Search Results (111)

Nucleic acid strand displacement reactions (SDRs) are fundamental building blocks of dynamic DNA nanotechnology. A detailed understanding of their kinetics is crucial for designing efficient sequences and regulating reaction networks with applications in biosensing, synthetic biology, biocomputing, and medical diagnostics. Since the development of toehold-mediated strand displacement, researchers have devised many strategies to adjust reaction kinetics. These efforts have expanded the available tools in DNA nanotechnology. This review summarizes the basic principles and recent advances in activation strategies, emphasizing the role of strand proximity as a central driving force. Proximity-based approaches include toehold docking, associative toeholds, remote toeholds, and allosteric designs, as well as strategies that operate without explicit toehold motifs. These methods enable flexible and scalable construction of DNA reaction networks. We further discuss how combining different activation and kinetic control approaches gives rise to dynamic networks with complex and dissipative behaviors, providing new directions for DNA-based nanotechnology. Full article

Nucleic acid amplification methods are widely used in science, medicine and forensics for molecular biological assays and for the detection of genetic material. The newly developed strand displacement chain reaction (SDCR) method is a hybrid amplification technique based on polymerase chain reaction (PCR) and isothermal nucleic acid amplification. Here, we compared conventional PCR, the “gold standard” for molecular diagnostic assays, with the SDCR method by performing real-time amplification assays using human, bacterial and viral genetic materials. In the assays, SDCR demonstrated very high sensitivity and amplification efficiency. We found that the SDCR method provided an amplification factor above three, which noticeably outperformed that of PCR amplification and enabled a marked reduction in the number of cycles in comparison with PCR. Therefore, the new hybrid amplification technique could be extremely useful for the detection of genetic material and the development of new diagnostic kits. Full article

Schiff base ligands prepared from salicylaldehyde and 2-, 3- and 4-methoxybenzylamine were used to prepare copper(II) complexes, characterized by spectral methods, elemental analysis and X-ray crystallography in the case of complex 4a derived from 2-methoxybenzylamine. The DNA cleavage activity of the prepared complexes was exceptional, with best activities of over 95% one-strand cleavage for 4c at 3 mM and full double-strand cleavage for complex 4a at 5 mM. Absorption titration studies with ct-DNA revealed good binding constants (at 10⁵ M⁻¹) with a decrease of up to 56% light absorption. Meanwhile, the EB–DNA displacement method and viscosity studies revealed groove binding as a possible binding mode. For BSA binding studies, all three complexes showed K_BSA values in the optimal range for reversible BSA binding (10⁴ M⁻¹). The copper(II) complexes showed significant cytotoxic effects (67–96% at 1 mM) in mitochondrial activity monitoring assays. Cytotoxicity was confirmed against cancer cell lines (A549 and HepG2) and HEL cells. The complexes 4a and 4c exhibited high activity against HepG2 cancer cells (IC50 < 22 μM), comparable to cisplatin. The radical scavenging activity was determined by the INT method with the best IC50 for 4c (189 ± 11 μM). Overall, complexes 4a and 4c with a methoxy group in the ortho and para positions show high potential in most determined activities, but mainly as DNA cleavers and as cytotoxic agents with selectivity against HepG2 cells. Full article

Polymerase-coupled nanopore sequencing requires DNA polymerases with strong strand displacement activity and high processivity to sustain continuous signal generation. In this study, we characterized two novel B family DNA polymerases, SRHS and BBum, isolated from Bacillus phages SRT01hs and BeachBum, respectively. Both enzymes exhibited robust strand displacement, 3′→5′ exonuclease activity, and maintained processivity under diverse reaction conditions, including across a broad temperature range (10–45 °C) and in the presence of multiple divalent metal cofactors (Mg²⁺, Mn²⁺, Fe²⁺), comparable to the well-characterized Phi29 polymerase. Through biochemical analysis of mutants designed using AlphaFold3-predicted structural models, we identified key residues (G96, M97, D486 in SRHS; S97, M98, A493 in BBum) that modulated exonuclease activity, substrate specificity and metal ion utilization. Engineered variants SRHS_F and BBum_Pro_L efficiently incorporated unnatural nucleotides in the presence of Mg²⁺—a function not observed in Phi29 and other wild-type strand-displacing B family polymerases. These combined biochemical features highlight SRHS and BBum as promising enzymatic scaffolds for nanopore-based long-read sequencing platforms. Full article

