Double-stranded Nucleic Acids Research Articles

Targeting double-stranded nucleic acids using the λExo-pDNA system.

Targeting double-stranded nucleic acids using the λExo-pDNA system.

Bacteriophage λ exonuclease and a 5'-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids.

Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustered regularly interspaced short palindromic repeats and Cas systems rely on protospacer-adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesired off-target effects. Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5'-phosphorylated single-stranded DNA (pDNA) to complementary regions on dsDNA and DNA-RNA duplexes, without the need for a PAM-like motif. Upon binding, the λExo-pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg2+. This process is sensitive to mismatches within a wide range of the pDNA-binding region, resulting in exceptional sequence specificity and reduced off-target effects in various applications. The absence of a requirement for a specific motif such as a PAM sequence greatly broadens the range of targets. We demonstrate that the λExo-pDNA system is a versatile tool for molecular diagnostics, DNA computing and gene imaging applications.

Crystal structure and nucleic acid binding mode of CPV NSP9: implications for viroplasm in Reovirales.

Cytoplasmic polyhedrosis viruses (CPVs), like other members of the order Reovirales, produce viroplasms, hubs of viral assembly that shield them from host immunity. Our study investigates the potential role of NSP9, a nucleic acid-binding non-structural protein encoded by CPVs, in viroplasm biogenesis. We determined the crystal structure of the NSP9 core (NSP9ΔC), which shows a dimeric organization topologically similar to the P9-1 homodimers of plant reoviruses. The disordered C-terminal region of NSP9 facilitates oligomerization but is dispensable for nucleic acid binding. NSP9 robustly binds to single- and double-stranded nucleic acids, regardless of RNA or DNA origin. Mutagenesis studies further confirmed that the dimeric form of NSP9 is critical for nucleic acid binding due to positively charged residues that form a tunnel during homodimerization. Gel migration assays reveal a unique nucleic acid binding pattern, with the sequential appearance of two distinct complexes dependent on protein concentration. The similar gel migration pattern shared by NSP9 and rotavirus NSP3, coupled with its structural resemblance to P9-1, hints at a potential role in translational regulation or viral genome packaging, which may be linked to viroplasm. This study advances our understanding of viroplasm biogenesis and Reovirales replication, providing insights into potential antiviral drug targets.

Increased levels of circulating cell-free double-stranded nucleic acids in the plasma of glioblastoma patients.

Circulating cell-free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell-free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double-stranded nucleic acids. Fluorescent staining of EVs isolated from cell-conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs' membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma-derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.

Motif-VI loop acts as a nucleotide valve in the West Nile Virus NS3 Helicase.

The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Piand ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471Nand D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development.

Liposomes-Encapsulating Double-Stranded Nucleic Acid (Poly I:C) for Head and Neck Cancer Treatment.

Polyriboinosinic acid-polyribocytidylic acid (Poly I:C) serves as a synthetic mimic of viral double-stranded dsRNA, capable of inducing apoptosis in numerous cancer cells. Despite its potential, therapeutic benefits, the application of Poly I:C has been hindered by concerns regarding toxicity, stability, enzymatic degradation, and undue immune stimulation, leading to autoimmune disorders. To address these challenges, encapsulation of antitumor drugs within delivery systems such as cationic liposomes is often employed to enhance their efficacy while minimizing dosages. In this study, we investigated the potential of cationic liposomes to deliver Poly I:C into the Head and Neck 12 (HN12) cell line to induce apoptosis in the carcinoma cells and tumor model. Cationic liposomes made by the hydrodynamic focusing method surpass traditional methods by offering a continuous flow-based approach for encapsulating genes, which is ideal for efficient tumor delivery. DOTAP liposomes efficiently bind Poly I:C, confirmed by transmission electron microscopy images displaying their spherical morphology. Liposomes are easily endocytosed in HN12 cells, suggesting their potential for therapeutic gene and drug delivery in head and neck squamous carcinoma cells. Activation of apoptotic pathways involving MDA5, RIG-I, and TLR3 is evidenced by upregulated caspase-3, caspase-8, and IRF3 genes upon endocytosis of Poly(I:C)-encapsulated liposomes. Therapeutic evaluations revealed significant inhibition of tumor growth with Poly I:C liposomes, indicating the possibility of MDA5, RIG-I, and TLR3-induced apoptosis pathways via Poly I:C liposomes in HN12 xenografts in J:NU mouse models. Comparative histological analysis underscores enhanced cell death with Poly I:C liposomes, warranting further investigation into the precise mechanisms of apoptosis and inflammatory cytokine response in murine models for future research.

