G-quadruplex DNA Research Articles

Werner helicase interacting protein 1 contributes to G-quadruplex processing in human cells

Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.

Accumulation of DNA G-quadruplex in mitochondrial genome hallmarks mesenchymal senescence.

Searching for biomarkers of senescence remains necessary and challenging. Reliable and detectable biomarkers can indicate the senescence condition of individuals, the need for intervention in a population, and the effectiveness of that intervention in controlling or delaying senescence progression and senescence-associated diseases. Therefore, it is of great importance to fulfill the unmet requisites of senescence biomarkers especially when faced with the growing global senescence nowadays. Here, we established that DNA G-quadruplex (G4) in mitochondrial genome was a reliable hallmark for mesenchymal senescence. Via developing a versatile and efficient mitochondrial G4 (mtG4) probe we revealed that in multiple types of senescence, including chronologically healthy senescence, progeria, and replicative senescence, mtG4 hallmarked aged mesenchymal stem cells. Furthermore, we revealed the underlying mechanisms by which accumulated mtG4, specifically within respiratory chain complex (RCC) I and IV loci, repressed mitochondrial genome transcription, finally impairing mitochondrial respiration and causing mitochondrial dysfunction. Our findings endowed researchers with the visible senescence biomarker based on mitochondrial genome and furthermore revealed the role of mtG4 in inhibiting RCC genes transcription to induce senescence-associated mitochondrial dysfunction. These findings depicted the crucial roles of mtG4 in predicting and controlling mesenchymal senescence.

Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands.

Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.

DNA G-quadruplexes in the genome of Trypanosoma cruzi as potential therapeutic targets for Chagas disease: Dithienylethene ligands as effective antiparasitic agents

Chagas disease is caused by the parasite Trypanosoma cruzi and affects over 7 million people worldwide. The two actual treatments, Benznidazole (Bzn) and Nifurtimox, cause serious side effects due to their high toxicity leading to treatment abandonment by the patients. In this work, we propose DNA G-quadruplexes (G4) as potential therapeutic targets for this infectious disease. We have found 174 PQS per 100,000 nucleotides in the genome of T. cruzi and confirmed G4 formation of three frequent motifs. We synthesized a family of 14 quadruplex ligands based in the dithienylethene (DTE) scaffold and demonstrated their binding to these identified G4 sequences. Several DTE derivatives exhibited micromolar activity against epimastigotes of four different strains of T. cruzi, in the same concentration range as Bzn. Compounds L3 and L4 presented remarkable activity against trypomastigotes, the active form in blood, of T. cruzi SOL strain (IC50 = 1.5–3.3 μM, SI = 25–40.9), being around 40 times more active than Bzn and displaying much better selectivity indexes.

Research progress in targeting DNA G-quadruplexes using natural products

DNA G-quadruplex(G4) is a guanine-rich single-stranded DNA sequence that spontaneously folds into a spherical four-stranded DNA secondary structure in oncogene promoter sequences and telomeres. G4s are highly associated with the occurrence and development of cancer and have emerged as promising anticancer targets. Natural products have long been important sources of anticancer drug development. In recent years, significant progress has been made in the discovery of natural drugs targeting DNA G4s, with many DNA G4s have been confirmed as promising targets of natural products, including MYC-G4, KRAS-G4, PDGFR-β-G4, BCL-2-G4, VEGF-G4, and telomeric G4. This review summarizes the research progress in discovering natural small molecules that target DNA G4s and their binding mechanisms. It also discusses the opportunities of and challenges in developing drugs targeting DNA G4s. This review will serve as a valuable reference for the research on natural products, particularly in the development of novel antitumor medications.

Curcumin Knoevenagel's Schiff Base as a Promising Stabilizer of G-Quadruplex Structure.

G-quadruplex DNA sequences present in the promoter and telomere regions of the genomic sequence are considered therapeutic targets for the treatment of cancer. Curcumin, derived from Curcuma longa, has been known as a quadruplex binder and has a potential role in the apoptosis of cancer cells. Here, we have reported the Schiff base ligand of curcumin synthesized through the condensation of the amino acid L-tryptophan and the knoevenagel derivative of curcumin (4-nitrobenzylidene curcumin (NBC)) as a potential G-quadruplex binder. Thus, spectroscopic and biophysical studies reveal a higher binding affinity of the ligand Sb-NBC towards the promoter and telomere G-quadruplex sequence as compared to the parent NBC. The ligand Sb-NBC highly stabilizes the parallel and hybrid G-quadruplex topologies to 10.5 °C-6.4 °C. Interestingly, the ligands also exhibit selective cytotoxicity toward cancer cells over normal cells. Taken together, this work provides evidence of the possibility of applying curcumin Schiff base in cancer therapy to regulate oncogene expression in cancer cells.

