Search Results (77)

Background: Guanine-rich DNA regions can fold into G-quadruplex (G4) structures, which are prevalent in telomeres and oncogene promoters, making them attractive targets for anticancer therapeutics. Small molecules capable of selectively stabilizing G4 DNA can disrupt telomerase activity and oncogene expression, offering a promising strategy for cancer intervention. Methods: A rationally designed series of DPPZ–anhydride-conjugated ligands (1 and 2) and their corresponding quaternized derivatives (1-q and 2-q) were synthesized to investigate the combined effects of π-extension, bromine substitution, and cationic modification on DNA recognition. The synthetic strategy relied on the incorporation of a highly planar DPPZ–anhydride scaffold to enhance π-surface area, followed by selective quaternization to introduce permanent positive charge and reinforce electrostatic interactions with the DNA backbone. All compounds were fully characterized by NMR and spectroscopic methods. The DNA-binding properties of the ligands were systematically evaluated toward duplex (ds-DNA) and G-quadruplex (G4-DNA) structures using UV–Vis absorption titration, fluorescence intercalator displacement (FID) assays, and competitive dialysis experiments. Quaternization markedly enhanced intrinsic binding constants and significantly reduced DC₅₀ values, particularly for G4-DNA. While bromine substitution increased overall binding affinity, it did not substantially improve topology selectivity. Among the series, compound 1-q exhibited the most favorable balance between affinity and G4 selectivity. Results: The interaction of the compounds with BSA was quantified using Stern–Volmer quenching constants, which demonstrated a clear trend of enhanced quenching efficiency upon modification. The binding strength followed a descending order of 1-q > 2-q > 1 > 2, highlighting the superior performance of the first series over the second. These findings indicate that the structural features of 1-q facilitate a more robust interaction within the hydrophobic pockets of the protein. Conclusions: Overall, the results demonstrate that strategic π-conjugation combined with electrostatic reinforcement provides an effective approach for the development of topology-selective DNA-binding ligands. Full article

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry. 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

Background/Objectives: DNA interstrand cross-links (ICLs) mark one of the most deleterious lesions that can preclude strand separation required for essential cellular processes. Efforts to discover ICL-inducing agents and endogenous substrates for ICL repair pathways have led to the identification of structurally diverse ICLs produced by reactive aldehydes and abasic sites, among others. While several studies point to UV rays as ICL-inducing agents, UV ray-induced ICL formation from biologically relevant DNA lesions has been rarely reported. We conjectured that solar radiation-induced reactive oxygen species may give rise to ICLs via further oxidation of DNA lesions with lower redox potential (e.g., 8-oxoadenine (oxoA)). Here, we present the discovery of ICL production via light-induced modification of the major oxidative adenine lesion oxoA. Methods/Results: In the absence of a photosensitizer, both UVC and UVB rays, but not UVA and visible rays, trigger the formation of oxoA-G ICLs, albeit in low yields. By contrast, the inclusion of the naturally occurring photosensitizer riboflavin in the cross-linking reaction makes UVA and visible rays readily generate oxoA-G ICLs, suggesting solar radiation facilitates the formation of oxoA ICLs in vivo. Conclusions: The plausible oxoA-G ICL formation mechanism concerns the further oxidation of oxoA into an iminoquinone, followed by the nucleophilic attack of the opposite guanine on the iminoquinone. OxoA-G ICLs represent rare examples of ICLs produced by photosensitization. These results will contribute to the discovery of a novel form of ICLs induced by solar radiation. Full article

Background: Cervical cancer is among the most prevalent malignancies in women worldwide, and early detection of epigenetic alterations such as Deoxyribose Nucleic Acid (DNA) methylation is of utmost significance for improving clinical results. This study introduces a novel deep learning-based framework for predicting DNA methylation in cervical cancer, utilizing a UNet architecture integrated with an innovative one-hot character encoding technique. Methods: Two encoding strategies, monomer and dimer, were systematically evaluated for their ability to capture discriminative features from DNA sequences. Experiments were conducted on Cytosine–Guanine (CG) sites using varying sequence window sizes of 100 bp, 200 bp, and 300 bp, and sample sizes of 5000, 10,000, and 20,000. Model validation was performed on promoter regions of five cervical cancer-associated genes: miR-100, miR-138, miR-484, hTERT, and ERVH48-1. Results: The dimer encoding strategy, combined with a 300-base pair window and 5000 CG sites, emerged as the optimal configuration. The proposed framework demonstrated better predictive performance, with an accuracy of 91.60%, sensitivity of 96.71%, specificity of 87.32%, and an Area Under the Receiver Operating Characteristic (AUROC) score of 96.53, significantly outperforming benchmark deep learning models, including Convolutional Neural Networks and MobileNet. Validation on promoter regions further confirmed the robustness of the model, as it accurately identified 86.27% of methylated CG sites and maintained a strong AUROC of 83.99, demonstrating its precision–recall balance and practical relevance during validation in promoter-region genes. Conclusions: These findings establish the potential of the proposed UNet-based approach as a reliable and scalable tool for early detection of epigenetic modifications. Thus, the work contributes significantly to improving biomarker discovery and diagnostics in cervical cancer research. Full article

