Nucleic acid-functionalized upconversion luminescence biosensor based on strand displacement-mediated signal amplification for the detection of trivalent chromium ions.

BACKGROUND: Rapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr3+) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs). RESULTS: The sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH2-Capture probe DNA (NH2-Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH2-Cp. The DNAzyme activated by Cr3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH2-Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr3+ by the change of I585/I545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5-75 nM and a detection limit of 0.135 nM for Cr3+. SIGNIFICANCE AND NOVELTY: Firstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids.

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria.

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Target-promoted activation of DNAzyme walker for in situ assembly of hemin/G-quadruplex nanowires enable ultrasensitive and label-free electrochemical myocardial microRNA assay.

The concentration elevation of myocardial microRNA (miRNA) biomarker is associated with the pathogenic process of acute myocardial infarction (AMI), and sensitive quantification of myocardial miRNA biomarker plays an important role for early AMI diagnosis and its treatment. In response, this work describes an ultrasensitive and non-label electrochemical biosensor for the assay of myocardial miRNA based on cascade signal amplifications integrated by DNAzyme walker and hemin/G-quadruplex nanowires. The DNAzyme walker is activated by presence of target miRNAs to move along the electrode surface to cyclically cleave the substrate hairpins to release G-quadruplex segments, which further trigger the in situ formation of many hemin/G-quadruplex nanowires. The large amounts of hemin intercalated into the DNA nanowires subsequently generate drastically magnified electrochemical current signals for highly sensitive label-free assay of myocardial miRNAs down to 15.7 fM within dynamic range of 100 fM to 10 nM. Such a biosensor also has high selectivity and can monitor myocardial miRNAs in diluted serums at low levels, providing a sensitive and reliable platform for diagnosing infarct-associated cardiovascular diseases.

Rapid and direct detection of m6A methylation by DNAzyme-based and smartphone-assisted electrochemical biosensor.

m6A methylation detection is crucial for understanding RNA functions, revealing disease mechanisms, guiding drug development and advancing epigenetics research. Nevertheless, high-throughput sequencing and liquid chromatography-based traditional methods still face challenges to rapid and direct detection of m6A methylation. Here we report a DNAzyme-based and smartphone-assisted electrochemical biosensor for rapid detection of m6A. We initially identified m6A methylation-sensitive DNAzyme mutants through site mutation screening. These mutants were then combined with tetrahedral DNA to modify the electrodes, creating a 3D sensing interface. The detection of m6A was accomplished by using DNAzyme to capture and cleave the m6A sequence. The electrochemical biosensor detected the m6A sequence at nanomolar concentrations with a low detection limit of 0.69 nM and a wide detection range from 10 to 104 nM within 60 min. As a proof of concept, the 3'-UTR sequence of rice was selected as the m6A analyte. Combined with a smartphone, our biosensor shows good specificity, sensitivity, and easy-to-perform features, which indicates great prospects in the field of RNA modification detection and epigenetic analysis.

A Methyl-Engineered DNAzyme for Endogenous Alkyltransferase Monitoring and Self-Sufficient Gene Regulation.

The on-demand gene regulation is crucial for extensively exploring specific gene functions and developing personalized gene therapeutics, which shows great promise in precision medicines. Although some nucleic acid-based gene regulatory tools (antisense oligonucleotides and small interfering RNAs) are devised for achieving on-demand activation, the introduction of chemical modifications may cause undesired side effects, thereby impairing the gene regulatory efficacy. Herein, a methyl-engineered DNAzyme (MeDz) is developed for the visualization of endogenous alkyltransferase (AGT) and the simultaneous self-sufficiently on-demand gene regulation. The catalytic activity of DNAzyme can be efficiently blocked by O6-methylguanine (O6MeG) modification and specifically restored via the AGT-mediated DNA-repairing pathway. This simply designed MeDz is demonstrated to reveal AGT of varying expression levels in different cells, opening the possibility to explore the AGT-related biological processes. Moreover, the AGT-guided MeDz exhibits cell-selective regulation on the human early growth response-1 (EGR-1) gene, with efficient gene repression in breast cancer cells and low effectiveness in normal cells. The proposed MeDz offers an attractive strategy for on-demand gene regulation, displaying great potential in biomedical applications.

A target-triggered colorimetric sensor for ultrasensitive detection of miRNAs based on self-powered three-dimensional DNA walker.

