysis of miRNAs

Pre-Folded G‑Quadruplex as a Tunable Reporter to Facilitate CRISPR/Cas12a-Based Visual Nucleic Acid Diagnosis

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or “signal-on” colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection

Zipper-Confined DNA Nanoframe for High-Efficient and High-Contrast Imaging of Heterogeneous Tumor Cell

Current study in the heterogeneity and physiological behavior of tumor cells is limited by the fluorescence in situ hybridization technology in terms of probe assembly efficiency, background suppression capability, and target compatibility. In a typically well-designed assay, hybridization probes are constructed in a confined nanostructure to achieve a rapid assembly for efficient signal response, while the excessively high local concentration between different probes inevitably leads to nonspecific background leakage. Inspired by the fabric zipper, we propose a novel confinement reaction pattern in a zipper-confined DNA nanoframe (ZCDN), where two kinds of hairpin probes are independently anchored respective tracks. The metastable states of the dual tracks can well avoid signal leakage caused by the nonspecific probe configuration change. Biomarker-mediated proximity ligation reduces the local distance of dual tracks, kinetically triggering an efficient allosteric chain reaction between the hairpin probes. This method circumvents nonspecific background leakage while maintaining a high efficiency in responding to targets. ZCDN is employed to track different cancer biomarkers located in both the cytoplasm and cytomembrane, of which the expression level and oligomerization behavior can provide crucial information regarding intratumoral heterogeneity. ZCDN exhibits high target response efficiency and strong background suppression capabilities and is compatible with various types of biological targets, thus providing a desirable tool for advanced molecular diagnostics

Visual Assay for Methicillin-Resistant <i>Staphylococcus aureus</i> Based on Rolling Circular Amplification Triggering G‑Quadruplex/Hemin DNAzyme Proximity Assembly

Nowadays, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have constituted a new challenge for anti-infective treatment. Precise identification and rapid clinical diagnostics of MRSA from other methicillin-sensitive strains entail assays with robust diagnostic efficiency and simple operation steps. Sensitive detection of MecA gene is promising to indicate MRSA infection, but it is challenged by the lack of isothermal and simple strategies. A visual assay based on isothermal rolling circular amplification and G-quadruplex/hemin (G4/hemin) DNAzyme proximity assembly was proposed for the immediate, efficient, and cost-effective detection of MecA in simple operation steps and in a single tube. The presence of MecA specifically drove the formation of circular templates, which further triggered isothermal amplification. The amplified product offered abundant binding sites for DNA-grafted hemin probes to form a novel proximity-assembled G4/hemin DNAzyme structure for colorimetric changing diagnosis. This tandem-repeated novel DNAzyme possessed higher catalytic activity and a lower background signal than traditional G4/hemin DNAzyme, ensuring sensitive discrimination of MRSA (limit of detection: 9.6 pM). Assay stability and antimatrix interference capability enable clinical application, which shows compared diagnostic ability with classic methods (100% sensitivity and 100% specificity) but possesses more simplified procedures and shorter turnaround time (<6 h). This colorimetric strategy in a nonsite-specific and hypersensitive manner holds foreseeable prospects in clinical diagnostic and research applications