RNA-cleaving DNAzymes Research Articles

A Rapid Sputum-based Lateral Flow Assay for Airway Eosinophilia using an RNA-cleaving DNAzyme Selected for Eosinophil Peroxidase.

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy.

Site-Specific Bioorthogonal Activation of DNAzymes for On-Demand Gene Therapy.

RNA-cleaving DNAzymes hold great promise as gene silencers, and spatiotemporal control of their activity through site-specific reactions is crucial but challenging for on-demand therapy. We herein report a novel design of a bioorthogonally inducible DNAzyme that is deactivated by site-specific installation of bioorthogonal caging groups on the designated backbone sites but restores the activity via a phosphine-triggered Staudinger reduction. We perform a systematical screening for installing the caging groups on each backbone site in the catalytic core of 10-23 DNAzyme and identify an inducible DNAzyme with very low leakage activity. This design is demonstrated to achieve bioorthogonally controlled cleavage of exogenous and endogenous mRNA in live cells. It is further extended to photoactivation and endogenous stimuli activation for spatiotemporal or targeted control of gene silencing. The bioorthogonally inducible DNAzyme is applied to a triple-negative breast cancer mouse model using a lipid nanoparticle delivery system, demonstrating high efficiency in knockdown of Lcn2 oncogenes and substantial suppression of tumor growth, thus highlighting the potential of precisely controlling the DNAzyme functions for on-demand gene therapy.

Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing.

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.

In Vitro Selection and Characterization of a DNAzyme Probe for Diverse Pathogenic Strains of Clostridium difficile.

Clostridium difficile frequently causes an infectious disease known as Clostridium difficile infection (CDI) and there are urgent needs for the development of more effective rapid diagnostic tests for CDI. Previously we have developed an RNA-cleaving fluorogenic DNAzyme (RFD) probe named RFD-CD1, which is capable of detecting a specific strain of C. difficile but is too specific to recognize other pathogenic C. difficile strains. To overcome this issue, herein we report RFD-CD2, another RFD that is not only highly specific to C. difficile but also capable of recognizing diverse pathogenic C. difficile strains. Extensive sequence and structure characterization establishes a pseudoknot structure and a significantly minimized sequence for RFD-CD2. As a fluorescence sensor, RFD-CD2 can detect C. difficile at a concentration as low as 100 CFU/mL, making this DNAzyme an attractive molecular probe for rapid diagnosis of CDI caused by diverse strains of C. difficile.

A Highly Selective Mn(II)-Specific DNAzyme and Its Application in Intracellular Sensing.

Manganese is an essential trace element in the human body that acts as a cofactor in many enzymes and metabolisms. It is important to develop methods to detect Mn2+ in living cells. While fluorescent sensors have been very effective in detecting other metal ions, Mn2+-specific fluorescent sensors are rarely reported due to nonspecific fluorescence quenching by the paramagnetism of Mn2+ and poor selectivity against other metal ions such as Ca2+ and Mg2+. To address these issues, we herein report in vitro selection of an RNA-cleaving DNAzyme with exceptionally high selectivity for Mn2+. Through converting it into a fluorescent sensor using a catalytic beacon approach, Mn2+ sensing in immune cells and tumor cells has been achieved. The sensor is also used to monitor degradation of manganese-based nanomaterials such as MnOx in tumor cells. Therefore, this work provides an excellent tool to detect Mn2+ in biological systems and monitor the Mn2+-involved immune response and antitumor therapy.

RNA-Cleaving DNAzyme-based Amplification Strategies for Biosensing and Therapy.

Since the first discovery in 1994, DNAzymes have been extensively applied in biosensing and therapy that acting as recognition elements and signal generators with the outstanding properties of good stability, simple synthesis and high sensitivity. One subset, RNA-cleaving DNAzymes, are widely employed for diverse applications, including as reporters capable of transmitting detectable signals. In this review, we focus on the recent advances of RNA-cleaving DNAzyme-based amplification strategies in scaled-up biosensing, the application in diagnosis and disease treatment are also disscussed. We highlight two major types of RNA-cleaving DNAzyme-based amplification strategies, namely direct response amplification strategies and combinational response amplification strategies. The direct response amplification strategies refer to those based on novel designed single-stranded DNAzyme, and the combinational response amplification strategies mainly include two-part assembled DNAzyme, cascade reactions, CHA/HCR/RCA, DNA walker, CRISPR-Cas12a and aptamer. Finally, we present the current status of DNAzymes, the challenges and prospects of DNAzyme-based biosensors. This article is protected by copyright. All rights reserved.