Guanine-rich nucleic acid sequences can adopt G-quadruplex (G4) structures, which pose barriers to DNA replication and repair. The FANCJ helicase contributes to genome stability by resolving these structures, a function linked to its G4-binding site that features an AKKQ amino acid motif. This site is thought to recognize oxidatively damaged G4, specifically those containing 8-oxoguanine (8oxoG) modifications. We hypothesize that FANCJ AKKQ recognition of 8oxoG-modified G4s (8oxoG4s) depends on the sequence context, the position of the lesion within the G4, and overall structural stability. Using fluorescence spectroscopy, we measured the binding affinities of a FANCJ AKKQ peptide for G4s formed by (GGGT)₄, (GGGTT)₄, and (TTAGGG)₄ sequences. G4 conformation and thermal stability were assessed by circular dichroism spectroscopy. Each sequence was modified to include a single 8oxoG at the first (8oxo1), third (8oxo3), or fifth (8oxo5) guanine position. In potassium chloride (KCl), the most destabilized structures were (GGGT)₄ 8oxo1, (GGGTT)₄ 8oxo1, and (TTAGGG)₄ 8oxo5. In sodium chloride (NaCl), the most destabilized were (GGGT)₄ 8oxo1, (GGGTT)₄ 8oxo5, and (TTAGGG)₄ 8oxo5. FANCJ AKKQ binding affinities varied according to damage position and sequence context, with notable differences for (GGGT)₄ in KCl and (TTAGGG)₄ in NaCl. These findings support a model in which FANCJ binding to G4 and 8oxoG4 structures is modulated by both the oxidative damage position and the G4 local sequence environment. 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

The development of electrochemical DNA biosensors occurred by applying different organically coated Mn-NPs such as MnCO₃@OAm, MnCO₃@TEG and MnO₂/Mn₂O₃@TEG, as well as naked MnCO₃ NPs (where OAm = oleylamine and TEG = tetraethylene glycol). The detection performances of PGEs were modified with different types of Mn-NPs, according to the guanine signal magnitudes obtained after double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) immobilization at these surfaces. DNA interaction studies were realized using UV-vis, circular dichroism (CD), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) techniques. In addition, a 3- to 5.4-fold increase in guanine response in the presence of dsDNA and a 2.3-fold increase in the presence of ssDNA were obtained with the developed biosensor. The increased signals in DNA immobilization at the electrode surfaces modified with Mn-NPs compared to bare PGE clearly show that the modification of Mn-NPs increases the electroactive surface area of the electrode. The detection limit (LOD) of dsDNA was calculated as 7.86 μg·L⁻¹ using the MnO₂/Mn₂O₃@TEG type of the Mn-NP-modified biosensor, while the detection limit of ssDNA was calculated as 3.49 μg·L⁻¹ with the MnCO₃@OAm type Mn-NP-modified biosensor. The proposed sensor was applied to a human DNA sample where the amount of dsDNA extract was found to be 0.62 ± 0.03 mg·L⁻¹ after applying the MnO₂/Mn₂O₃@TEG type of Mn-NP-modified biosensor. Full article

Guanine-rich sequences of nucleic acids, including DNA and RNA, are known to fold into non-canonical structures named G-quadruplexes (G4s). Such arrangements of these macromolecular polymers are mainly located in telomeres and in promoter regions of oncogenes and, for this reason, they represent a potential target for compounds with therapeutic applications. In fact, the ligand-mediated stabilization of G4s inhibits telomerase and the activity of transcriptional machinery and counteracts cancer cell immortalization. Flavonoids, along with other classes of small molecules, have been previously tested for their ability to stabilize G4s, but the mechanism of their interaction has not been fully elucidated. In the current work, we report a multi-technique investigation on the binding of tosylated isoflavones obtained by the B-ring modification of compounds from Maclura pomifera to a telomeric DNA sequence. Our study demonstrates that such derivatization leads to compounds showing lower binding affinity but with an increased selectivity toward G4 with respect to double-stranded DNA. The binding mode to the macromolecular target G4 was studied by combining results from electrospray mass spectrometry binding studies, nuclear magnetic resonance experiments and computational simulations. Overall, our findings show that tosylation influences the selectivity toward the macromolecular target by affecting the interaction mode with the nucleic acid. Full article