MicroRNAs (miRNAs) play an important role in the process of heart failure (HF) and are emerging biomarkers that can be used for the auxiliary diagnosis of HF. However, it is very challenging to accurately analyze the expression levels of trace miRNAs in complex clinical samples. Here, we developed an enzyme-free colorimetric sensor for the ultrasensitive detection of miRNA-423-5p (HF-associated miRNA) based on three-dimensional DNA walkers constructed from functional nucleic acids and gold nanoparticles (AuNPs). DNAzyme with cleavage activity was specifically activated by miRNA-423-5p to sustainably cleave the substrate, thereby releasing the trigger sequence to initiate the subsequent mismatched catalytic hairpin assembly (MCHA) cycle. Then, as the MCHA cycle proceeded to continuously expose the G-quadruplex (GQ) sequence, the sequence bound with hemin to form a large amount of GQ/hemin DNAzyme on the surface of the AuNPs, which rapidly catalyzed the chromogenic oxidation of 3,3',5,5'-tetramethylbenzidine to yield an amplified colorimetric signal readout. The colorimetric sensor exhibited an ultralow detection limit (32 fM), showed excellent specificity and performed well in serum samples. The sensor was applied to detect miRNA-423-5p in clinical plasma samples from healthy individuals and HF patients, and the results revealed its good clinical application in HF diagnosis. Thus, the developed colorimetric sensor provides a convenient detection tool for early screening and diagnosis of HF, as well as for pathophysiological studies.

3D DNAzyme Motor Nanodevice With Self-Powered FRET Amplifier and Self-Supplied H2O2 for Enhancing Human Neutrophil Elastase Profiling and Chemodynamic Therapy in Lung Tumor.

The development of theragnostic nanosystems integrating FRET (fluorescence resonance energy transfer) imaging and chemodynamic therapy (CDT) for accurate diagnosis and effective treatment of lung tumors is still a big challenge. Herein, a peptide-assembled 3D DNAzyme motor nanodevice is engineered for a self-powered FRET amplifier profiling human neutrophil elastase (HNE) and self-supplied H2O2 enhancing CDT. The nanodevice is prepared by depositing AuNPs on ZIF-8, in which ZIF-8 co-loaded the lysosomal targeting peptide-modified copper peroxides (PCPs) and hairpins (H1, H2, and H3), AuNPs are co-labeled by DNAzyme-peptide (DP) conjugate and H3. In the tumor micro-environment, HNE driven 3D DNAzyme walker followed by an exponential amplification constructed by a synergistic cross-activation between hybridization chain reaction and DNAzyme, generating a self-powered FRET amplifier. The FRET amplifier specifically measures HNE with a sensitivity of 0.026 pM, and successfully images exogenous HNE in living cells and monitors HNE in mouse models. Moreover, the PCPs can target lysosomes, reducing lysosome escape. The self-supplying H2O2 undertaken by PCPs improves the Cu (II)-catalyzed Fenton-like reaction, effectively causing cell apoptosis to inhibit tumor growth. Significantly, the nanodevice successfully screens inhibitors and discriminates the HNE level in normal and lung cancer tissues, suggesting that the nanodevice provides an effective tool for the diagnosis and treatment of lung tumors.

Proximity ligation-triggered DNAzyme for selective fluorescent aptasensing of methicillin-resistant Staphylococcus aureus.

There is an urgent need for novel strategies to accurately and reliably detect pathogenic bacteria to address the global epidemic of antibiotic resistance. This study proposes an innovative approach combining dual aptamer-based target recognition and proximity ligation assay (PLA) triggered DNAzyme recycling cleavage. This method allows for the precise identification and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA). The fluorescence probe labeled with a fluorophore is modified on gold nanoparticles (AuNPs), resulting in the quenching of the fluorescent signal by the AuNPs. The interaction between MRSA and two aptamers leads to forming a Mg2+-dependent DNAzyme. The DNAzyme cleaves the fluorescence probe, causing the fluorescent fragment to detach from the surface of the AuNPs, in which the quenched fluorescence signal in the fluorescence probe reappears. The DNAzyme-assisted cleavage and rebinding process generates a processive strolling along the surface track of AuNPs. Consequently, the fluorescence intensity experiences a substantial recovery. A strong linear correlation is observed between the fluorescence intensity and MRSA concentration within 50 cfu/mL to 106 cfu/mL. We believe that implementing the novel integrated strategy will broaden the range of bacterial detection methods in the battlefield environment and stimulate the creation of potential new drugs in the future.

Functional DNA-Zn2+ coordination nanospheres for sensitive imaging of 8-oxyguanine DNA glycosylase activity in living cells.

Sensitive monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn2+, where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn2+. The released Zn2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn2+, DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical studies.

Fluorescence assay for aflatoxin B1 based on aptamer-binding triggered DNAzyme activity.

