Rapid and specific detection of thiabendazole: enzymatic digestion-enabled fluorescent aptasensor.

Thiabendazole, a widely used broad-spectrum fungicide in agriculture, poses risks to human health. To monitor its presence in water, we propose a fluorescent aptasensor utilizing Escherichia coli exonuclease I (Exo I). The findings demonstrate a linear correlation between thiabendazole concentrations and digestion percentage, with a detection limit (LOD) exceeding 1 µM and a determination coefficient (R2) of 0.959. This aptamer-based fluorescence spectroscopy detection system holds promise for a rapid, specific, and sensitive analysis of thiabendazole in environmental waters and food matrices.

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling.

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63- reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63- for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63- and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

Dual-mode aptasensor based on P-CeO2NR@Mxene and exonuclease I-assisted target recycling for malachite green detection.

Malachite green (MG) has been illicitly employed in aquaculture as a parasiticide, however, its teratogenic and carcinogenic effects pose a significant human health threat. Herein, a dual-mode colorimetric and electrochemical aptasensor was fabricated for MG detection, capitalizing on the robust catalytic and peroxidase-like activity of P-CeO2NR@Mxene and good capture efficiency of a tetrahedral DNA nanostructure (TDN) designed with multiple aptamers (m-TDN). P-CeO2NR@Mxene-modified complementary DNA (cDNA) served as both colorimetric and electrochemical probe. m-TDN was attached to AuE to capture MG and P-CeO2NR@Mxene/cDNA. The superior aptamer and MG binding to cDNA regulated signals and enabled precise MG quantification. The further introduced Exo I enabled aptamer hydrolysis, releasing MG for further binding rounds, allowing target recycling amplification. Under the optimal conditions, the aptasensor reached an impressively low detection limit 95.4 pM in colorimetric mode and 83.6 fM in electrochemical mode. We believe this dual-mode approach holds promise for veterinary drug residue detection.

Arbitrary Digital DNA Computing: A Programmable Molecular Perceptron Driven by Lambda Exonuclease for Lighting up Concatenated Circuits.

DNA circuits, as a type of biochemical system, have the capability to synchronize the perception of molecular information with a chemical reaction response and directly process the molecular characteristic information in biological activities, making them a crucial area in molecular digital computing and smart bioanalytical applications. Instead of cascading logic gates, the traditional research approach achieves multiple logic operations which limits the scalability of DNA circuits and increases the development costs. Based on the interface reaction mechanism of Lambda exonuclease, the molecular perceptron proposed in this study, with the need for only adjusting weight and bias parameters to alter the corresponding logic expressions, enhances the versatility of the molecular circuits. We also establish a mathematical model and an improved heuristic algorithm for solving weights and bias parameters for arbitrary logic operations. The simulation and FRET experiment results of a series of logic operations demonstrate the universality of molecular perceptron. We hope the proposed molecular perceptron can introduce a new design paradigm for molecular circuits, fostering innovation and development in biomedical research related to biosensing, targeted therapy, and nanomachines.

Crystal structure of the 3'→5' exonuclease from Methanocaldococcus jannaschii.

RecJ exonucleases are members of the DHH phosphodiesterase family ancestors of eukaryotic Cdc45, the key component of the CMG (Cdc45-MCM-GINS) complex at the replication fork. They are involved in DNA replication and repair, RNA maturation and Okazaki fragment degradation. Bacterial RecJs resect 5'-end ssDNA. Conversely, archaeal RecJs are more versatile being able to hydrolyse in both directions and acting on ssDNA as well as on RNA. In Methanocaldococcus jannaschii two RecJs were previously characterized: RecJ1 is a 5'→3' DNA exonuclease, MjaRecJ2 works only on 3'-end DNA/RNA with a preference for RNA. Here, I present the crystal structure of MjaRecJ2, solved at a resolution of 2.8 Å, compare it with the other RecJ structures, in particular the 5'→3' TkoGAN and the bidirectional PfuRecJ, and discuss its characteristics in light of the more recent knowledge on RecJs. This work adds new structural data that might improve the knowledge of these class of proteins.

The dual ECL signal enhancement strategy of Pd nanoparticles attached covalent organic frameworks and exonuclease cycling reaction for the ultrasensitive detection of progesterone.

Nowadays, novel and efficient signal amplification strategy in electrochemiluminescence (ECL) platform is urgently needed to enhance the sensitivity of biosensor. In this work, the dual ECL signal enhancement strategy was constructed by the interactions of Pd nanoparticles attached covalent organic frameworks (Pd NPs@COFs) with tris (bipyridine) ruthenium (RuP) and Exonuclease III (Exo.III) cycle reaction. Within this strategy, the COFs composite was generated from the covalent reaction between 2-nitro-1,4-phenylenediamine (NPD) and trialdehyde phloroglucinol (Tp), and then animated by glutamate (Glu) to attach the Pd NPs. Next, the "signal on" ECL biosensor was constructed by the coordination assembly of thiolation capture DNA (cDNA) onto the Pd NPs@COFs modified electrode. After the aptamer recognition of progesterone (P4) with hairpin DNA 1 (HP1), the Exo. III cycle reaction was initiated with HP2 to generate free DNA, which hybridized with cDNA to form double-stranded DNA (dsDNA). For that, the RuP was embedded into the groove of dsDNA and achieved the ultrasensitive detection of P4 with a lower limit of detection (LOD) down to 0.45 pM, as well as the excellent selectivity and stability. This work expands the COFs-based materials application in ECL signal amplification and valuable DNA cyclic reaction in biochemical testing field.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

SERS-electrochemical dual-mode detection of microRNA on same interface assisted by exonuclease III signal transformation.

