Search Results (169)

The role of G-quadruplexes (G4s) in gene regulation has been widely documented, especially in gene promoters. However, the transcriptional mechanisms involving G4s in other regulatory regions remain largely unexplored. In this study, we integrated the G4-DNA data derived from 22 breast cancer patient-derived tumor xenograft (PDTX) models and MCF7 cell line as potential breast cancer-associated G4s (BC-G4s). Genome-wide analysis showed that BC-G4s are more prevalent in gene promoters and the first introns. The genes accommodating promoter or intronic BC-G4s show significantly higher transcriptional output than their non-G4 counterparts. The biased distribution of BC-G4s in close proximity to the transcription start site (TSS) is associated with an enrichment of transcription factor (TF) interactions. A significant negative correlation was detected between the G4–TF interactions within the first introns and their cognate promoters. These different interactions are complementary rather than redundant. Furthermore, the differentially expressed genes (DEGs) harboring promoter and first intron BC-G4s are significantly enriched in the cell cycle pathway. Notably, promoter BC-G4s of DEGs could be a central hub for TF–TF co-occurrence. Our analysis also revealed that G4-related single nucleotide variants (SNVs) affect the stability of G4 structures and the transcription of disease-related genes. Collectively, our results shed light on how BC-G4s within promoters and first introns regulate gene expression and reinforce the critical role of G4s and G4-related genes in breast cancer-associated processes. Full article

Tuberculosis (TB) poses a persistent global health threat exacerbated by the emergence of drug-resistant strains; hence, there is a continuous quest for novel antimicrobial agents. Despite efforts to develop effective therapies, existing treatments require a relatively long duration of therapy to eradicate the pathogen due to its virulence factors, pathogenesis patterns, and ability to enter dormant states. This can lead to a higher risk of treatment failure due to poor patient adherence to the complex regimen. As a result, considerable research is necessary to identify alternative antituberculosis agents. The marine environment, particularly marine-derived fungi, has recently gained interest due to its potential as an abundant source of bioactive natural products. This review covers 19 genera of marine-derived fungi and 139 metabolites, 131 of which exhibit antimycobacterial activity. The integrated dataset pinpoints the fungal genera and chemical classes that most frequently yield potent antimycobacterial hits while simultaneously exposing critical gaps, such as the minimal evaluation of compounds against dormant bacilli and the presence of underexplored ecological niches and fungal genera. Several compounds exhibit potent activity through uncommon mechanisms, including the inhibition of mycobacterial protein tyrosine phosphatases (MptpB/MptpA), protein kinase PknG, ATP synthase and the disruption of mycobacterial DNA via G-quadruplex stabilization. Structure–activity relationship (SAR) trends are highlighted for the most potent agents, illuminating how specific functional groups underpin target engagement and potency. This review also briefly proposes a dereplication strategy and approaches for toxicity mitigation in the exploration of marine-derived fungi’s natural products. Through this analysis, we offer insights into the potency and challenges of marine-derived fungi’s natural products as hit compounds or scaffolds for further antimycobacterial research. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease’s molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes. Full article

Tetrahelical DNA structures, such as G-quadruplexes (G4s) or i-motifs (iMs), are adopted by sequences comprising several G/C tracts, exist in equilibria with respective duplexes, and may contribute to genomic instability upon helicase deficiency. To understand genomic rearrangements resulting from the juxtaposition of G/C-rich DNA duplexes, models of possible intermediate structures are needed. In this study, a general strategy for creating and verifying in silico 3D models of tetrahelical DNA was proposed. This strategy was used to investigate contacts of two or more duplexes with n G₃/C₃ tracts (n = 2–6) separated by T/A nucleotides. The revealed viable structures of DNA–DNA contacts include stacks of right-handed and left-handed G-quadruplexes (G4s), Holliday structure-resembling assemblies with the G4 and iM opposite each other on the borders of the central “hole”, etc. Based on molecular dynamic simulations, the most probable variants were determined by estimating the contributions to the free energy. The results may be used to clarify the mechanisms of strand exchange and other rearrangements upon DNA breaks near prolonged G/C-rich sites in living systems. Additionally, they provide a balanced view on the polymorphic versus programmed DNA assemblies in artificial systems. Full article

Background/Objectives: Aberrant expression of c-MYC drives aggressive cancers. A guanine-rich promoter sequence (Pu27) folds into a transcriptionally repressive G-quadruplex (G4). Epigallocatechin gallate (EGCG), the main green tea polyphenol, displays anticancer activity, but clear, easily replicated evidence for direct binding to the c-MYC G4 is lacking. We therefore obtained the first biophysical confirmation of an EGCG–c-MYC G4 interaction using routine UV–visible spectroscopy. Methods: A pre-annealed Pu27 G4 (5 µM) in potassium-rich buffer was titrated with freshly prepared EGCG (0–20 µM) at 25 °C. Full-range UV–Vis spectra (220–400 nm) were recorded after each addition, and absorbance variations at the DNA (260 nm) and ligand (275 nm) maxima were quantified across three independent replicates. Results: EGCG induced pronounced, concentration-dependent hyperchromicity at 260 nm, reaching ~8–10% above baseline at a 4:1 ligand/DNA ratio and exhibiting saturable binding behaviour. Concurrently, the 275 nm band displayed relative hypochromicity coupled with a subtle bathochromic shift. These reciprocal perturbations—absent in buffer-only controls—constitute definitive evidence of a specific EGCG•G4 complex most consistent with external π-stacking or groove engagement rather than intercalation. Conclusions: This study delivers the first rigorous, quantitative UV–Vis confirmation that a readily consumed dietary polyphenol directly targets the c-MYC promoter G4. By marrying conceptual elegance with methodological accessibility, it provides a compelling molecular rationale for EGCG’s anti-oncogenic repertoire, inaugurates an expedient platform for screening G4-reactive nutraceuticals, and paves the way for structural and cellular investigations en route to next-generation c-MYC-directed therapies. Full article

AS1411 is a G-quadruplex (G4) aptamer that binds tightly to nucleolin (NCL) on the cell surface and has shown strong anticancer effects. However, this aptamer is highly polymorphic, presenting different types of G4s, which may hinder its preclinical application. Several modifications have been made to decrease the polymorphism of this aptamer. In this work, we designed six AS1411 derivatives by substituting guanine with thymine in the central linker and modifying the number of thymines either in the linker itself and/or at both ends of the sequence. The G4 formation, stability, and NCL binding were evaluated by several biophysical techniques and computational and cell studies. Overall, a decrease in polymorphism of G4-forming sequences compared to AS1411 is observed by size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy in the presence of potassium salt. The melting experiments reveal a higher ability of the derivatives without thymine at both sequence ends to form a G4, consistent with the G4H score predictions. Additionally, it is possible to conclude that deletions of T in the central core increase the ability to form G4. Moreover, the AS1411 derivatives bind NCL with high affinity (K_D values in the 10⁻⁹ M range), particularly the sequences with only thymine modifications in the central linker. In silico studies reveal structural insights and demonstrate that AS1411 derivatives interact with NCL, establishing multiple interactions with the different domains, thereby further supporting the experimental findings. By using a lung cancer cell line with high cell surface NCL expression, we evaluate the internalization and uptake of AS1411 derivatives, identifying the derivative-lacking thymines in the central core as the ones with the highest internalization and cellular uptake. Full article