As a kind of mycotoxin, aflatoxin B1 (AFB1), which is often found in agricultural products, poses a threat to human health. Developing a simple sensitive method for AFB1 detection is in great demand. Here, we reported an aptamer-based fluorescence assay for AFB1 detection by using DNAzyme to generate and amplify a signal. We redesigned a pair of DNA sequences, which originated from the anti-AFB1 aptamer and RNA-cleaving DNAzyme 10-23. In the absence of AFB1, the aptamer hybridized with the region of the substrate-binding arm of the DNAzyme, inhibiting the activity of the DNAzyme. In the presence of AFB1, the binding of AFB1 to the aptamer led to the displacement of the DNAzyme from the aptamer. The substrate-binding arm was unblocked, and the activity of the DNAzyme was restored for the hydrolysis of the fluorophore and quencher-labeled substrate, causing a significant fluorescence increase. This assay could detect AFB1 in the dynamic range from 0.98 to 2000 nmol/L with high selectivity, and the detection limit was 0.98 nmol/L. Moreover, the assay was able to detect AFB1 in a complex sample matrix. This work provides a useful tool for the analysis of AFB1.

Enhancing Sensitivity in Nucleic Acid Detection via Collaborative Multiple Catalytic Cores in DNAzyme Nanomachines.

We introduce a novel multicore DNA nanomachine (MDNM), utilizing four binary DNAzymes for nucleic acid detection without the need for a preamplification step. This innovation remarkably yields a reduction in limit of detection (LOD), over 5-fold, as compared to single-core systems. This reduces the required test time, highlighting the potential of MDNM in advancing nucleic acid detection.

Hydrogen Sulfide-Triggered Artificial DNAzyme Switches for Precise Manipulation of Cellular Functions.

The development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H2S). To address this, we present the design of Gas signaling molecule-Responsive Artificial DNAzyme-based Switches (GRAS) to manipulate cellular functions via H2S-sensitive synthetic DNAzymes. By incorporating stimuli-responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H2S-responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H2S-reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H2S-induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H2S-triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering.

Sensitive and Enzyme-Free Pseudomonas aeruginosa Detection and Isolation via DNAzyme Cascade Triggered DNA Tweezer.

Effective isolation and sensitive detection of Pseudomonas aeruginosa (P. aeruginosa) is crucial for the early diagnosis and prognosis of various diseases, such as urinary tract infections. However, efficient isolation and simultaneous detection of P. aeruginosa remains a huge challenge. Herein, we depict a novel fluorescence assay for sensitive, enzyme-free detection of P. aeruginosa by integrating DNAzyme cascade-induced DNA tweezers and magnetic nanoparticles (MNPs)-based separation. The capture probe@MNPs is capable of accurately identifying target bacteria and transporting the bacteria signal to nucleic acid signals. Based on the DNAzyme cascade-induced DNA tweezers, the nucleic acid signals are extensively amplified, endowing the method with a high sensitivity and a low detection limit of 1 cfu/mL. In addition, the method also exhibits a wide detection of six orders of magnitudes. The proposed method could be extended to other bacteria detection by simply changing the aptamer sequence. Taking the merit of the high sensitivity, greatly minimized detection time (less than 1.5 h), enzyme-free characteristics, and stability, the proposed method could be potentially applied to diagnosing and preventing diseases caused by pathogenic bacteria.

Detection and simultaneous imaging of acrylamide, miR-21 and miR-221 based on multicolor aggregation-induced emission nanoparticles and DNAzyme walker.

Acrylamide (AA) in heat-processed foods has emerged as a global health problem, mainly carcinogenic, neurotoxic, and reproductive toxicity, and an increasing number of researchers have delved into elucidating its toxicological mechanisms. Studies have demonstrated that exposure of HepG2 by AA in a range of concentrations can induce the upregulation of miR-21 and miR-221. Monitoring the response of intracellular miRNAs can play an important role in unraveling the mechanisms of AA toxicity. Here, multicolor aggregation induced emission nano particle (AIENP) probes were constructed from three AIE dyes for simultaneous imaging of intracellular AA and AA-induced miR-21/miR-221 by combining the recognition function of AA aptamers and the signal amplification of a DNAzyme walker. The surface of these nanoparticles contains carboxyl groups, facilitating their linkage to a substrate chain modified with a fluorescent quencher group via an amide reaction. Optimization experiments were conducted to determine the optimal substrate-to-DNAzyme ratio, confirming its efficacy as a walker for signal amplification. Sensitive detection of AA, miR-21 and miR-221 was achieved in extracellular medium, with detection limits of 0.112 nM for AA, 0.007 pM and 0.003 pM for miR-21 and miR-221, respectively, demonstrating excellent selectivity. Intracellularly, ZIF-8 structure collapsed, releasing Zn2+, activating DNAzyme cleavage activity, and the fluorescence of multicolor AIENPs within HepG2 cells gradually recovered with increasing stimulation time (0-12 h) and concentrations of AA (0-500 µM). This dynamic response unveiled the relationship between AA exposure and miR-21/miR-221 expression, further validating the carcinogenicity of AA.