The DNA of all living organisms is a common matrix for both replication and transcription processes. This sometimes leads to inevitable collisions between DNA replication and transcription machinery. There is plethora of evidence demonstrating that such collisions (or TRCs) are one of the most common and significant reasons for genomic instability. One of the key outcomes of TRCs is the accumulation of non-canonical DNA secondary structures, including R-loops. R-loops are three-stranded DNA–RNA hybrids with a displaced third single-stranded DNA fragment. Although R-loops are thought to play several functional roles in biological processes, an imbalance in their metabolism has been proven to have severe consequences. In this review, we attempt to summarize the current knowledge of the participants in the process of R-loop regulation in cells, with an emphasis on eukaryotic systems. We also touch upon the conditions favoring TRCs and the possible ways of dealing with these conflicts. Full article

Niacinamide was recently shown to directly interact with bacterial DNA and interfere with cell replication; niacinamide mode of interaction and efficacy as a natural anti-microbial molecule were also described. The aim of this study is to elucidate the exact binding mechanism of niacinamide to microbial DNA. Intercalation is a binding mode where a small planar molecule, such as niacinamide, is inserted between base pairs, causing structural changes in the DNA. Melting curve analysis with various intercalating dyes demonstrated that niacinamide interaction with bacterial DNA reduces its melting temperature in a linear dose-dependent manner. Niacinamide’s effect on the melting temperature was found to be % GC-dependent, while purine stretches were also found to influence the binding kinetics. Finally, fluorescent intercalator displacement (FID) assays demonstrated that niacinamide strongly reduces SYBR Safe signal in a dose-dependent manner. Interestingly, competition assays with a minor groove binder also reduced Hoechst signal but in a non-linear manner, which can be attributed to strand lengthening and unwinding following niacinamide intercalation. Taken altogether; our results suggest a “disruptive intercalation” as the mode of interaction of niacinamide with bacterial DNA. Formation of locally destabilized DNA portions by niacinamide might interfere with protein–DNA interaction and potentially affect several crucial bacterial cellular processes, e.g., DNA repair and replication, subsequently leading to cell death. Full article

DNA methylation, as a critical epigenetic modification, plays a central role in gene regulation and has emerged as a powerful biomarker for early disease diagnostics, particularly in cancer. Owing to the limitations of traditional bisulfite sequencing—such as high cost, complexity, and chemical degradation—electrochemical biosensors have gained substantial attention as promising alternatives. This review summarizes recent advancements in electrochemical platforms for bisulfite-free detection of DNA methylation, encompassing direct oxidation strategies, enzyme-assisted recognition (e.g., restriction endonucleases and methyltransferases), immunoaffinity-based methods, and a variety of signal amplification techniques such as rolling circle amplification and catalytic hairpin assembly. Additional approaches, including strand displacement, magnetic enrichment, and adsorption-based detection, are also discussed. These systems demonstrate exceptional sensitivity, often down to the attomolar or femtomolar level, as well as high selectivity, reproducibility, and suitability for real biological matrices. The integration of nanomaterials and redox-active probes further enhances analytical performance. Importantly, many of these biosensing platforms have been validated using clinical samples, reinforcing their translational relevance. The review concludes by outlining current challenges and future directions, emphasizing the potential of electrochemical biosensors as scalable, cost-effective, and minimally invasive tools for real-time epigenetic monitoring and early-stage disease diagnostics. Full article

R-loops are nucleic acid structures composed of an RNA-DNA hybrid (RDH) duplex and a displaced single-stranded DNA (ssDNA), which are fundamentally involved in key biological functions, including transcription and the preservation of genome stability. In an R-loop, the RDH duplex is bent by the folded secondary structures of the displaced ssDNA. Previous experiments and simulations indicated the high bendability of DNA below the persistence length. However, the bendability of a short RDH duplex remains unclear. Here, we report that an RDH duplex exhibits higher bendability than a DNA duplex on the short length scale using single-molecule cyclization experiments. Our molecular dynamics simulations show that an RDH duplex has larger intrinsic curvature and structural fluctuations and more easily forms kinks than DNA, which promote the bending flexibility of RDH from unlooped structures. Interestingly, we found that an RDH duplex composed of a C-rich DNA strand and a G-rich RNA strand shows significantly higher bendability than that composed of a G-rich DNA strand and a C-rich RNA strand in the same CpG island promoter regions, which may contribute to the formation of an R-loop. These findings shape our understanding towards biological processes involving R-loops through the high and sequence-dependent bendability of an RDH duplex. Full article