Dual-mode sensing has attracted more attentions which provide more accurate and reliable approach of cancer-related biomarkers. Herein, we developed a novel SERS/electrochemical dual-mode biosensor for miRNA 21 detection based on Exo III-assisted signal transformation. Firstly, the Au NPs were deposited on electrode as SERS substrate and Mn3O4/S4(DNA signal strand) was modified on Au NPs/S5 by the DNA strands S5-S4 pairing principle as hydrogen peroxide catalyst, leading to an obviously high DPV electrical signal without Raman signal. Subsequently, the presence of miRNA 21 will activate the Mn3O4/S4 to be decomposed under exonuclease III-assisted process, then the S3' chains modified with Raman molecular Cy3(Cy3-S3') is continuously connected to the Au NPs/S5 by DNA stands S5-S3' pairing principle, leading to the Raman signal response and DPV signal reduction. The biosensor shows good linear calibration curves of both SERS and electrochemical sensing modes with the detection limit of 3.98 × 10-3 nM and 6.89 × 10-5 nM, respectively. This work finds an ingenious mode for dual detection of microRNA on a same interface, which opens a new strategy for SERS and electrochemical analysis.

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

Facile and sensitive detection of mercury ions based on fluorescent structure-switching aptamer probe and exonuclease â ¢-assisted signal amplification.

Hg2+ is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg2+-T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg2+ is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg2+-T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg2+. Under the optimal reaction conditions, there is an excellent linear relationship between Hg2+ concentration and fluorescence intensity in the range of 7.5-200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg2+ in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection.

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L-1 to µmol L-1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub.

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

Ratiometric fluorescent detection of miRNA-21 via pH-regulated adsorption of DNA on polymer dots and exonuclease III-assisted amplification.

This work proposed a simple and sensitive method for ratiometric fluorescent detection of nucleic acids via pH-dependent adsorption of dye-labeled DNA on polymer dots. The polymer dots (Pdots) could be conveniently prepared with nanoprecipitation in water. The mixture of dye-labeled DNA and Pdots at neutral pH showed the fluorescence of Pdots, while the adsorption of dye-labeled DNA on Pdots at acidic pH led to fluorescence resonance energy transfer from the Pdots to dye, and thus the fluorescence of dye. As a result, a signal switch could be designed for the detection of nucleic acids complementary to the DNA after combining with exonuclease III-assisted digestion of DNA. Using miRNA-21 as a target model and Cy3-labeled DNA as the probe, the hybridization of DNA with miRNA-21 provided active sites for EXO III, which released the hybridized miRNA-21 for cyclic digestion of DNA, and thus decreased the adsorption of Cy3-labeled DNA on Pdots and the fluorescence of Cy3. The ratio of fluorescent intensity of Pdots to Cy3 showed linear increase upon increasing miRNA-21 concentration ranging from 0.01 to 2.5 nM. The limit of detection at 3σ was 4.0 pM. The excellent performance and good extendability of the proposed strategy demonstrated its promising application in bioanalysis.

A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 gene via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies.

A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 (ORAOV1) gene was constructed via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. Capture DNA with a sulfhydryl group at its 3' terminus was modified onto the surface of a bare gold electrode via an Au-S bond. Assisted DNA hybridized with basal DNA to form hybrid DNA in advance, and ORAOV1 gene hybridized continuously with such a hybrid DNA from the other terminus to construct intact double-stranded DNA. Exonuclease III digested basal DNA in such intact double-stranded DNA specifically, and both ORAOV1 gene and assisted DNA were released into solution. ORAOV1 gene induced another intact double-stranded DNA digestion for target recycling, while assisted DNA hybridized with the capture DNA to form double-stranded DNA on the modified electrode surface. Unhybridized capture DNA on the modified electrode surface was hydrolyzed by RecJf exonuclease to reduce the background electrochemical signal. The 3' terminus of double-stranded DNA on the modified electrode surface was prolongated to be guanine-rich oligonucleotides under the catalysis of terminal deoxynucleotidyl transferase. In the presence of K+ ions, hemin adsorbed onto guanine-rich oligonucleotides to construct a G-quadruplex/hemin complex with a large steric hindrance effect to efficiently avoid the charge transfer of the [Fe(CN)6]3-/4- probe toward the electrode surface. The electrochemical impedance value was increased significantly after the addition of ORAOV1 gene via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. The electrochemical impedance value was linearly related to the logarithmic concentration of ORAOV1 gene in the range from 0.05 fM to 20 pM, and the detection limit of ORAOV1 gene was low to 0.019 fM. This biosensor was used to detect ORAOV1 gene in complicated human saliva samples with satisfactory results.