Liquid NanoBiosensors Enable One-Pot Electrochemical Detection of Bacteria in Complex Matrices.

There is a need for point-of-care bacterial sensing and identification technologies that are rapid and simple to operate. Technologies that do not rely on growth cultures, nucleic acid amplification, step-wise reagent addition, and complex sample processing are the key for meeting this need. Herein, multiple materials technologies are integrated for overcoming the obstacles in creating rapid and one-pot bacterial sensing platforms. Liquid-infused nanoelectrodes are developed for reducing nonspecific binding on the transducer surface; bacterium-specific RNA-cleaving DNAzymes are used for bacterial identification; and redox DNA barcodes embedded into DNAzymes are used for binding-induced electrochemical signal transduction. The resultant single-step and one-pot assay demonstrates a limit-of-detection of 102 CFUmL-1 , with high specificity in identifying Escherichia coli amongst other Gram positive and negative bacteria including Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. Additionally, this assay is evaluated for analyzing 31 clinically obtained urine samples, demonstrating a clinical sensitivity of 100% and specify of 100%. When challenging this assay with nine clinical blood cultures, E. coli-positive and E. coli-negative samples can be distinguished with a probability of p<0.001.

Induced ultrasensitive electrochemical biosensor for target MDA-MB-231 cell cytoplasmic protein detection based on RNA-cleavage DNAzyme catalytic reaction

Herein, we implemented RNA-cleaving DNAzymes specific for the endogenous protein of breast cancer cells (MDA-MB -231) and programmed for electrochemical detection. Thionine-modified gold nanoparticles and modified magnetic nanoparticles are attached to the two ends of the DNAzyme molecule. The prepared probe is pulled to the surface of the electrode with the help of a magnetic field, and the signal caused by the electrochemical activity of thionine is observed on the surface of the electrode. The presence of a covalent gold nanoparticle-thionine hybrid as a highly electroactive/enhanced electrochemical label ensures a strong detection signal. After addition of the enzyme activator cofactor (MDA-MB -231 cytoplasmic cell protein), it reacts with the catalytic core of the enzyme sequence in the DNAzyme molecule and triggers the cleavage reaction in the substrate sequence of the DNAzyme molecule. During this process, the gold nanoparticle-thionine labels are detached from the probe and released into the solution. Inductive removal of gold nanoparticles leads to a decrease in the current related to the reduction of thionine on the electrode surface. The results show that this biosensor can detect this protein marker in the linear range of (1.0E-06 to 1.0E+01) pg/ml, with a detection limit (1.0129E-07 pg/ml), using differential pulse voltammetry as a measuring technique. As well as, electrochemical impedance spectroscopy (EIS).

DNAzyme-Based Microscale Thermophoresis Sensor.

Microscale thermophoresis (MST) technology has emerged as a powerful growing method in a molecular interaction study by measuring fluorescence responses of molecules inside a capillary to infrared (IR) laser heating with the benefits of rapid ratiometric measurement, separation-free, no immobilization, and low sample consumption. Combining the advantages of RNA-cleaving DNAzymes in target recognition and enzymatic catalysis and the strength of MST technology for fluorescence signaling, here, we reported a DNAzyme-based MST method for sensitive target detection. We introduced a fluorescein terminal label at the RNA-cleaving DNAzyme, and the substrate was linked to DNAzyme together with a poly-T sequence in a unimolecular design or not conjugated with DNAzyme in a bimolecular design. The presence of the cofactor activated DNAzyme to catalytically cleave the substrate, causing molecular structure alteration and significant changes in MST signals. This DNAzyme MST sensor enabled sensitively detecting activator targets Pb2+ and l-histidine, with a detection limit of 49 pM Pb2+ and 3.9 μM l-histidine. This biosensing strategy is universal and promising for wide applications.