Enzyme-Free Exponential Amplification via Growth and Scission of Crisscross Ribbons from Single-Stranded DNA Components.

The self-assembly of DNA-based monomers into higher-order structures has significant potential for realizing various biomimetic behaviors including algorithmic assembly, ultrasensitive detection, and self-replication. For these behaviors, it is desirable to implement high energetic barriers to undesired spurious nucleation, where such barriers can be bypassed via seed-initiated assembly. Joint-neighbor capture is a mechanism enabling the construction of such barriers while allowing for algorithmic behaviors, such as bit-copying. Cycles of polymerization with division could accordingly be used for implementing exponential growth in self-replicating materials. Previously, we demonstrated crisscross polymerization, a strategy that attains robust seed-dependent self-assembly of single-stranded DNA and DNA-origami monomers via joint-neighbor capture. Here, we expand the crisscross assembly to achieve autonomous, isothermal exponential amplification of ribbons through their concurrent growth and scission via toehold-mediated strand displacement. We demonstrate how this crisscross chain reaction, or 3CR, can be used as a detection strategy through coupling to single- and double-stranded nucleic acid targets and introduce a rule-based stochastic modeling approach for simulating molecular self-assembly behaviors such as crisscross-ribbon scission.

GRASSERIE - A BACULOVIRUS DISEASE OF THE SILKWORM BOMBYX MORI L.

Grasserie is a major infectious disease of silkworms caused by the Bombyx mori nuclear polyhedrosis virus. The virus belongs to the family Baculoviridae and is composed of a polyhedron with a single molecule of circular supercoiled double-stranded deoxyribose nucleic acid. The disease causes massive mortality (more than 60% of the total crop loss caused by different diseases), causing significant great crop loss to the sericulturists by reducing the cocoon crop and silk produce. There has been no effective solution for preventing and treating the disease. In the present review, the authors explore the subject in detail and inspire the researchers to search for molecular mechanisms of gene action and promote disease-resistant silkworm breeding.. KEYWORDS :Bombyx mori,,Baculoviridae, Disease resistance, Nuclear polyhedrosis virus, Polyhedron, Silkworm

Zα domain proteins mediate the immune response.

The Zα domain has a compact α/β architecture containing a three-helix bundle flanked on one side by a twisted antiparallel β sheet. This domain displays a specific affinity for double-stranded nucleic acids that adopt a left-handed helical conformation. Currently, only three Zα-domain proteins have been identified in eukaryotes, specifically ADAR1, ZBP1, and PKZ. ADAR1 is a double-stranded RNA (dsRNA) binding protein that catalyzes the conversion of adenosine residues to inosine, resulting in changes in RNA structure, function, and expression. In addition to its editing function, ADAR1 has been shown to play a role in antiviral defense, gene regulation, and cellular differentiation. Dysregulation of ADAR1 expression and activity has been associated with various disease states, including cancer, autoimmune disorders, and neurological disorders. As a sensing molecule, ZBP1 exhibits the ability to recognize nucleic acids with a left-handed conformation. ZBP1 harbors a RIP homotypic interaction motif (RHIM), composed of a highly charged surface region and a leucine-rich hydrophobic core, enabling the formation of homotypic interactions between proteins with similar structure. Upon activation, ZBP1 initiates a downstream signaling cascade leading to programmed cell death, a process mediated by RIPK3 via the RHIM motif. PKZ was identified in fish, and contains two Zα domains at the N-terminus. PKZ is essential for normal growth and development and may contribute to the regulation of immune system function in fish. Interestingly, some pathogenic microorganisms also encode Zα domain proteins, such as, Vaccinia virus and Cyprinid Herpesvirus. Zα domain proteins derived from pathogenic microorganisms have been demonstrated to be pivotal contributors in impeding the host immune response and promoting virus replication and spread. This review focuses on the mammalian Zα domain proteins: ADAR1 and ZBP1, and thoroughly elucidates their functions in the immune response.

DNA mimic foldamers affect chromatin composition and disturb cell cycle progression.