Construction of Molecular Transporter Based on DNA Structure Regulation.

This study aims to build a molecular transporter machine that is based on the microstructure regulation of DNA triplets, which can automatically search, load, target delivery, and unload target protein molecules. The design of the molecular transporter includes: (1) a DNA triplet, which can recognize and load of the target protein; (2) a similar DNA triplet realizing the target transport; and (3) the signal-indicating DNA, which is connected at the target destination to achieve fixation of the target protein at the target destination. The molecular transporter machine would provide research practice and theoretical guidance for the development of DNA-based molecular machines.

Highly sensitive and efficient fluorescent sensing for Hg2+ detection based on triple-helix molecular switch and exonuclease III-assisted amplification.

Here, a novel fluorescent sensing for simple, highly sensitive and efficient detection of Hg2+ was developed as joint result of triple-helix molecular switch (THMS) and exonuclease III (Exo III)-assisted signal amplification. In this study, the special structure of THMS was used to realize efficient fluorescence quenching and excellent signal unit transformation to complete the output of signal FAM. In the absence of Hg2+, hairpin probe (HP) containing thymine-rich (T-rich) ssDNA strand can induce the dissociation of the THMS, causing FAM far away from BHQ1 and increasing fluorescence intensity. Nevertheless, Hg2+ could bind to the thymine (T) base to form the dsDNA with T-Hg2+-T structure that stimulates Exo III to digest it from the blunt 3'-terminus to 5'-terminus, causing Hg2+ to be released from the dsDNA. The released Hg2+ could initiate the next cycling, allowing a large number of hairpin probes to be cleaved by Exo III to form ssDNA. These ssDNA could inhibit the switch dissociation of THMS, causing a dramatic decrease in the fluorescence signal. This allowed for the highly sensitive detection of Hg2+ at concentrations as low as 1.04 pM. In addition, the sensing showed a linear detection range of 0.01-50 nM and was used for the assay of Hg2+ in real samples of Xiangjiang river water and tap water. These results showed that the provided fluorescent sensing has a good application prospect in environmental and food monitoring.

Target-Mediated 5'-Exonuclease Digestion of DNA Aptamers with RecJ to Modulate Rolling Circle Amplification for Biosensing.

We report a new method for biosensing based on the target-mediated resistance of DNA aptamers against 5'-exonuclease digestion, allowing them to act as primers for rolling circle amplification (RCA). A target-bound DNA strand containing an aptamer region on the 5'-end and a primer region on the 3'-end is protected from 5'-exonuclease digestion by RecJ exonuclease in a target-dependent manner. As the protected aptamer is at the 5'-end, the exposed primer on the 3'-end can participate in RCA in the presence of a circular template to generate a turn-on sensor. Without target, RecJ digests the primer and prevents RCA from occurring, allowing quantitative fluorescence detection of both thrombin, a protein, and ochratoxin A (OTA), a small molecule, at picomolar concentrations.

5'-Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A.

Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.

Could ß-Lactam Antibiotics Block Humoral Immunity?

It has been reported that treatment with ß-lactam antibiotics induces leukopenia and candidemia, worsens the clinical response to anticancer immunotherapy and decreases immune response to vaccination. ß-lactamases can cleave ß-lactam antibiotics by blocking their activity. Two distincts superfamilies of ß-lactamases are described, the serine ß-lactamases and the zinc ion dependent metallo-ß-lactamases. In human, 18 metallo-ß-lactamases encoding genes (hMBLs) have been identified. While the physiological role of most of them remains unknown, it is well established that the SNM1A, B and C proteins are involved in DNA repair. The SNM1C/Artemis protein is precisely associated in the V(D)J segments rearrangement, that leads to immunoglobulin (Ig) and T-cell receptor variable regions, which have a crucial role in the immune response. Thus in humans, SNM1C/Artemis mutation is associated with severe combined immunodeficiency characterized by hypogammaglobulinemia deficient cellular immunity and opportunistic infections. While catalytic site of hMBLs and especially that of the SNM1 family is highly conserved, in vitro studies showed that some ß-lactam antibiotics, and precisely third generation of cephalosporin and ampicillin, inhibit the metallo-ß-lactamase proteins SNM1A & B and the SNM1C/Artemis protein complex. By analogy, the question arises as to whether ß-lactam antibiotics can block the SNM1C/Artemis protein in humans inducing transient immunodeficiency. We reviewed here the literature data supporting this hypothesis based on in silico, in vitro and in vivo evidences. Understanding the impact of ß-lactam antibiotics on the immune cell will offer new therapeutic clues and new clinical approaches in oncology, immunology, and infectious diseases.

FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.

The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain.