Search Results (450)

Ochratoxin A (OTA) is a highly toxic mycotoxin commonly detected in food and agricultural products, requiring sensitive analytical methods for reliable monitoring. Herein, we report an ultrasensitive turn-on electrochemical aptasensor for OTA detection based on a target-induced displacement of an aptamer–complementary DNA (cDNA) duplex assembled on an electrochemically reduced graphene oxide (ERGO)-modified glassy carbon electrode (GCE). In the absence of OTA, a methylene blue (MB)-labeled aptamer hybridized with cDNA is immobilized on the ERGO surface via π–π stacking interactions, forming a rigid duplex that suppresses electron transfer and yields a low electrochemical signal. Upon OTA binding, the aptamer undergoes a conformational transition into a G-quadruplex structure, leading to dissociation of the cDNA strand. This target-induced folding brings the MB redox tag into close proximity to the ERGO surface, markedly accelerating electron transfer and enhancing the cathodic reduction current of MB, thereby producing a pronounced signal-on response in square-wave voltammetry (SWV). The ERGO-modified electrode provides a conductive and stable interface without chemical linkers. Under optimized conditions, the aptasensor shows a linear response to OTA from 10 fM to 100 pM with an ultralow LOD of 0.67 fM, together with high selectivity, good reproducibility, and satisfactory stability. This work demonstrates a simple and effective turn-on aptasensing strategy for sensitive electrochemical detection of OTA. Full article

The objective of this study is to develop a label-free fluorescent sensor for the quantitative detection of hypochlorite ions (ClO⁻) and validate its applicability in biological samples, particularly exploring the potential of ClO⁻ as a biomarker for stress-induced hypertension (SIH). Male Sprague-Dawley rats (8 weeks old, 250–300 g) were used to establish the SIH model. A guanine-rich (G-rich) signal DNA sequence (S-DNA) was rationally designed, with a ClO⁻-responsive phosphorothioate (PS) moiety integrated into the probe architecture. In the absence of ClO⁻, the S-DNA folds into a stable G-quadruplex structure, which specifically binds to ThT and triggers a significant enhancement of the dye’s fluorescence intensity. Upon introduction of ClO⁻, the specific hydrolysis reaction between the PS moiety and ClO⁻ induces cleavage of the S-DNA into two discrete fragments, thereby abrogating G-quadruplex formation and resulting in a remarkable quenching of ThT fluorescence. This proposed method exhibits excellent anti-interference capability against other reactive oxygen species (ROS) and achieves a low detection limit of 41.2 nM for ClO⁻. Furthermore, this strategy was successfully applied to the quantitative determination of endogenous ClO⁻ in human cells and the plasma of stress-induced hypertensive (SIH) rats, highlighting its substantial potential for clinical and physiological research. Full article

G-quadruplexes (G4s) are specialized nucleic acid structures extensively formed throughout the genome, with particular enrichment in regulatory regions such as telomeres, promoters, and transcriptional enhancers. These four-stranded assemblies are involved in multiple chromosomal processes, including DNA replication, transcription, maintenance of genomic stability, and epigenetic regulation, and are closely associated with cancer biology. Due to their unusual thermodynamic stability, G4s serve as physical barriers to DNA/RNA unwinding, thereby impeding replication, transcription, and translation and compromising genome integrity. To mitigate this threat, cells have evolved dedicated helicases that can actively resolve G4 structures. In this review, we summarize recent structural advances—primarily derived from protein crystallography—regarding the mechanisms by which helicases unwind G4 quadruplexes. The insights presented herein establish a framework for elucidating the molecular basis of G4 unfolding and for the rational design of small-molecule G4 ligands and therapeutic agents. Additionally, we explore the applications of G4 helicases in nanopore sequencing, which aim to enhance sequencing accuracy, throughput, and continuity. Full article

G-quadruplex (G4) DNA structures have recently emerged as promising chiral scaffolds for enantioselective catalysis. This study investigates how thymidine loop modifications influence the catalytic performance of the telomeric G4 sequence HT21 in the asymmetric sulfoxidation of thioanisole. To this end, several singly or doubly modified HT21 derivatives were synthesized by using β-L-2′-deoxythymidine, 5-hydroxymethyl-2′-deoxyuridine, and 5-bromo-2′-deoxyuridine instead of a T residue, or β-L-2′-deoxyadonesine instead of an A residue, in specific positions within the TTA loops. The catalytic activity of these analogues was evaluated in the Cu(II)-mediated oxidation of thioanisole using hydrogen peroxide as oxidant. All modified sequences maintained complete substrate conversion, but their enantioselectivities varied markedly. Whereas the highest enantiomeric excess (84% ee) had previously been achieved with the HT21 analogue bearing a β-L-2′-deoxyadenosine in the first loop, the thymidine-based modifications, either alone or in combination, resulted in lower ee values, suggesting that loop alterations critically affect the chiral microenvironment, not all loop positions are functionally equivalent, and single substitutions within the same loop can result in different enantioselectivities. These findings highlight new insights on how individual loop residues contribute to asymmetric induction and offer further details for tuning G4-based catalytic scaffolds. Full article