Simple and sensitive detection of Pseudomonas aeruginosa in neonatal infection based on a both-end blocked peroxidase-mimicking DNAzyme.

Developing a simple and highly sensitive approach for Pseudomonas aeruginosa (P. aeruginosa) detection is crucial, as it is closely associated with various disorders, such as newborn infections. Nevertheless, few of techniques have the capability to accurately identify P. aeruginosa with a high level of sensitivity and significantly improved stability. The employment of the both-end blocked peroxidase-mimicking DNAzyme significantly diminished the interferences from background signals, so conferring the approach with a high degree of selectivity and reproducibility. The proposed method is demonstrated with exceptional discernment capacity in differentiating interfering microorganisms. The simplicity, elevated sensitivity and high discerning capability make the method a highly promising alternative instrument for pathogenic bacteria detection.

This research presents a novel method for detecting P. aeruginosa using a combination of a simple molecular beacon (MB), duplex-specific nuclease (DSN), and both-end blocked peroxidase-mimicking DNAzyme. The MB probe utilized in this method can be shielded from DSN hydrolysis without requiring any additional modifications by regulating the number of stem bases to five. This assay is simple yet precise in its ability to quantitatively detect P. aeruginosa with a high level of sensitivity and specificity. In addition, the beacon enabled the identification of P. aeruginosa without the need for labeling, exhibiting a higher sensitivity over the conventional hairpin fluorescence beacon based methods.

Functionalized primer initiated signal cycles and personal glucose meter for sensitive and portable miRNA analysis.

MicroRNA (miRNA) has garnered considerable attention due to its diagnostic capabilities, such as in hypoxic cognitive impairment and cancers. However, the existing miRNA detection methods are commonly criticized for the drawbacks of low sensitivity and false-positive detection derived from interfering molecules. Here, we provide a novel, sensitive and portable method for miRNA detection by combining target identification based cyclization of padlocks, immobilized primer-based signal amplification and a personal glucose meter. The proposed method exhibits several advantages, including precise identification of specific sites, exceptional sensitivity and instrument-free feature. These attributes hold great promise for the diagnosis and clinical investigation of various diseases, such as cancer and hypoxic cognitive impairment, enabling a deeper understanding of their pathological and physiological aspects.

With miRNA-155 as detective target, the feasibility of the method has been demonstrated. The padlock sequences are cyclized by miRNA-155, which subsequently hybridize with primer sequence that is immobilized on the surface of a 96-well plate, and the interfering molecules are removed. This DNA polymerase triggers a chain extension process on the terminus of primer sequence, activating DNAzyme based cleavage. Consequently, a multitude of linker sequences are generated to facilitate the formation of the 'e/linker/f/sucrase' on magnetic bead, thereby enabling the catalysis of sucrose into glucose. This enzymatic reaction may be identified and measured using the personal glucose meter.

Colorimetric detection of furin based on enhanced catalytic activity of G-quadruplex/hemin DNAzyme.

BACKGROUND: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. RESULTS: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SIGNIFICANCE: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades.

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

Regulating nucleic acid catalysis using active droplets.

Cells use transient membraneless organelles to regulate biological reaction networks. For example, stress granules selectively store mRNA to downregulate protein expression in response to heat or oxidative stress. Models mimicking this active behavior should be established to better understand in vivo regulation involving compartmentalization. Here we use active, complex coacervate droplets as a model for membraneless organelles to spatiotemporally control the activity of a catalytic DNA (DNAzyme). Upon partitioning into these peptide-RNA droplets, the DNAzyme unfolds and loses its ability to catalyze the cleavage of a nucleic acid strand. We can transiently pause the DNAzyme activity upon inducing droplet formation with fuel. After fuel consumption, the DNAzyme activity autonomously restarts. We envision this system could be used to up and downregulate multiple reactions in a network, helping understand the complexity of a cell's pathways. By creating a network where the DNAzyme could reciprocally regulate the droplet properties, we would have a powerful tool for engineering synthetic cells.

Dual-mode detection of glucose based on pistol-like DNAzyme-mediated exonuclease-assisted signal cycle.

Detecting glucose accurately and sensitively from clinical samples like tears and saliva is still difficult. We have created a sensor that can detect glucose with high sensitivity and accuracy by combining the use of glucose oxidase (GOx) to catalyze glucose, a pistol-like DNAzyme (PLDz) to transform the signal, gold nanoparticles (AuNPs) to enhance the optical properties and the exonuclease-III (Exo-III) to amplify the signal. As a result, the proposed method exhibits a low detection limit of 7.5 pM and a wide detection range covering seven orders of magnitude. The suggested dual-mode strategy provides a sensitive, precise and specific detection method for glucose. Another advantage is that the dual-mode technique significantly improves the precision and consistency of the measurements, demonstrating its immense potential for use in biomedical research and clinical diagnostics.

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