Background/Objectives: Some G-quadruplex (G4)-forming nucleic acid sequences bind a hemin cofactor to enhance its peroxidase-like activity. This has been implemented in a variety of bioanalytical assays benefiting from analyte-dependent peroxidation of a chromogenic organic substrate (e.g., ABTS) to produce a color change. Adenine and cytosine nucleotides in the vicinity of the G4 hemin-binding site promote the peroxidation reaction. In this work, the effect of G4 loop and flanking nucleotides on the colorimetric signal of split hybridization probes utilizing hemin-G4 signal reporters was tested. Methods: G4s varying by loop sequences and flanking nucleotides were tested with hemin for ABTS peroxidation (A₄₂₀), and the signal was compared with that produced by the most catalytically efficient complexes reported in the literature using one-way ANOVA and post hoc pairwise comparison with Tukey’s HSD test. The best G4s were used as signal transducers in the split peroxidase deoxyribozyme (sPDz) probes for sensing two model nucleic acid analytes, as well as in a cascade system, where the analyte-dependent assembly of an RNA-cleaving deoxyribozyme 10–23 results in G4 release. Results: Intramolecular G4s (G₃T)₃G₃TC or G₃T₃G₃ATTG₃T₃G₃ were found to be the most efficient hemin PDzs. When splitting intramolecular G4 for the purpose of sPDz probe design, the addition of a flanking d(TC) sequence at one of the G4 halves or d(ATT) in a loop connecting the second and third G-tracts helps boost analyte-dependent signal intensity. However, for the cascade system, the effect of d(TC) or d(ATT) in the released G4 was not fully consistent with the data reported for intramolecular G4-hemin complexes. Conclusions: Our findings offer guidance on the design of split hybridization probes utilizing the peroxidase-like activity of G4-hemin complexes as a signal transducer. Full article

Within the framework of rational drug design, this study introduces a novel approach to enhance the specificity of small molecules in targeting cancer cells. This approach starts from the use of dyads merging into a single entity, a naphthalene diimide (NDI) and core-extended NDI (ceNDI), both known as G-quadruplex (G4) ligands and fluorescent probes. The strategy aims to leverage the unique diagnostic strengths of the ceNDI moiety featuring red emission by improving its binding affinity and target selectivity through inclusion in dyads built with different linkers. The newly developed NDI-ceNDI dyads are promising probes, as they exhibit fluorescence turn-on upon DNA recognition and induced circular dichroism signals dependent on DNA conformation. Both dyads have an excellent affinity for hybrid G4, with two orders of magnitude higher binding constants than those for ds DNA. Their high cytotoxicity on cancer cell lines further demonstrates their potential as therapeutic agents, highlighting the role of the linker in target selectivity. Specifically, only the dyad with the rigid triazole linker exhibits selectively induced DNA damage in transformed cells, compared to normal cells primarily targeting telomeric regions. Our findings shed light on DIPAC’s potential as a promising theranostic agent, offering insights into future developments in precision medicine. Full article

G-quadruplexes (G4s) are unique nucleic acid structures composed of guanine-rich (G-rich) sequences that can form diverse topologies based on the arrangement of their four strands. G4s have attracted attention for their potential roles in various biological processes and human diseases. In this review, we focus on the G4 structures formed by human telomeric sequences, (GGGTTA)_n, and the hexanucleotide repeat expansion, (GGGGCC)_n, in the first intron region of the chromosome 9 open reading frame 72 (C9orf72) gene, highlighting their structural diversity and biological significance. Human telomeric G4s play crucial roles in telomere retention and gene regulation. In particular, we provide an in-depth summary of known telomeric G4s and focus on our recently discovered chair-type conformation, which exhibits distinct folding patterns. The chair-type G4s represent a novel folding pattern with unique characteristics, expanding our knowledge of telomeric G4 structural diversity and potential biological functions. Specifically, we emphasize the G4s formed by the (GGGGCC)_n sequence of the C9orf72 gene, which represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The thorough structural analysis in this review advances our comprehension of the disease mechanism and provides valuable insights into developing targeted therapeutic strategies in ALS/FTD. Full article