RNA-cleaving DNAzymes for accurate biosensing and gene therapy.

RNA-cleaving DNAzymes, which are single-stranded catalytic DNA, have attracted considerable attention in bioanalysis and biomedical applications because of their high stability, high catalytic activity, easy synthesis, easy functionalization, and modification. By incorporating these DNAzymes with amplification systems, the sensing platforms can be used to detect a series of targets with high sensitivity and selectivity. In addition, these DNAyzmes possess therapeutic potential by cutting the mRNA in cells and viruses to regulate the expression of the corresponding proteins. This review systematically summarizes the applications of RNA-cleaving DNAzymes in recent years, explaining the uniqueness and superiority of RNA-cleaving DNAzymes in biosensing and gene therapy. Finally, this review extends the discussion to the challenges and perspectives of applying RNA-cleaving DNAzyme as a diagnostic and therapeutic agent. This review provides the researchers with valuable suggestions and promotes the development of DNAzymes for accurate analysis, early diagnosis, and effective therapy in medicine and their broader applications beyond biomedicine.

Activity-enhanced DNAzyme for design of label-free copper(II) biosensor.

Metal ion-driven, DNA-cleaving DNAzymes are characterised by high selectivity and specificity. However, their use for metal ion sensing remains largely unexplored due to long reaction times and poor reaction yields relative to RNA-cleaving DNAzymes and other sensing strategies. Herein we present a study demonstrating a significant rate enhancement of a copper-selective DNA cleaving DNAzyme by both polydopamine (PDA) and gold (Au) nanoparticles (NPs). PDA NPs enhance the reaction through the production of hydrogen peroxide, while for AuNPs the enhancement is aided by the presence of citrate surface moeities, both of which drive the oxidative cleavage of the substrate. A 50-fold enhancement for PDA NPs makes the combination of PDA and DNAzyme suitable for a practical application as a sensitive biosensor for Cu(II) ions. Using DNAzyme deposition onto a gold electrode followed by Polydopamine Assisted DNA Immobilisation (PADI), we achieve a cost-effective, label-free and fast (within 15 min) electrochemical biosensor with a limit of detection of 180 nmol (11 ppm), thus opening a route for the rational design of a new generation of hybrid DNAzyme-based biosensors.

Controllable DNA hybridization by host–guest complexation-mediated ligand invasion

Dynamic regulation of nucleic acid hybridization is fundamental for switchable nanostructures and controllable functionalities of nucleic acids in both material developments and biological regulations. In this work, we report a ligand-invasion pathway to regulate DNA hybridization based on host–guest interactions. We propose a concept of recognition handle as the ligand binding site to disrupt Watson–Crick base pairs and induce the direct dissociation of DNA duplex structures. Taking cucurbit[7]uril as the invading ligand and its guest molecules that are integrated into the nucleobase as recognition handles, we successfully achieve orthogonal and reversible manipulation of DNA duplex dissociation and recovery. Moreover, we further apply this approach of ligand-controlled nucleic acid hybridization for functional regulations of both the RNA-cleaving DNAzyme in test tubes and the antisense oligonucleotide in living cells. This ligand-invasion strategy establishes a general pathway toward dynamic control of nucleic acid structures and functionalities by supramolecular interactions.

Paper-based LRET sensor for the detection of total heavy rare-earth ions.

Based on the mechanism of luminescence resonance energy transfer (LRET) and using a special single strand DNA as the recognition element, a portable paper-based sensor for the accurate detection of total heavy rare-earth ions (mainly Gd3+, Tb3+ and Dy3+) concentration was proposed. The RNA cleaving-DNAzyme should recognize rare-earth ions to cleave RNA on DNA duplexes linking UCNPs and AuNPs, causing UCNPs and AuNPs to approach each other, inducing LRET, which attenuated the green upconversion luminescence (UCL) triggered by the 980 nm laser. UCL was captured by a charge-coupled device (CCD) image sensor and processed with the red-green-blue (RGB) image to quantitatively analyze heavy rare-earth ions in the samples. In the range of 5–50 μmol·L-1, the sensor has good sensitivity, with the limit of detection of 1.26 μmol L−1.