The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Long noncoding RNA MALAT1 is dynamically regulated in leader cells during collective cancer invasion

Cancer cells collectively invade using a leader-follower organization, but the regulation of leader cells during this dynamic process is poorly understood. Using a dual double-stranded locked nucleic acid (LNA) nanobiosensor that tracks long noncoding RNA (lncRNA) dynamics in live single cells, we monitored the spatiotemporal distribution of lncRNA during collective cancer invasion. We show that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is dynamically regulated in the invading fronts of cancer cells and patient-derived spheroids. MALAT1 transcripts exhibit distinct abundance, diffusivity, and distribution between leader and follower cells. MALAT1 expression increases when a cancer cell becomes a leader and decreases when the collective migration process stops. Transient knockdown of MALAT1 prevents the formation of leader cells and abolishes the invasion of cancer cells. Taken together, our single-cell analysis suggests that MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

Noncovalent Adsorption of Single-Stranded and Double-Stranded DNA on the Surface of Gold Nanoparticles

Understanding the patterns of noncovalent adsorption of double-stranded nucleic acids (dsDNA) on gold nanoparticles (GNPs) was the aim of this study. It was found that the high-affinity motifs in DNA can and do act as an “anchor” for the fixation of the whole molecule on the GNP (up to 98 ± 2 single-stranded (ss)DNA molecules per particle with diameter of 13 ± 2 nm). At the same time, the involvement of an “anchor” in the intramolecular DNA interaction can negatively affect the efficiency of the formation of ss(ds)DNA–GNP structures. It has been shown that the interaction of GNP with DNA duplexes is accompanied by their dissociation and competitive adsorption of ssDNAs on GNP, wherein the crucial factor of DNA adsorption efficiency is the intrinsic affinity of ssDNA to GNP. We propose a detailed scheme for the interaction of dsDNA with GNPs, which should be taken into account in studies of this type. Researchers focused on this field should accept the complicated nature of such objects and take into account the many competing processes, including the processes of adsorption and desorption of DNA on gold as well as the formation of secondary structures by individual DNA strands.

Enhancement of the thermal stability of G-quadruplex structures by urea

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5′-GGGTGGGTGGGTGGG-3′]), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

Specific Adsorption of Magnetic Fe3O4@hydroxyapatite Nanocomposites to a Hybridized Duplex for Immobilization and Label-Free Electrochemical Biosensing of MicroRNA

The unique discriminating ability of hydroxyapatite (HAP) to single- and double-stranded nucleic acid implies its potential as a biosensing material for nucleic acid analysis. Herein, a novel immobilization- and label-free electrochemical strategy has been developed for miRNA let-7a detection based on the specific adsorption of magnetic Fe3O4@hydroxyapatite (Fe3O4@HAP) to a duplex structure. Typically, the highly efficient homogeneous reaction between probe DNA (pDNA) and target miRNA let-7a produces the nucleic acid duplex, which is specifically adsorbed on the surface of Fe3O4@HAP. Thereafter, the electroactive intercalator Nile blue (NB) is introduced as the label-free signal molecule and inserts between the stacked base pairs of the pDNA–miRNA duplex. Thus, the Fe3O4@HAP bearing pDNA–miRNA and numerous intercalated NBs are adsorbed by the magnetic glassy carbon electrode, giving rise to an intense sensing signal without the need of nuclease-assisted cyclic amplification. With this advantage, the sensing system shows a wide linear range from 1.0 fM to 100 nM and a low detection limit of 0.051 fM for miRNA let-7a analysis. What is more, the synergy of the base-pairing-driven hybridization, the specific intercalation of NB to the duplex structure, and the selective adsorption of Fe3O4@HAP contribute to a high sensing specificity of the biosensing strategy. The sensing method provides a state-of-the-art strategy for the sensitive and facile bioanalysis and clinical diagnosis of miRNA.

Integrated analysis of circulating cell free nucleic acids for cancer genotyping and immune phenotyping of tumor microenvironment.

