Search Results (25)

Dengue virus (DENV), which comprises four antigenically distinct serotypes (DENV-1 to DENV-4), remains a major global public health concern and continues to expand geographically; however, the structural features of the viral genome remain incompletely understood. Although G-quadruplexes (G4s) have previously been reported in coding regions of DENV, their presence within the 3′ untranslated region (3′ UTR) has not been experimentally characterized. Here, we focused on selected guanine-rich motifs within the 3′ UTRs of DENV-1 to DENV-4 and investigated their ability to form RNA G4 structures. Using bioinformatic analysis, we identified comparable G-rich regions in the 3′ UTRs of the four serotypes, with serotype-dependent differences in conservation. We then examined the propensity of the selected putative quadruplex-forming sequences (PQSs) to adopt G4 structures using circular dichroism spectroscopy, UV melting analysis, ¹H NMR spectroscopy, ligand-binding analysis, and reverse transcription stop (RT-stop) assays. Our results provided in vitro evidence that the 3′ UTR oligonucleotides from DENV-1 to DENV-4 are capable of forming ligand-responsive G4 structures, with serotype-dependent differences in conservation, stability, and conformational homogeneity. In addition, reverse transcription (RT)-stop analysis revealed ligand-dependent arrest at the corresponding PQS sites in the presence of the G4 ligand 6OTD, which stabilizes G4 structures. These findings suggest the DENV 3′ UTR as an additional source of ligand-responsive RNA G4-forming elements and support future studies on their possible roles in DENV RNA regulation. Full article

G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that help maintain genomic stability and regulate gene transcription. Although the genome contains a vast number of putative G4-forming sequences (PQSs, sequences with intrinsic in vitro G4-forming potential), only a small fraction fold stably into G4 structures within the complex chromatin environment of living cells. Existing deep learning approaches improve predictive accuracy by incorporating cell line–specific epigenetic data; however, their heavy reliance on costly, large-scale sequencing assays (e.g., ChIP-seq) limits broader application to clinical samples and newly profiled cell lines. To address this challenge, we propose iDualG4, an interpretable dual-channel deep learning framework that uses DNA sequence as the only input at inference time. By leveraging a pretrained Enformer module, iDualG4 infers epigenomic proxy features directly from DNA sequence and integrates them with local sequence features, thereby replacing the need for newly measured cell-specific epigenomic assays during prediction. Evaluations across multiple cell lines, including K562, demonstrate that iDualG4 significantly outperforms existing methods, particularly in handling imbalanced data (achieving an AUPR of 0.981 on K562). Interpretability analysis based on DeepSHAP indicates that iDualG4 provides an in vivo G4 prediction tool combining high precision and interpretability without the need for additional experimental sequencing data, and offers a novel computational framework for elucidating how sequence and the epigenetic environment jointly determine genomic G4 formation. 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

Translation of mRNAs is a tightly regulated process in gene expression. In mRNA, the 5′ untranslated region (5′ UTR) controls ribosome recruitment and frequently contains structured elements that modulate translation efficacy. This study investigates stable structural motifs within the 5′ UTR of DDX23 mRNA, encoding a protein relevant for anticancer therapy, as potential regulators and targets for antisense oligonucleotides (ASOs). Despite bioinformatic predictions and transcriptomic validations suggesting RNA G-quadruplex (rG4) formation, comprehensive structural analysis using a light-up assay and CD, UV, and NMR spectroscopy revealed that most putative rG4-forming sequences do not fold into stable rG4 structures, although one of them exists in an equilibrium between rG4 and an alternative, likely hairpin, conformation. Reporter assays using a robust G4 stabilizer also argue against the significant regulatory role of rG4s in DDX23 mRNA translation. Instead, we identified and characterized a stable hairpin structure with potential regulatory function. Based on these findings, we designed fully locked nucleic acid (LNA)-modified ASOs to target this hairpin and regions flanking the upstream open reading frame (uORF) and start codon of the coding sequence. A reporter assay demonstrated that cap-proximal targeting achieved robust translation inhibition up to 80%. In contrast, targeting the efficiently translated uORF was ineffective, presumably due to steric hindrances from the ribosomal complex. The study yields crucial design principles for translation-regulating ASOs: avoid targeting regions shielded by efficient uORF translation and carefully tune ASO-RNA duplex stability to surpass endogenous structures without disrupting regulatory mechanisms. These findings provide insights into the regulation of DDX23 expression and establish a framework for developing ASO-based therapeutics with broad implications for mRNA targeting in anticancer applications. Full article

Symbiosis establishment between Trichoderma atroviride and plant roots triggers the priming of defense responses, among other effects. Currently, there is no clear evidence regarding the molecular mechanisms that allow the plant to remain alert to future stimulus, either by pathogen attack or any other abiotic stress. Epigenetic modifications have emerged as a strategy to explain the increased defense response of plants in a priming state conferred by Trichoderma. Recently, various non-canonical structures of nucleic acids, especially G-quadruplex structures (G-quadruplexes or G4s), have been identified as potential targets during the establishment or maintenance of plant signals. In the present study, we developed a screening test for the identification of putative G4-forming sequences (PQSs) in previously identified Z. mays priming genes. Bioinformatic analysis revealed the presence of PQSs in the promoter region of five essential genes playing a critical role in priming in maize. Biophysical and spectroscopy studies showed the formation of G4s by these PQSs in vitro, and ChIP assays demonstrate their formation in vivo. Therefore, G4 formation could play a role as an epigenetic regulatory mechanism involved in the long-lasting primed state in maize plants. Full article

Long non-coding RNAs (lncRNAs) have emerged as auxiliary regulators of gene expression influencing tumor microenvironment, metastasis and radio-resistance in cancer. The presence of lncRNA in extracellular fluids makes them promising diagnostic markers. LncRNAs deploy higher-order structures to facilitate a complex range of functions. Among such structures, G-quadruplexes (G4s) can be detected or targeted by small molecular probes to drive theranostic applications. The in vitro identification of G4 formation in lncRNAs can be a tedious and expensive proposition. Bioinformatics-driven strategies can provide comprehensive and economic alternatives in conjunction with suitable experimental validation. We propose a pipeline to identify G4-forming sequences, protein partners and biological functions associated with dysregulated lncRNAs in cervical cancer. We identified 17 lncRNA clusters which possess transcripts that can fold into a G4 structure. We confirmed in vitro G4 formation in the four biologically active isoforms of SNHG20, MEG3, CRNDE and LINP1 by Circular Dichroism spectroscopy and Thioflavin-T-assisted fluorescence spectroscopy and reverse-transcriptase stop assay. Gene expression data demonstrated that these four lncRNAs can be potential prognostic biomarkers of cervical cancer. Two approaches were employed for identifying G4 specific protein partners for these lncRNAs and FMR2 was a potential interacting partner for all four clusters. We report a detailed investigation of G4 formation in lncRNAs that are dysregulated in cervical cancer. LncRNAs MEG3, CRNDE, LINP1 and SNHG20 are shown to influence cervical cancer progression and we report G4 specific protein partners for these lncRNAs. The protein partners and G4s predicted in lncRNAs can be exploited for theranostic objectives. Full article

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17–26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections. Full article

The recently introduced semi-orthogonal system of nucleic acid imaging offers a greatly improved method of identifying DNA sequences that are capable of adopting noncanonical structures. This paper uses our newly developed G-QINDER tool to identify specific repeat sequences that adopt unique structural motifs in DNA: TG and AG repeats. The structures were found to adopt a left-handed G-quadruplex form under extreme crowding conditions and a unique tetrahelical motif under certain other conditions. The tetrahelical structure likely consists of stacked AGAG-tetrads but, unlike G-quadruplexes, their stability does not appear to be dependent on the type of monovalent cation present. The occurrence of TG and AG repeats in genomes is not rare, and they are also found frequently in the regulatory regions of nucleic acids, so it is reasonable to assume that putative structural motifs, like other noncanonical forms, could play an important regulatory role in cells. This hypothesis is supported by the structural stability of the AGAG motif; its unfolding can occur even at physiological temperatures since the melting temperature is primarily dependent on the number of AG repeats in the sequence. Full article

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302. Full article

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesis in vivo during zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to fold in vitro as G4. MiR-150 controls the expression of myb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos with in vitro transcribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels of myb mRNA and stronger phenotypes associated with myb knock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related to myb knock-down. Overall, results suggest that the G4 formed in pre-miR-150 functions in vivo as a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis. Full article

Sequences of nucleic acids with the potential to form four-stranded G-quadruplex structures are intensively studied mainly in the context of human diseases, pathogens, or extremophile organisms; nonetheless, the knowledge about their occurrence and putative role in plants is still limited. This work is focused on G-quadruplex-forming sites in two gene sets of interest: drought stress-responsive genes, and genes related to the production/biosynthesis of phenolic compounds in the model plant organism Arabidopsis thaliana. In addition, 20 housekeeping genes were analyzed as well, where the constitutive gene expression was expected (with no need for precise regulation depending on internal or external factors). The results have shown that none of the tested gene sets differed significantly in the content of G-quadruplex-forming sites, however, the highest frequency of G-quadruplex-forming sites was found in the 5′-UTR regions of phenolic compounds’ biosynthesis genes, which indicates the possibility of their regulation at the mRNA level. In addition, mainly within the introns and 1000 bp flanks downstream gene regions, G-quadruplex-forming sites were highly underrepresented. Finally, cluster analysis allowed us to observe similarities between particular genes in terms of their PQS characteristics. We believe that the original approach used in this study may become useful for further and more comprehensive bioinformatic studies in the field of G-quadruplex genomics. Full article

G-quadruplexes (G4s) have been long considered rare and physiologically unimportant in vitro curiosities, but recent methodological advances have proved their presence and functions in vivo. Moreover, in addition to their functional relevance in bacteria and animals, including humans, their importance has been recently demonstrated in evolutionarily distinct plant species. In this study, we analyzed the genome of Pisum sativum (garden pea, or the so-called green pea), a unique member of the Fabaceae family. Our results showed that this genome contained putative G4 sequences (PQSs). Interestingly, these PQSs were located nonrandomly in the nuclear genome. We also found PQSs in mitochondrial (mt) and chloroplast (cp) DNA, and we experimentally confirmed G4 formation for sequences found in these two organelles. The frequency of PQSs for nuclear DNA was 0.42 PQSs per thousand base pairs (kbp), in the same range as for cpDNA (0.53/kbp), but significantly lower than what was found for mitochondrial DNA (1.58/kbp). In the nuclear genome, PQSs were mainly associated with regulatory regions, including 5′UTRs, and upstream of the rRNA region. In contrast to genomic DNA, PQSs were located around RNA genes in cpDNA and mtDNA. Interestingly, PQSs were also associated with specific transposable elements such as TIR and LTR and around them, pointing to their role in their spreading in nuclear DNA. The nonrandom localization of PQSs uncovered their evolutionary and functional significance in the Pisum sativum genome. Full article

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K⁺ and Na⁺ affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K⁺ and Na⁺ + PEG conditions in vitro. We found that K⁺-specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na⁺-specific IP-G4s. Moreover, K⁺- and Na⁺-specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K⁺-specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na⁺-specific IP-G4s; thus, K⁺-specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K⁺- and Na⁺-specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K⁺ and Na⁺ on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future. Full article

In the past two decades, significant efforts have been put into designing small molecules to target selected genomic sites where DNA conformational rearrangements control gene expression. G-rich sequences at oncogene promoters are considered good points of intervention since, under specific environmental conditions, they can fold into non-canonical tetrahelical structures known as G-quadruplexes. However, emerging evidence points to a frequent lack of correlation between small molecule targeting of G-quadruplexes at gene promoters and the expression of the associated protein, which hampers pharmaceutical applications. The wide genomic localization of G-quadruplexes along with their highly polymorphic behavior may account for this scenario, suggesting the need for more focused drug design strategies. Here, we will summarize the G4 structural features that can be considered to fulfill this goal. In particular, by comparing a telomeric sequence with the well-characterized G-rich domain of the KIT promoter, we will address how multiple secondary structures might cooperate to control genome architecture at a higher level. If this holds true, the link between drug–DNA complex formation and the associated cellular effects will need to be revisited. Full article

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4_mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4_mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4_mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt², qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics. Full article