Triggered amplification of gene theranostics with high accuracy and efficacy using metallo-nanoassemblies

RNA-cleaving DNAzymes hold great promise for gene regulation, yet the low in vivo catalytic efficacy and the unavoidable nonspecific response in normal tissues hinder their practical applications. Herein, we develop a DNA prodrug-laden metallo-nanoassembly for sensitive orthogonal activation of DNAzymes to regulate disease gene with high precision and efficacy. With rich positive charges, metallo-nanoassemblies efficiently load DNA prodrugs (functional DNAzyme subunits), and thus improve drug stability and cellular uptake efficacy. Under tumor microenvironment, the cancer-specific biomarker triggers the amplified assembly of DNA prodrugs into active DNAzymes to efficiently cleave tumor-involved mRNA. Meanwhile, these glutathione-responsive metallo-nanoassemblies decompose and release metal ions as self-sufficient DNAzyme cofactors, which are accommodated into the afore-assembled DNAzymes to enhance the gene cleavage activity. Moreover, upon laser stimulus, these photothermal metallo-nanoassemblies can also remarkably promote the catalytic reaction of DNAzymes via the elevated local temperature. Such a smart delivery approach allows disease diagnosis via sensitive fluorescence and photothermal imaging, and achieves the precise and robust gene regulation with photothermal ablation as an auxiliary therapy. These results provide a new route to design programmable and activatable prodrugs for precision nanomedicine.

Ultrasensitive electrochemiluminescence biosensors based on programmable aptazyme-induced hybridization chain reaction for detecting adenosine triphosphate and quinine

Biosensors with programmability and universal applicability that simplify the detection of biomolecules are highly valuable. Herein, a programmable-component electrochemiluminescence biosensor based on an aptazyme-induced hybridization chain reaction and DNA-templated silver nanoclusters has been developed. An aptazyme-induced hybridization chain reaction system was designed, wherein aptazymes composed of a split aptamer and RNA-cleaving DNAzyme could recognize the target and regulate the activity of DNAzyme. Aptazymes activated by the target cyclically cleaved the substrate to produce short-ssDNA as converted signals, which were captured by engineering tetrahedral-DNA to trigger hybridization chain reaction, hence forming long dsDNA with nicks on the surface electrode. These dsDNA molecules were pre-programmed with cytosine (C)-rich sequences at the nick site, and silver nanoclusters could be synthesized in situ, generating an amplified electrochemiluminescence signal. Thus, the constructed multilevel-amplified adenosine triphosphate (ATP) electrochemiluminescence aptasensor showed good sensitivity, selectivity, and reconfiguration with a linear range from 0.1 pM to 10 nM and a limit of detection of 38.2 fM. Subsequently, an electrochemiluminescence biosensor for the detection of quinine was constructed using the same design, by replacing the ATP aptamer of aptazymes with the quinine aptamer. The quinine electrochemiluminescence biosensor was also significantly sensitive with a linear range from 10 pM to 1 μM and a limit of detection of 3.7 pM, indicating flexibility of the proposed strategy, which can be extended to fabricate other aptasensors.

Label-free DNAzyme for highly sensitive detection of multiple biomolecules in real samples through target-triggered catalytic cleavage reactions with auramine O's discriminated fluorescence emission.

A universal enzyme strand (E-DNA) recyclable L-histidine (L-His), melamine (MA), and cisplatin (CP) biosensor was fabricated on the basis of a target-specific RNA-cleaving DNAzyme with specific auramine O (AuO) dye instead of thioflavin T. In this strategy, the substrate strand (S-DNA) of the RNA-cleaving site was constructed as an intramolecular stem-loop structure, and a GT-rich sequence was imprisoned in the double-stranded stem which inhibits the formation of stable G-quadruplex (G4cpx) with AuO. The presence of L-His initiates a catalytic reaction for cleaving the RNA site of the S-DNA hydrolytically releasing the GT-rich portion, which subsequently combines with AuO and forms a G4cpx for enhanced fluorescent signal. The subsequent addition of MA uncoils the G4cpx to form T-MA-T dsDNA, or addition of CP unwound the G4cpx to form CP-DNA leading to an intensive decrease of AuO emission. Remarkably, the liberated L-His can ultimately cause several rounds of cleavage, and the liberated E-DNA can catalyze the subsequent reaction with the other S-DNA. The use of L-His and E-DNA repeatedly induces S-DNA cleavage and intensifies the emission signal. The results show that the proposed biosensor is extremely sensitive to L-His, MA, and CP with a detection limit of 0.98, 10, and 3.4nM respectively. To the best of our knowledge, the utilization of AuO as the G4cpx inducer and stabilizer for L-His, MA, and CP detection in real milk and urine samples has never been reported.