In mammals, specificity protein 1 (SP1) was the first Cys2-His2 zinc finger transcription factor to be isolated within the specificity protein and Krüppel-like factor (Sp/KLF) gene family. SP1 regulates gene expression by binding to Guanine–Cytosine (GC)-rich sequences on promoter regions of target genes, affecting various cellular processes. Additionally, the activity of SP1 is markedly influenced by posttranslational modifications, such as phosphorylation, acetylation, glycosylation, and proteolysis. SP1 is implicated in the regulation of apoptosis, cell hypertrophy, inflammation, oxidative stress, lipid metabolism, plaque stabilization, endothelial dysfunction, fibrosis, calcification, and other pathological processes. These processes impact the onset and progression of numerous cardiovascular disorders, including coronary heart disease, ischemia-reperfusion injury, cardiomyopathy, arrhythmia, and vascular disease. SP1 emerges as a potential target for the prevention and therapeutic intervention of cardiac ailments. In this review, we delve into the biological functions, pathophysiological mechanisms, and potential clinical implications of SP1 in cardiac pathology to offer valuable insights into the regulatory functions of SP1 in heart diseases and unveil novel avenues for the prevention and treatment of cardiovascular conditions. Full article

In the fight against cancer, researchers have turned their attention to the eukaryotic initiation factor eIF4E, a protein whose increased level is strongly correlated with the development and progression of various types of cancer. Among the numerous strategies devised to tackle eIF4E overexpression, the use of 5′ end mRNA cap analogues has emerged as a promising approach. Here, we present new candidates as potent m⁷GMP analogues for inhibiting translation and interfacing with eIF4E. By employing an appropriate strategy, we synthesized doubly modified mono- and dinucleotide cap analogues, introducing simultaneous substituents at both the N7 and N2 positions of the guanine ring. This approach was identified as an effective and promising combination. Our findings reveal that these dual modifications increase the potency of the dinucleotide analogue, marking a significant advancement in the development of cancer therapeutics targeting the eIF4E pathway. Full article

Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine–cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC. Full article

Transglutaminase type 2 (TG2) is the most ubiquitously expressed and well characterized member of the transglutaminase family. It is a ubiquitous multifunctional enzyme implicated in the regulation of several cellular pathways that support the survival, death, and general homeostasis of eukaryotic cells. Due to its multiple localizations both inside and outside the cell, TG2 participates in the regulation of many crucial intracellular signaling cascades in a tissue- and cell-specific manner, making this enzyme an important player in disease development and progression. Moreover, TG2 is capable of modulating the tumor microenvironment, a process of dynamic tissue remodeling and biomechanical events, resulting in changes which influence tumor initiation, growth, and metastasis. Even if generally related to the Ca²⁺-dependent post-translational modification of proteins, a number of different biological functions have been ascribed to TG2, like those of a peptide isomerase, protein kinase, guanine nucleotide binder, and cytosolic–nuclear translocator. With respect to cancer, TG2′s role is controversial and highly debated; it has been described both as an anti- and pro-apoptotic factor and is linked to all the processes of tumorigenesis. However, numerous pieces of evidence support a tissue-specific role of TG2 so that it can assume both oncogenic and tumor-suppressive roles. Full article

Numerous studies have shown that oxidative modifications of guanine (7,8-dihydro-8-oxoguanine, 8-oxoG) can affect cellular functions. 7,8-Dihydro-8-oxoadenine (8-oxoA) is another abundant paradigmatic ambiguous nucleobase but findings reported on the mutagenicity of 8-oxoA in bacterial and eukaryotic cells are incomplete and contradictory. Although several genotoxic studies have demonstrated the mutagenic potential of 8-oxoA in eukaryotic cells, very little biochemical and bioinformatics data about the mechanism of 8-oxoA-induced mutagenesis are available. In this review, we discuss dual coding properties of 8-oxoA, summarize historical and recent genotoxicity and biochemical studies, and address the main protective cellular mechanisms of response to 8-oxoA. We also discuss the available structural data for 8-oxoA bypass by different DNA polymerases as well as the mechanisms of 8-oxoA recognition by DNA repair enzymes. Full article

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription. Full article