The circulating cell-free nucleic acids (ccfNAs) consist of a heterogenous cocktail of both single (ssNA) and double-stranded (dsNA) nucleic acids. These ccfNAs are secreted into the blood circulation by both healthy and malignant cells via various mechanisms including apoptosis, necrosis, and active secretion. The major source of ccfNAs are the cells of hematopoietic system under healthy conditions. These ccfNAs include fragmented circulating cell free DNA (ccfDNA), coding or messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), and mitochondrial DNA/RNA (mtDNA and mtRNA), that serve as prospective biomarkers in assessment of various clinical conditions. For, e.g., free fetal DNA and RNA migrate into the maternal plasma, whereas circulating tumor DNA (ctDNA) has clinical relevance in diagnostic, prognostic, therapeutic targeting, and disease progression monitoring to improve precision medicine in cancer. The epigenetic modifications of ccfDNA as well as circulating cell-free RNA (ccfRNA) such as miRNA and lncRNA show disease-related variations and hold potential as epigenetic biomarkers. The messenger RNA present in the circulation or the circulating cell free mRNA (ccf-mRNA) and long non-coding RNA (ccf-lncRNA) have gradually become substantial in liquid biopsy by acting as effective biomarkers to assess various aspects of disease diagnosis and prognosis. Conversely, the simultaneous characterization of coding and non-coding RNAs in human biofluids still poses a significant hurdle. Moreover, a comprehensive assessment of ccfRNA that may reflect the tumor microenvironment is being explored. In this review, we focus on the novel approaches for exploring ccfDNA and ccfRNAs, specifically ccf-mRNA as biomarkers in clinical diagnosis and prognosis of cancer. Integrating the detection of circulating tumor DNA (ctDNA) for cancer genotyping in conjunction with ccfRNA both quantitatively and qualitatively, may potentially hold immense promise towards precision medicine. The current challenges and future directions in deciphering the complexity of cancer networks based on the dynamic state of ccfNAs will be discussed.

A Case Series of Amicrobial Pustulosis of Folds: An Eye-opener for the Diagnosis of Autoimmune Disorders

Amicrobial Pustulosis of Folds (APF) is a rare, chronic, relapsing cutaneous disease seen exclusively in younger women with a history of autoimmune disease, most commonly Systemic Lupus Erythematosus (SLE), or who simply have circulating autoantibodies. This case series highlights the occurrence of an unusual manifestation of APF as a marker of autoimmune disorders and emphasises the importance of double-stranded Deoxyribose Nucleic Acid (dsDNA) positivity in such cases. A total of five female patients with APF were included in this case series. All five cases presented with pustular lesions lasting 10-12 weeks, involving conchal bowls, eyes, perineal, and perianal regions, either as an initial presentation or as a flare-up of pre-existing autoimmune disease. All patients underwent screening for autoimmune disorders, which revealed dsDNA positivity and met the diagnostic criteria for APF. Therefore, a diagnosis of APF with SLE was made. All patients showed significant improvement with oral steroids. Although SLE is a complex multisystem disorder where patients may not always present with malar rash, photosensitivity, arthritis, and arthralgia, APF could serve as an eye-opener for diagnosing underlying autoimmune diseases.

Force-dependent elasticity of nucleic acids.

The functioning of double-stranded (ds) nucleic acids (NAs) in cellular processes is strongly mediated by their elastic response. These processes involve proteins that interact with dsDNA or dsRNA and distort their structures. The perturbation of the elasticity of NAs arising from these deformations is not properly considered by most theoretical frameworks. In this work, we introduce a novel method to assess the impact of mechanical stress on the elastic response of dsDNA and dsRNA through the analysis of the fluctuations of the double helix. Application of this approach to atomistic simulations reveals qualitative differences in the force dependence of the mechanical properties of dsDNA with respect to those of dsRNA, which we relate to structural features of these molecules by means of physically-sound minimalistic models.

TripDesign: A DNA Triplex Design Approach Based on Interaction Forces.

A DNA triplex has the advantages of improved nanostructure stability and pH environment responsiveness compared with single-stranded and double-stranded nucleic acids. However, sequence stability and low design efficiency hinder the application of DNA triplexes. Therefore, a DNA triplex design approach (TripDesign) based on interaction forces is proposed. First, we present the stacking force constraint, torsional stress constraint, and G-quadruplex motif constraint and then use an improved memetic algorithm to design triplex sequences under combinatorial constraints. Finally, to quantify the process of triplex formation, we also explore the minimum length of the triplex-forming oligos (TFOs) required to form the triplex and the factors that produce depletion in cyclic pH-jump experiments. The experimental results show that the sequences produced by TripDesign have high stability and reversibility, and the proposed approach achieves efficient and automatic sequence design. In addition, this study characterizes multiple basic parameters of DNA triplex formation and promotes the wider application of DNA triplexes in nanotechnology.

Kinetic Characterization of SARS-CoV-2 nsp13 ATPase Activity and Discovery of Small-Molecule Inhibitors.

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5′-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 μM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.

