Search Results (682)

R-loops, three-stranded nucleic acid structures formed by an RNA-DNA hybrid, have emerged as important regulators of transcription and genome stability. Although advances in high-throughput sequencing have revealed widespread R-loop landscapes, platform-specific biases hinder the identification of conserved R-loops in specific cell types. Mouse embryonic stem cells, which are transcriptionally active, provide an ideal system for investigating the potential roles of stable R-loops in RNA biology. Here, we integrated 13 independent R-loop profiling datasets from four experimental platforms to define 27,950 Common R-loop regions in mouse embryonic stem cells and characterized their chromatin environment and associated biological functions. Common R-loop regions were reproducibly detected across methods and were preferentially localized to promoter-proximal and genic regions enriched in CpG islands. Genes associated with Common R-loops were highly and stably expressed, showing strong functional enrichment in RNA metabolic processes such as mRNA processing, RNA splicing, and ribonucleoprotein complex biogenesis. Chromatin state analysis revealed that Common R-loops are enriched in transcriptionally active and regulatory contexts. Sequence feature analysis further identified GC skew as a prominent signature of Common R-loops, particularly within transcribed chromatin states. Transcription factor motif analyses have identified distinct regulatory environments in Common R-loop regions, including pluripotency-associated OCT4-SOX2-TCF-NANOG motifs in enhancers, CTCF motifs in open chromatin, and YY1 motifs in promoters. Together, this study provides the first integrated analysis of conserved R-loop regions in mouse embryonic stem cells, revealing their preferential localization at regulatory loci linked to RNA metabolism and highlighting R-loops as structural and functional nodes in RNA biology. Full article

G-quadruplexes (G4s) are emerging as potential antiviral targets. SARS-CoV-2 genomic RNA contains 42 G-rich regions harboring putative G-quadruplex-forming sequences (PQSs). Here, we performed a systematic genomic and structural analysis of SARS-CoV-2 PQSs. It was proposed that non-G-tetrads or different triads may stabilize most G4s in this RNA. Many G4s may include the most stable U·A-U triad. Several G-quadruplexes may be significantly stabilized by 3′ U-tetrad. Large-scale mutational analysis of RNA structural elements containing PQSs showed that most PQSs are highly conserved, while persistent G4-destroying mutations were observed only for one PQS and were transient for two others. Based on G4 position and structural context, we propose that: (i) G4 370 in nsp1 may contribute to cap-independent translation initiation; (ii) certain putative G4s in different genes may assist in co-translational folding of viral proteins; (iii) G4 13385, located upstream of the frameshift stimulation element, may promote formation of a pseudoknot competent for −1 frameshifting. For putative G4s at positions 3467, 13385 and 28903, we analyzed binding to 13 compounds by molecular docking and selected four candidates for molecular dynamics simulations. The ligand EKM emerged as a promising antiviral candidate due to its specific binding to G4 3467. Full article

Chemotherapeutic agents targeting ribosome biogenesis induce profound reorganization of nucleolar architecture, yet how the tumor suppressor p53 governs these structural responses remains unclear. Here, we show that loss of p53 leads to NF-κB-dependent disappearance of nucleolar caps induced by doxorubicin (DOXO). Under these conditions, fibrillarin (FBL), which is normally confined to the nucleolus, relocates to the nucleoplasm and forms foci that partially associate with G-quadruplex (G4) structures, non-canonical nucleic acid secondary structures enriched at transcriptionally active genomic regions. To examine whether this redistribution is linked to transcriptional changes, we integrated publicly available transcriptomic datasets and identified genes that were upregulated in p53-deficient cells under DOXO treatment and downregulated upon FBL depletion. Given that casein kinase 2 alpha (CK2α) is a nuclear binding partner of FBL, we further analyzed CK2α-dependent gene programs. This analysis revealed that a fraction of FBL-responsive genes overlapped with CK2α-dependent signatures and were enriched for promoter-proximal G4 structures. Among candidate regulators, the G4-binding transcription factor MAZ emerged as a potential mediator linking nucleoplasmic FBL and CK2α to G4-associated transcriptional regulation. Together, our findings identify a mechanism linking loss of p53 to G4-associated transcriptional reprogramming through nucleolar architectural disruption mediated by an FBL–CK2α–MAZ axis during DOXO treatment. 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

Post-transcriptional regulation of gene expression is influenced by RNA-binding proteins (RBPs) and small non-coding RNAs that bind to conserved mRNA sequences to modulate mRNA processing. These regulatory molecules affect the structural conformation of mRNAs, creating formations like G-quadruplexes (G4s), which alter translation initiation and regulatory-factor site accessibility. Recent studies have highlighted Nuclear factor erythroid 2–related factor 2 (NRF2) as a key regulator of cellular redox homeostasis and cellular response to oxidative stress. An intriguing feature of NRF2 is the structural formation of its 5′ untranslated region (UTR), which may promote or inhibit translation initiation depending on the cellular context. In this study with minigenes, we provide in vitro evidence of RNA G4s in the NRF2 mRNA’s 5′ UTR under basal (no stress) conditions. Achieved via electrophoretic mobility shift assay and fluorescence spectra in the presence of Pyridostatin. Understanding how structural motifs within NRF2 5′ UTRs influence mRNA function provides insights into a common molecular mechanism underlying diseases where NRF2 is dysregulated, like cancers, cardiovascular disease, and neurodegeneration, and highlights potential therapeutic avenues through regulation of NRF2. 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