Split G-quadruplex DNAzymes offer unique opportunities for building gated biosensors with a wide range of applications. Splitting G4 DNAzymes involves separating guanine tracts in the G-quadruplex DNA sequence into two non-functional sequences that reconstitute into a functional G-quadruplex with peroxidase activity upon hybridisation of the aptamer probe region within the split system with the target molecule. Several studies have demonstrated the reassembly of split G4 DNAzymes and their applications in the detection of various analytes. This approach offers unique opportunities for modular biosensor construction, target-dependent activation, lack of requirement for labelling, amplification-free high sensitivity, and specificity over traditional G4 sensing. In this review, we explore the strategies of splitting G-quadruplex and their applications in biomedical diagnosis, environmental sensing, food safety monitoring, cell detection, and the integration of the technology with nanomaterials for enhanced stability and sensitivity. We considered the classical intermolecular split strategies that utilise binary probes and intramolecular split systems, which integrate the spacer DNA that allow for single probes as the model G4 sequence. Finally, we explore the current challenges required to develop split G-quadruplex DNAzymes into tools for routine practical applications. Full article

The role of alternative nucleic acid structures (ANS) in biology is an area of increasing interest. These non-canonical structures include the Z-DNA and Z-RNA duplexes (ZNA), the three-stranded triplex, the four-stranded G-quadruplex (GQ), and i-motifs. Previously, the biological relevance of ANS was dismissed. Their formation in vitro often required non-physiological conditions, and there was no genetic evidence for their function. Further, structural studies confirmed that sequence-specific transcription factors (TFs) bound B-DNA. In contrast, ANS are formed dynamically by a subset of repeat sequences, called flipons. The flip requires energy, but not strand cleavage. Flipons are enriched in promoters where they modulate transcription. Here, computational modeling based on AlphaFold V3 (AF3), under optimized conditions, reveals that known B-DNA-binding TFs also dock to ANS, such as ZNA and GQ. The binding of HLH and bZIP homodimers to Z-DNA is promoted by methylarginine modifications. Heterodimers only bind preformed Z-DNA. The interactions of TFs with ANS likely enhance genome scanning to identify cognate B-DNA-binding sites in active genes. Docking of TF homodimers to Z-DNA potentially facilitates the assembly of heterodimers that dissociate and are stabilized by binding to a cognate B-DNA motif. The process enables rapid discovery of the optimal heterodimer combinations required to regulate a nearby promoter. Full article

Expansion of d(GGGGC)_n repeat in the C9ORF72 gene is causal for Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Dementia (FTD). Proposed mechanisms include Repeat-Associated Non-AUG translation or the formation of G-quadruplexes (GQ) that disrupt translation, induce protein aggregation, sequester RNA processing factors, or alter RNA editing. Here, I show, using AlphaFold V3 (AF3) modeling, that the TAR DNA-binding protein (TDP-43) docks to a complex of GQ and hemin. TDP-43 methionines lie over hemin and likely squelch the generation of superoxide by the porphyrin-bound Fe. These TDP-43 methionines are frequently altered in ALS patients. Tau protein, a variant of which causes ALS, also binds to GQ and heme and positions methionines to detoxify peroxides. Full-length Tau, which is often considered prone to aggregation and a prion-like disease agent, can bind to an array composed of multiple GQs as a fully folded protein. In ALS and FTD, loss-of-function variants cause an uncompensated surplus of superoxide, which sparks neuronal cell death. In Alzheimer’s Disease (AD) patients, GQ and heme complexes bound by β-amyloid 42 (Aβ4) are also likely to generate superoxides. Collectively, these neuropathologies have proven difficult to treat. The current synthesis provides a framework for designing future therapeutics. Full article

A novel, label-free chemiluminescence sensing platform for CpG methylation was developed, leveraging the G-quadruplex (G4) structural sensitivity of G4–protein interactions to eliminate bisulfite conversion. This sensing system is based on the enhancement of luminol chemiluminescence generated from myoglobin upon binding to the G4-forming DNA. At the core of this biosensor is the G4-structure-dependent modulation of the peroxidase-like activity generating luminol chemiluminescence of myoglobin. The structural change by CpG methylation within the G4-forming sequence of the B cell lymphoma 2 (BCL2) gene promoter altered its binding to myoglobin, transducing the methylation state into a measurable signal catalyzed by myoglobin. This principle was validated in a practical assay using immobilized probes to capture the target DNA for methylation analysis. This system demonstrated the capability to distinguish methylation differences of 50% when the target DNA concentration was over 25 nM. Versatility was further confirmed using the sequence from the dopamine receptor D2 (DRD2) gene promoter, where the methylation similarly induced distinct topological and functional changes. This is the first study to directly link the epigenetic state of a G4-forming DNA sequence to a protein-mediated enzymatic output, offering a framework for simple, rapid, and highly adaptable biosensors for research and clinical applications. Full article