Genetic mutation detection for colorectal cancer (CRC) is crucial for precision diagnosis and treatment, yet current methods often suffer from challenges such as low sensitivity, time consumption, and high costs. In our preliminary bioinformatic analysis of 751 CRC cases from The Cancer Genome Atlas and 131 Chinese patient samples, APC, TP53, and KRAS were identified as the most frequently mutated genes. Among them, KRAS missense mutations emerged as key diagnostic biomarkers. In this study, we applied a fluorescence-based long block displacement amplification (LBDA) sensing method for the rapid, high-throughput, and cost-effective detection of KRAS genetic mutations. In the LBDA system, SYBR Green dye binds to the amplified double-stranded DNA, generating a fluorescence signal that directly reflects the abundance of mutant types (MTs). This real-time signal output enables the enrichment and sensitive detection of MTs, establishing LBDA as an efficient biosensing platform for KRAS genotyping. Using this technique, a detection limit of 0.08% variant allele frequency was achieved with 20 ng of synthetic DNA input. To evaluate clinical performance, the LBDA method was applied to 118 tissue samples from 59 CRC patients, including tumor and matched peritumoral tissues. For 59 CRC tumor samples, LBDA successfully identified KRAS mutations in 37.29% of cases, closely matching results (42.37%) obtained by next-generation sequencing and achieving 88% sensitivity and 100% specificity. In conclusion, this study presents a rapid and cost-effective mutation detection method based on optical biosensing, offering strong potential for advancing personalized CRC diagnosis and treatment. Full article

The rational design of multifunctional drug delivery systems capable of achieving precise drug release remains a huge challenge. Herein, we designed a stimuli-responsive dendritic-DNA-based nanohydrogel as a nanocarrier to achieve the co-delivery of doxorubicin and HMGN5 mRNA-targeting antisense oligonucleotides, thus achieving dual therapeutic effects. The nanocarrier, constructed from dendritic DNA with three crosslinking branches and one loading branch, formed biocompatible and programmable DNA nanohydrogels. The C-rich sequences in the crosslinking branches conferred pH sensitivity, while the loading strand enabled efficient incorporation of a shielding DNA/ASO complex. DOX encapsulation yielded a chemo–gene co-delivery platform. Upon cellular uptake by cancer cells, the nanocarrier disassembled in the acidic tumor microenvironment, releasing DOX for chemotherapy and ASOs via toehold-mediated strand displacement (TMSD) for targeted gene silencing. Cellular studies demonstrated significantly enhanced cancer cell inhibition compared to single-agent treatments, highlighting strong combined effects. This study provides a novel strategy for tumor-microenvironment-responsive co-delivery, enabling precise, on-demand release of therapeutic agents to enhance combined chemo–gene therapy. Full article

DNA is an exceptional building block for the fabrication of dynamic supramolecular systems with switchable geometries. Here, a self-assembled, tunable plasmonic–fluorescent nanostructure was developed. A precise sliding motion mechanism was operated through the control of strand displacement reactions, shifting two single-strand DNA (ssDNA) rails connected by a ssDNA quasi-ring structure. The system was reconfigured as a nano-mechanical structure, generating six discrete configurations, and setting specific distances between a tethered gold nanoparticle (AuNP) and a fluorophore, Sulfo-Cyanine3 (Cy3). Each configuration produced a distinct fluorescence emission intensity via plasmonic quenching/enhancement effects, and therefore the structure behaved as a nano-ruler. To optimize the system, the reversible distance-dependent fluorescence quenching or enhancement phenomena were investigated by testing AuNPs with diameters of 5, 10, and 15 nm, yielding the best performances with 10 nm AuNPs. Furthermore, a geometric model of the system was produced, confirming the observed results. The fluorophore–plasmonic surface positioning, conferred by the DNA ruler, led to a finite state nano-machine with six alternative signal outputs. This mechanism, working as a fluorescent reporter, could find application in a multiple-responsive detection system of single-strand nucleic acids, such as viruses or microRNAs. Full article

DNA (Deoxyribonucleic Acid) logic circuit systems provide a powerful arithmetic architecture for the development of molecular computations. DNA nanotechnology, particularly DNA origami, provides a nanoscale addressable surface for DNA logic circuit systems. Although molecular computations based on DNA origami surfaces have received significant attention in research, there are still obstacles to constructing localized scalable DNA logic circuit systems. Here, we developed elementary DNA logic circuits on a DNA origami surface by employing the strand displacement reaction (SDR) to realize the localized scalable DNA logic circuit systems. We showed that the constructed elementary logic circuits can be scaled up to the localized DNA logic circuit systems that perform arbitrary digital computing tasks, including square root functions, full adder and full subtractor. We used a 50% reduction in the number of localized DNA logic components, compared to localized logic systems based on the threshold strategy. We further demonstrated that the localized DNA logic circuit systems for three-satisfiability (3-SAT) problem solving and disease classification can be implemented using the constructed elementary DNA logic circuits. We expect our approach to provide a new design paradigm for the development of molecular computations and their applications in complex mathematical problem solving and disease diagnosis. Full article