In recent years, harmful algal blooms have led to frequent occurrences of shellfish toxin contamination, posing a significant threat to the safety of aquatic products and public health. As a potent neurotoxin, domoic acid (DA) can accumulate in shellfish, highlighting the urgent need for rapid and highly sensitive detection methods. In this study, we developed a fluorescent aptasensor based on a dual-signal amplification system by combining G-quadruplex (G4) dimers with multi-walled carbon nanotubes (CNTs). The sensor is designed with a hairpin-structured aptamer as the recognition probe, where short multi-walled CNTs serve as both a fluorescence quencher and platform, and G4 dimers are incorporated into the sensing interface to enhance signal output. In the absence of the target, the hairpin-structured aptamer remains closed, keeping the fluorescence signal “off”. Upon binding to DA, the aptamer undergoes a specific conformational change that exposes the G4-dimer sequence. The exposed sequence then binds to thioflavin T (ThT), which in turn generates a greatly enhanced fluorescence signal, leading to a substantial fluorescence enhancement and completing the second stage of the cascade amplification. Under optimal conditions, the constructed sensor achieves rapid detection of DA within 5 min, with a low detection limit of 1.1 ng/mL. This work presents a valuable tool for the rapid and sensitive detection of DA in shellfish, with promising applications in marine environmental monitoring and food safety regulation. Full article

G-quadruplex (G4) DNAzymes, guanine-rich sequences that fold into four-stranded structures and bind hemin to mimic peroxidase activity, are widely used in biosensing. Split G4 DNAzymes offer conditional activation upon target recognition, enabling high specificity and modularity. However, achieving low OFF-state leakage remains a major challenge. Here, we systematically characterized four representative G4 scaffolds, C-myc, Bcl2, PS5.M, and C-kit, under standardized ABTS/H₂O₂ conditions to assess their kinetic properties and suitability for split designs. C-myc exhibited the highest sustained activity and near-linear concentration dependence, making it ideal for quantitative sensing, while Bcl2 showed durable catalysis suited for extended read windows. C-kit produced rapid bursts with early plateaus, favoring binary outputs, and PS5.M initiated quickly but inactivated rapidly, suggesting potential application of systems requiring fast response. Split-mode analysis revealed that symmetric 2:2 partitions often retained significant activity, whereas asymmetric 3:1 splits reduced but did not eliminate leakage. Among the four G4 DNAzymes, PS5.M demonstrated the most promising OFF-state suppression. Design strategies to minimize leakage including non-classical splits, loop/flank edits, and template-assisted assembly could be used to optimize biosensor functionalities. These findings identify essential factors critical for designing robust split DNAzyme biosensors, advancing applications in diagnostics and molecular logic gates. Full article

Temozolomide (TMZ) remains foundational in the management of adult-type diffuse gliomas in general, and glioblastoma specifically. However, its efficacy harbors an evolutionary trade-off. TMZ drives its cytotoxicity through generating O⁶-methylguanine lesions, especially active in MGMT-silenced, mismatch repair (MMR)-proficient tumors. By selecting for acquired MMR-deficient subclones, often via MSH6 inactivation, this process escalates into a hypermutator phenotype, generating thousands of de novo alterations. This is a hallmark of the mutational signature known as SBS11, characterized by C>T transitions, which is associated with TMZ treatment. The hypermutator phenotype drives heterogeneity, therapeutic resistance, spatial diversification, and distant recurrence. Despite harboring a mutational burden comparable to melanoma and lung cancer, TMZ-induced hypermutation does not sensitize gliomas to immune checkpoint blockade. This resistance reflects the profoundly immunosuppressive brain microenvironment, impaired antigen presentation, marked transcriptional plasticity, and perhaps also the frequent use of corticosteroids. Emerging strategies aim to exploit vulnerabilities created by TMZ-mediated genomic instability, including PARP, ATR, WEE1, and AURKA inhibition; alternative alkylators; metabolic rewiring; and G-quadruplex stabilization. Notably, the real-time detection of evolving mutational signatures via CSF-based liquid biopsies may enable adaptive therapy before radiographic progression. By reframing TMZ as a potent evolutionary agent rather than a conventional chemotherapy, this review synthesizes recent mechanistic insights and translational opportunities to guide a next-generation, evolution-informed treatment paradigm for glioma. 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