GC-rich sequences affect DNA replication, recombination and repair, as well as RNA transcription in vivo. Such sequences may also impede site-directed mutagenesis in vitro. P3a site-directed mutagenesis is a highly efficient method, but it has not been tested with plasmids possessing GC-rich sequences. Here we report that it is very efficient with a BRPF3 expression vector but unsuccessful with that for KAT2B. Because two GC-rich regions located within the synthetic CAG promoter and the KAT2B coding region may form guanine (G)-quadruplexes and hinder plasmid denaturation during PCR, we developed P3b site-specific mutagenesis, achieving an average efficiency of 97.5% in engineering ten KAT2B mutants. Importantly, deletion mutagenesis revealed that either of the two GC-rich regions is sufficient for rendering the plasmid incompatible with P3a mutagenesis. Consistent with this, only P3b mutagenesis worked efficiently with several widely used sgRNA/Cas9 expression vectors, which contain the CAG promoter, and with an expression vector for CDK13, which possesses an intrinsically disordered domain encoded by a GC-rich DNA fragment. Thus, this study highlights serious challenges posed by GC-rich sequences to site-directed mutagenesis and provides an effective remedy to address such challenges. The findings support that G-quadruplex formation is one mechanism whereby such sequences impede regular PCR-based mutagenesis methods. Full article

For decades, 8-oxoguanine (8-oxoG) has been recognized as a pervasive and pro-mutagenic oxidative DNA lesion. In human cells, 8-oxoG is removed from DNA via the base excision repair pathway initiated by 8-oxoguanine–DNA glycosylase (OGG1). However, emerging evidence over the past twenty years suggests a more complex, regulatory role for this DNA modification. Here, we discuss findings that 8-oxoG, particularly when present in gene promoters, can act as a signal to modulate transcription, establishing an 8-oxoG/OGG1 axis in the inflammatory response. Proposed mechanisms include the generation of 8-oxoG during chromatin remodeling processes involving histone demethylases, the recruitment of transcription factors (NF-κB, HIF1α, Myc, SMAD, etc.) by OGG1, and the lesion’s enrichment in guanine-rich sequences prone to forming G-quadruplex structures. The pro-mutagenic nature of 8-oxoG and the lack of dedicated, functionally separate writer and reader proteins challenge its classification as a true epigenetic DNA mark, distinguishing it from canonical epigenetic nucleobases like 5-methylcytosine and 5-hydroxymethylcytosine. On the other hand, 8-oxoG is well suited for the role of a regulatory signal localized to DNA and involved in the cellular response to oxidative stress and the associated physiological stimuli. Full article

R-loops are three-stranded nucleic acid structures implicated in genome regulation and stability. In Arabidopsis thaliana, the chloroplast-localized RNase H1 enzyme (AtRNH1C) is important for chloroplast development and genome integrity; however, its molecular activity has not been experimentally verified. In the present study, we characterized the enzymatic activity of recombinant AtRNH1C toward model R-loops of various structures. Using a set of synthetic R-loop substrates, we demonstrate that AtRNH1C cleaves the RNA within DNA/RNA hybrids with a strong preference for purine-rich sequences, most notably at G↓X dinucleotides. Kinetic assays showed that the enzyme’s efficiency is highly dependent on the length of the hybrid duplex but is not affected by a G-quadruplex structure in the single-stranded DNA flap of the R-loop. The most rapid degradation was observed for an R-loop with an 11 nt DNA/RNA hybrid region. This study provides a comparative analysis of chloroplast-localized RNase H1 activity and elucidates its substrate preferences, suggesting that an R-loop with a heteroduplex length closest to the native size found in transcription elongation complexes is the most efficient substrate. These findings suggest that the enzymatic activity of AtRNH1C is sufficient to perform its function in maintaining chloroplast genome stability by the degradation of R-loops in DNA. Full article