DNAzyme-Immobilizing Microgel Magnetic Beads Enable Rapid, Specific, Culture-Free, and Wash-Free Electrochemical Quantification of Bacteria in Untreated Urine.

Rapid, ultrasensitive, and specific detection and identification of bacteria in unprocessed clinical specimens is critically needed to enable point-of-care diagnosis of infectious diseases. However, existing systems require sample processing and/or analyte enrichment for direct bacterial analysis in clinical samples, which significantly adds to the assay time and complexity. Herein, we integrate RNA-cleaving DNAzymes specific to Escherichia coli (E. coli) and programmed for electrochemical signal transduction, multifunctional microgel magnetic beads for immobilizing the DNAzyme into a hydrated and three-dimensional scaffold, and hierarchical electrodes for ultrasensitive electrochemical readout to achieve rapid bacterial analysis in undiluted and unprocessed urine collected from symptomatic patients suspected of having urinary tract infections (UTIs). The microgel magnetic bead assay enables highly efficient conjugation and hydration of the immobilized DNAzymes, resulting in low limits-of-detection of 6 CFU/mL in buffer and 138 CFU/mL in unprocessed urine with high specificity against multiple urinary pathogens within a 1 hour assay time. The assay successfully identifies which patients are infected with E. coli as the causative organism for their UTI symptoms, indicating the clinical relevance of this assay.

Recent Advances on DNAzyme-Based Sensing.

Owing to the high sensitivity, excellent programmability, and flexible obtainment through in vitro selection, RNA-cleaving DNAzymes have attracted increasing interest in developing DNAzyme-based sensors. In this review, we summarize the recent advances on DNAzyme-based sensing applications. We initially conclude two general strategies to expand the library of DNAzmes, in vitro selection to discover new DNAzymes towards different targets of interest and chemical modifications to endue the existing DNAzymes with new function or properties. We then discuss the recent applications of DNAzyme-based sensors for the detection of a variety of important biomoleucles both in vitro and in vivo. Finally, perspectives on the challenges and future directions in the development of DNAzyme-based sensors are provided.

Selection of M2+ -Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine.

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+ ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dUga TP) and 5-aminoallyl-2'-dCTP (dCaa TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

Primer Exchange Reaction-Synthesized DNAzyme for the Sensitive Determination of an Oral Cancer Protein Biomarker

A novel biosensing method is reported for the fluorescence determination of an oral cancer protein biomarker using primer exchange reaction (PER)-synthesized DNAzyme. Specifically, the target protein is captured by antibody-functionalized magnetic beads and recruits antibody-nucleic acid probes through immunoreactions to form sandwich-like complexes. After magnetic separation, the nucleic acid part of the antibody-nucleic acid probe participates in the PER as a catalytic hairpin, mediating the extension of primers. The extended primer forms the complete sequence of RNA-cleaving DNAzyme to effectively catalyze the cleavage of molecular beacons and produce a significantly amplified fluorescence signal. Taking interleukin 6 as a model biomarker, the method allows quantitative determination of the target in a wide linear range from 5 fg/mL to 500 pg/mL with a detection limit of 2.3 fg/mL and shows desirable specificity and usability in biological samples. More importantly, the complete DNAzyme is synthesized by the target-powered PER, which not only ensures the detection specificity, but also enables the decrease of background signals, thus greatly improving the sensitivity. Therefore, the method may provide a valuable tool for the determination of oral cancer protein biomarkers and may expand applications of DNAzyme in biosensing.