Monitoring of immune factors, including interferon-gamma (IFN-γ), holds great importance for understanding immune responses and disease diagnosis. Wearable sensors enable continuous and non-invasive detection of immune markers in sweat, drawing significant attention to their potential in real-time health monitoring and personalized medicine. Among these, electrochemical sensors are particularly advantageous, due to their excellent signal responsiveness, cost-effectiveness, miniaturization, and broad applicability, making them ideal for constructing wearable sweat sensors. In this study, we present a flexible and sensitive wearable platform for the detection of IFN-γ, utilizing a DNA hydrogel with favorable loading performance and sample collection capability, and the application of mobile software achieves immediate data analysis and processing. This platform integrates three-dimensional DNA hydrogel functionalized with IFN-γ-specific aptamers for precise target recognition and efficient sweat collection. Signal amplification is achieved through target-triggered catalytic hairpin assembly (CHA), with DNA hairpins remarkably enhancing sensitivity. Ferrocene-labeled reporting strands immobilized on a screen-printed carbon electrode are displayed via CHA-mediated strand displacement, leading to a measurable reduction in electrical signals. These changes are transmitted to a custom-developed mobile application via a portable electrochemical workstation for real-time data analysis and recording. This wearable sensor platform combines the specificity of DNA aptamers, advanced signal amplification, and the convenience of mobile data processing. It offers a high-sensitivity approach to detecting low-abundance targets in sweat, paving the way for new applications in point-of-care diagnostics and wearable health monitoring. Full article

Gene II Protein (Gp2/P2) is a nicking enzyme of the M13 bacteriophage that plays a role in the DNA replication of the viral genome. P2 recognizes a specific sequence at the f1 replication origin and nicks one of the strands and starts replication. This study was conducted to address the limitations of previous experiments, improve methodologies, and precisely determine the biochemical activity conditions of the P2 enzyme in vitro. For these purposes, the gene encoding P2 was cloned in Escherichia coli and expressed as a hybrid protein together with a green fluorescent protein (P2-GFP). P2-GFP was purified via metal affinity chromatography, and its nicking activity was determined by conversion of supercoiled DNA to open circular or linear forms. We discovered that, among the two loops of the f1 origin defined previously, P2 can recognize just the A1 loop. When a supercoiled plasmid containing the f1 origin was treated with P2-GFP, the plasmid was present in an open circular form, indicating that a nick was created on only one of the strands. However, when the A1 loop sequence was inserted into the 3′ ends of both strands by cloning a PCR product obtained by primers with the A1 loop sequence, the plasmid was linearized by treatment with P2-GFP, indicating that nicks were created on both strands. Certain infectious diseases are caused by single-stranded DNA viruses, and some of them have specific nicking enzymes that enable strand displacement and free 3′ end of a single strand that works as a primer for their replication mechanisms like M13 bacteriophages, such as parvovirus B19. Despite there being different host viruses such as bacteria and humans, their DNA replication mechanisms are very similar in this concept. Investigating the features of the P2-nicking enzyme may deepen the understanding of human pathogenic single-stranded viruses and facilitate the development of drugs that inhibit viral replication. Full article

DNA chemical reaction networks can perform complex information processing through careful design of reaction kinetics, which involves the reaction network structure, rate constants, and initial concentrations. The toehold-mediated strand displacement reaction (TMSDR) is a key mechanism in creating DNA circuits, offering a rational design approach by integrating individually designed TMSDRs. Tools such as VisualDSD and NUPACK facilitate the efficient design of these systems by allowing precise tuning of reaction parameters. However, discrepancies between simulated and experimental results can occur, often due to the modification of reporter molecules. Recently, fluorophore dyes and quenchers were found to significantly impact the dynamics of irreversible TMSDRs, altering them by nearly two orders of magnitude. The impact on reaction dynamics varies with the modification site of these reporters. This study examines the mechanisms of reporter modifications affecting reversible TMSDRs, influencing transient and steady-state characteristics. This is crucial for DNA circuit design, which integrates reversible and irreversible TMSDRs. Our findings indicate that modifying fluorescent dye and quencher an appropriate distance apart (e.g., toehold length) can minimize adverse effects on the DNA reaction dynamics while ensuring effective FRET, therefore improving the accuracy of experimental verification for DNA reaction systems. Full article