In this study, a sliding microfluidic biosensor integrating RPA-SDA cascaded amplification was developed for the rapid, visual detection of Shigella. A novel RPA primer targeting the specific ipaH gene was designed to include a 5′-end G-quadruplex (G4) sequence and the complementary sequence of an Nt.BstNBI endonuclease recognition site. The RPA product templates a subsequent SDA reaction, generating abundant G4 structures that form peroxidase-mimicking DNAzymes with hemin, catalyzing a TMB reaction that produces a distinct blue color for visual readout (on-chip detection at OD₃₇₀, distinct from conventional tube assays at OD₄₅₀). The core on-chip detection process was completed within 13 min (10 min for SDA and 3 min for color development), achieving a limit of detection of 3.5 × 10⁻⁴ ng/μL for Shigella genomic DNA. This timing explicitly excludes the preceding, off-chip steps of nucleic acid extraction and RPA amplification. Validation using spiked lettuce samples confirmed the platform’s high specificity and sensitivity. This work establishes a proof-of-concept for a portable screening tool, highlighting its potential for on-site food safety applications. However, further validation in diverse food matrices and under real-world field conditions is required to fully establish its practical utility. Full article

Computational and machine learning approaches play a pivotal role in identifying, characterizing, and targeting noncanonical DNA structures, including G-quadruplexes, Z-DNA, hairpins, and triplexes. These configurations play critical roles in maintaining genomic stability, facilitating DNA repair, and regulating chromatin organization. Although the human genome predominantly adopts the B DNA conformation, evidence indicates that non-B DNA forms exert significant influence on gene expression and disease development. This highlights the need for dedicated computational frameworks to systematically investigate these alternative structures. Machine learning model, encompassing supervised and unsupervised algorithms such as K Nearest Neighbors, Support Vector Machines, and deep learning architectures including Convolutional Neural Networks, have shown considerable potential in predicting sequence motifs predisposed to forming non-B DNA conformations. These predictive tools contribute to identifying genomic regions associated with disease susceptibility. Complementary bioinformatics platforms and molecular docking tools, notably Auto Dock, along with chemical libraries like ZINC, facilitate the virtual screening of small molecules targeting specific DNA structures. Stabilizers of G quadruplexes, exemplified by CX 5461, have demonstrated therapeutic promise in BRCA-deficient cancers, highlighting the translational impact of computational methods on drug discovery. Anticipating DNA structural shifts opens new avenues in personalized medicine for complex diseases, with computational chemistry and machine learning deepening our understanding of DNA topology and guiding smarter ligand design. The integrated approach proposed in this review addresses the previous studies performed in this field and highlights the current limitations in structural genomics and advances the development of precision therapeutics aligned with individual genomic profiles. Full article

Interest in non-canonical DNA structures continues to grow, in part fueled by the recent discovery of a new structure, G-triplex DNA. Originally proposed as folding intermediates for G-quadruplex DNA, G-triplex DNA has more recently been shown to form from truncated G-quadruplex sequence oligonucleotides and other, specifically designed sequences. In this review, we provide the first, comprehensive survey of G-triplex DNA and RNA, covering the literature up to 2024. We include reports of G-triplex DNA from bulk solution and single-molecule approaches, the structural characterization of G-triplex DNA, and the breadth of oligonucleotide sequences that have been reported to form these structures. The formation of G-triplex RNA structures is also reviewed. The evolving understanding of sequence and environmental effects on G-triplex formation are presented together with challenges due to structural polymorphism and competing formation of multimeric G-quadruplex structures. Hints of the biological relevance of G-triplexes are provided by reports of protein recognition of these structures and their effects on DNA replication in vitro. Interaction of G-triplex DNA with a variety of ligands has been reported, although the search for selective ligands that can distinguish G-triplex from G-quadruplex is on-going. The vast majority of publications in the area have focused on the utilization of G-triplex in biosensing applications, which has shown some advantages compared to G-quadruplex-based systems. These results highlight the potential utility of G-triplex structures in a variety of domains and show its promise in applications in biotechnology, medicine, and research. Full article

G-quadruplexes (G4s) are four-stranded DNA or RNA structures formed by guanine-rich sequences. They occur in functional regions of the genomic material, including the promoter part of genes, regulatory region, and telomeric threads. G4s play a key role in various biological processes, including transcription, replication, and telomere maintenance. Guanidine-containing derivatives can bind to G-quadruplexes, either by intercalating into the structure or by interacting with the grooves or loops. The binding can stabilize the G-quadruplex, potentially affecting its biological function. In this paper, the ability of guanidinium-containing hordatines to interact with G4 was evaluated. Analogues lacking the guanidinium group or showing the benzofuran system instead of the dihydrobenzofuran core were prepared and tested as well. NMR titration and docking calculations were used to probe the binding of the compounds to G4 of c-MYC oncogene. Spectroscopic analyses were consistent with a significant interaction of benzofurans 3 and 4 at the 5′-end and 3′-end tetrads and with the formation of ligand/G-quadruplex complexes with a 2:1 stoichiometry. The resulting data were supported by docking simulations. Cytotoxic activity was evaluated on a model of U2OS osteosarcoma (ATCC HTB-96) and breast cancer (MDA-MB-231) cell lines, further highlighting the key role of the guanidinium fragment and the benzofuran core in the G-quadruplex stabilization. Full article