DNA G-quadruplexes (G4) are non-canonical DNA structures that play key roles in various biological processes. Antibody-dependent sequencing is an important tool for identifying intracellularly formed DNA G4s, and peak calling is a crucial step in processing the sequencing data. As the applicability of existing peak calling algorithms to intracellular G4 data has not been previously assessed, we systematically compared and evaluated these algorithms to determine those best suited for G4 detection. We selected seven representative candidates from 43 published peak calling algorithms for detailed evaluation. The performance of each candidate on six published intracellular G4 sequencing datasets (GSE107690, GSE145090, GSE133379, GSE178668ChIP-seq, GSE178668CUT&Tag, GSE221437) were assessed by precision and recall against customized benchmarks integrating results from multiple algorithms, as well as consistency with known G4 information (pG4 predicted by pqsfinder, oG4 from GSE63874, and multi-cell-line conserved G4s) and epigenetic signals. We identified MACS2, PeakRanger, and GoPeaks as the most effective algorithms for analyzing intracellular G4 sequencing data, and attributed their superior performance partially to the distribution model of sequencing reads/fragments used in the hypothesis testing step of the peak calling procedures. These findings provide guidance and rationale for selecting peak callers appropriate for intracellular G4 data. Full article

RNA G-quadruplexes (rG4s) in 5′-UTRs represent complex regulatory elements capable of both inhibiting and activating mRNA translation through diverse mechanisms in eukaryotes. This review analyzes the evolution of our understanding of 5′-UTR rG4-mediated translation regulation, from early discoveries of simple translation inhibitors to the current recognition of their multifaceted regulatory roles. We discuss canonical and non-canonical rG4 structures, their interactions with regulatory proteins, including helicases and FMRP, and their function in both cap-dependent and IRES-mediated translation. Special attention is given to the synergistic effects between rG4s and upstream open reading frames (uORFs), stress-responsive translation regulation, and their role in repeat-associated non-AUG (RAN) translation linked to neurodegenerative diseases. We critically evaluate methodological challenges in the field, including limitations of current detection methods, reporter system artifacts, and the necessity to verify rG4 presence in endogenous transcripts. Recent technological advances, including genome editing and high-throughput sequencing approaches, have revealed that rG4 effects are more complex and context-dependent than initially thought. This review highlights the importance of developing more robust methodologies for studying rG4s at endogenous levels and carefully reevaluating previously identified targets, while emphasizing their potential as therapeutic targets in various diseases. Full article

Background: The methylation of cytosine residues at CpG sites within the O6-methylguanine-DNA methyltransferase (MGMT) promoter is a key biomarker in glioblastoma therapy. The MGMT promoter (MGMTp) contains multiple guanine-rich sequences capable of folding into G-quadruplexes (G4s), but their relevance for MGMTp methylation is poorly understood. Objectives: Our study explores the impact of potential G-quadruplex-forming sequences (PQS) in the MGMT promoter CpG island on the activity of de novo DNA methyltransferase Dnmt3a. Additionally, we investigate their influence on the accuracy of methylation pattern detection using nanopore sequencing. Methods: Nanopore sequencing was employed to analyze the methylation of 94 clinically significant CpG sites in the human MGMTp using an in vitro de novo methylation system. Circular dichroism spectroscopy was used to identify G4 structures within the MGMTp CpG island. Interactions between the catalytic domain of Dnmt3a and the PQS from the MGMTp were examined by biolayer interferometry. Results: Guanine-rich DNA strands of the PQSs in the MGMTp were hypomethylated, while the complementary cytosine-rich strands were methylated by DNA methyltransferase Dnmt3a with higher efficiency. The accuracy of detecting modified bases in the PQS was significantly lower compared to surrounding sequences. Single-stranded guanine-rich DNA sequences from the MGMTp exhibited strong binding to Dnmt3a-CD, with an affinity approximately 10 times higher than their cytosine-rich complements (K_d = 3 × 10⁻⁸ M and 3 × 10⁻⁷ M, respectively). By binding to Dnmt3a, G4-forming oligonucleotides from MGMTp effectively inhibited the methylation reaction (IC₅₀ 6 × 10⁻⁷ M). Conclusions: The obtained data indicate the role of PQSs in establishing de novo methylation of the MGMT promoter. They also highlight the challenges of sequencing guanine-rich regions and the impact of specific de novo methylation patterns on clinical data interpretation. Full article