Biosensor for a Rapid and Sensitive Detection and Quantification of Nuclease Activity

Nucleases are family of hydrolytic enzymes which cleave the phosphodiesteric bonds between the nucleotides producing 3'-phospho or 5'-phospho end products. These enzymes can be classified as endo- and exo-nucleases, cleaving inside of a polynucleotide chain or at the ends respectively, however, some enzymes may have both activities. Typically, some nucleases prefer double-stranded nucleic acid (e.g. DNase I), while other cleave single-stranded (e.g. Nuclease P1) or RNA-DNA hybrid (RNase H) polynucleotides. Deoxyribonucleases (DNases) are important objects in biotechnology presenting both as tools for manipulations with DNA1 as well as contaminants where nuclease-like activity is undesired.2 In addition, data emerges indicating the importance of DNase activity detection for medical applications, i.e., DNases may be related with various diseases and could be used to monitor the progress of various cancers3 , 4 as well as could be a rapid and fast biomarker of bacterial infections.5 Typically, DNases are being detected and quantified using classical approach: fluorescence detection or gel electrophoresis techniques. Though those methods are highly sensitive and reliable, they are costly, time-consuming as well as require toxic markers such as ethidium bromide or radiolabeled molecules. As alternative methods novel detection techniques for DNase detection were reported, mostly based on fluorescence or chemiluminescence detection incorporating nanomaterials for signal enhancement.6 In addition, electrochemical biosensors, provide simple and inexpensive platform for biomolecule detection while maintaining a high degree of accuracy and sensitivity. However, to this day the absolute majority of electrochemical biosensors are being developed from small biomolecules, e.g., glucose, glycerol, lactate while the development of large marcomolecules, i.e., proteins or enzymes is still lagging. In a case of DNase detection, a few biosensors have been reported. Sato et al. reported electrochemical biosensor for DNase I detection based on ferrocene-modified DNA immobilized on the electrode surface with the detection limit (LOD) up to 10–2 U mL–1. Ding and Qin have reported a potentiometric biosensor for the detection of DNAses based on polycation-sensitive membrane, and for DNase I the LOD was 0.45 U mL–1.7 Despite advances, the developed electrochemical biosensors for DNase detection still lack sensitivities comparable to fluorescence- or radiolabeling-based detection methods and reliability required for the analysis of real samples.In this work we present a rapid and sensitive bioelectrochemical device for the determination of nuclease activity in various fluids. The device system consists of a sensor electrode, a special design DNA target to maximize the nuclease cleavage rate, signal analysis algorithm and the supporting electronics. The developed sensor allows to determine DNase activity in range 0.0003–0.03 U mL–1 with the limit of detection up to 0.00034 U mL–1 over 15 min measurement (calibrated against DNase I standard) as well as has a high storage stability. The system was also implemented using inexpensive single-used electrodes. Moreover, the sensor was tested measuring nuclease activity in real unmodified human saliva samples and demonstrated high accuracy compared to the industry standard DNaseAlert™ QC System. The developed technology could significantly improve nuclease quality control processes in pharma/biotech industry and give new insights into nuclease importance for medical applications. References (1) Pan, Y.; Xiao, L.; Li, A. S. S.; Zhang, X.; Sirois, P.; Zhang, J.; Li, K. Mol Biotechnol 2013, 55 (1), 54–62.(2) Senavirathne, G.; Liu, J.; Lopez, M. A.; Hanne, J.; Martin-Lopez, J.; Lee, J.-B.; Yoder, K. E.; Fishel, R. Nat Methods 2015, 12 (10), 901–902.(3) Patel, P. S.; Patel, B. P.; Rawal, R. M.; Raval, G. N.; Patel, M. M.; Patel, J. B.; Jha, F. P.; Patel, D. D. Tumor Biol 2000, 21(2), 82–89.(4) Balian, A.; Hernandez, F. J. Biomark Res 2021, 9 (1), 86.(5) Flenker, K. S.; Burghardt, E. L.; Dutta, N.; Burns, W. J.; Grover, J. M.; Kenkel, E. J.; Weaver, T. M.; Mills, J.; Kim, H.; Huang, L.; Owczarzy, R.; Musselman, C. A.; Behlke, M. A.; Ford, B.; McNamara, J. O. Molecular Therapy 2017, 25 (6), 1353–1362.(6) Mozioğlu, E.; Akgoz, M.; Kocagöz, T.; Tamerler, C. Anal. Methods 2016, 8 (20), 4017–4021.(7) Ding, J.; Qin, W. Biosensors and Bioelectronics 2013, 47, 559–565. Figure. The general operation scheme of nuclease biosensor. (A) In a case a sample does not contain nuclease the Target nucleotide is not cleaved and its hybridization rate is high. (B) In a case sample contains nucleases the Target nucleotide is cleaved and its hybridization rate is lower and relates to the nuclease activity. Figure 1