Apurinic/apyrimidinic (AP) sites are endogenous DNA lesions widespread in human cells. Having no nucleobases, they are noncoding and promutagenic. AP site repair is generally initiated through strand incision by AP endonuclease 1 (APE1). Although AP sites’ repair in regular B-DNA has been studied extensively, their processing in G-quadruplexes (G4s) has received much less attention. Here, we used the hTERT promoter region that is capable of forming three stacked parallel G4s to understand how AP sites can influence higher-order quadruplex folding and stability and how a G4 affects the efficiency of human APE1-mediated AP site processing. We designed a series of synthetic single- and double-stranded DNA constructs of varying lengths containing a stable AP site analog in both G- and C-rich strands at positions corresponding to somatic driver mutations. Using circular dichroism, we studied the effect of the AP site on hTERT G4 structure and stability. Bio-layer interferometry and gel-based approaches were employed to characterize APE1 binding to the designed DNA substrates and AP site processing. It was shown that (i) an AP site leads to G4 destabilization, which depends on the lesion location in the G4 scaffold; (ii) APE1 binds tightly to hTERT G4 structure but exhibits greatly reduced cleavage activity at AP sites embedded in the quadruplex; and (iii) a clear correlation was revealed between AP site-induced hTERT G4 destabilization and APE1 activity. We can hypothesize that reduced repair of AP sites in the hTERT G4 is one of the reasons for the high mutation rate in this promoter region. Full article

G-quadruplexes (G4s) are distinctive four-stranded nucleic acid structures formed by guanine-rich sequences, making them attractive targets for drug repurposing efforts. Modulating their stability and function holds promise for treating diseases like cancer. To identify potential drug candidates capable of interacting with these complex DNA formations, docking studies and molecular dynamics (MDs) simulations were conducted. Our analysis revealed kanamycin’s ability to bind to various G4 structures, offering valuable insights into its potential as a modulator of G4 activity. Kanamycin exhibited favorable interactions with both parallel and hybrid G4 topologies in human structures, suggesting a broader mechanism of action for aminoglycosides. These findings may also shed light on aminoglycoside-associated toxicities, indicating that their effects might extend to binding non-ribosomal RNA structures. In summary, this research highlights kanamycin’s potential as a promising tool for influencing G4 dynamics, paving the way for innovative therapeutic strategies targeting G4-related pathways. Full article

G-quadruplexes (G4s) are non-canonical secondary structures that play a crucial role in the regulation of genetic expression. This study explores the interaction between G4s and a small family of oligostyrylbenzene (OSB) derivatives, characterized by tris(styryl)benzene and tetrastyrylbenzene backbones, functionalized with either trimethylammonium or 1-methylpyridinium groups. Initially identified as DNA ligands, these OSB derivatives have now been recognized as potent G4 binders, surpassing in binding affinity commercially available ligands such as pyridostatin and displaying good selectivity for G4s over duplex DNA. Furthermore, OSB derivatives 1 and 2 demonstrated significant antiparasitic activity against bloodstream forms of T. brucei and extracellular L. major, with high selectivity indices when compared to MRC-5 healthy control cells. Derivatives 1 and 2 exhibited moderate biocidal effects against a range of Gram-positive and Gram-negative bacterial strains. Notably, a synergistic antibacterial effect was observed when these compounds were combined with traditional antibiotics, particularly against Acinetobacter baumannii, highlighting their potential utility in addressing drug-resistant bacterial infections. The differences in bioactivity among the OSB derivatives can be attributed to variations in cellular uptake, as proved by flow cytometry analysis. This suggests that the degree of cellular internalization plays a pivotal role in the observed antiparasitic and antibacterial efficacy. Full article