A Cost-Effective Hemin-Based Artificial Enzyme Allows for Practical Applications.

Nanomaterials excel in mimicking the structure and function of natural enzymes while being far more interesting in terms of structural stability, functional versatility, recyclability, and large-scale preparation. Herein, the story assembles hemin, histidine analogs, and G-quadruplex DNA in a catalytically competent supramolecular assembly referred to as assembly-activated hemin enzyme (AA-heminzyme). The catalytic properties of AA-heminzyme are investigated both in silico (by molecular docking and quantum chemical calculations) and in vitro (notably through a systematic comparison with its natural counterpart horseradish peroxidase, HRP). It is found that this artificial system is not only as efficient as HRP to oxidize various substrates (with a turnover number kcat of 115 s-1) but also more practically convenient (displaying better thermal stability, recoverability, and editability) and more economically viable, with a catalytic cost amounting to <10% of that of HRP. The strategic interest of AA-heminzyme is further demonstrated for both industrial wastewater remediation and biomarker detection (notably glutathione, for which the cost is decreased by 98% as compared to commercial kits).

A non-B DNA binding peptidomimetic channel alters cellular functions

DNA binding transcription factors possess the ability to interact with lipid membranes to construct ion-permeable pathways. Herein, we present a thiazole-based DNA binding peptide mimic TBP2, which forms transmembrane ion channels, impacting cellular ion concentration and consequently stabilizing G-quadruplex DNA structures. TBP2 self-assembles into nanostructures, e.g., vesicles and nanofibers and facilitates the transportation of Na+ and K+ across lipid membranes with high conductance (~0.6 nS). Moreover, TBP2 exhibits increased fluorescence when incorporated into the membrane or in cellular nuclei. Monomeric TBP2 can enter the lipid membrane and localize to the nuclei of cancer cells. The coordinated process of time-dependent membrane or nuclear localization of TBP2, combined with elevated intracellular cation levels and direct G-quadruplex (G4) interaction, synergistically promotes formation and stability of G4 structures, triggering cancer cell death. This study introduces a platform to mimic and control intricate biological functions, leading to the discovery of innovative therapeutic approaches.

Heme oxygenase-1 protects cells from replication stress

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress.Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation.Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.

K+-Specification with Flavone P0 Probe in a G-Quadruplex DNA.

G-quadruplex (G4) DNA is considered as a prospective therapeutic target due to its potential biological significance. To understand G4 biological roles and function, a G4-specific fluorescent probe is necessary. However, it is difficult for versatile G4 to precisely recognize without perturbing their folding dynamics. Herein, we reported that flavone P0 can be a fluorescent probe for G4 DNA-specific recognition and have developed a highly selective detection of K+ ion by dimeric G4/P0 system. When comparing various nucleic acid structures, including G4, i-motif, ss/ds-DNA, and triplex, an apparent fluorescence enhancement is observed in the presence of G4 DNA for 85-fold, but only 8-fold for non-G4 DNA. Furthermore, based on fluorescent probe of flavone P0 for G4 DNA screening, the noncovalent dimeric G4/P0 system is exploited as a K+ sensor, that selectively responds to K+ with a 513-fold fluorescence enhancement and a detection range for K+ ion concentration from 0 to 500 mM. This K+ sensor also has a remarkably anti-interference ability for other metal cations, especially for a high concentration of Na+. These results have demonstrated that flavone P0 is an efficient tool for monitoring G-quadruplex DNA and endows flavone P0 with bioanalytical and medicinal applications.

Structural Optimization of Azacryptands for Targeting Three-Way DNA Junctions.

Transient melting of the duplex-DNA (B-DNA) during DNA transactions allows repeated sequences to fold into non-B-DNA structures, including DNA junctions and G-quadruplexes. These noncanonical structures can act as impediments to DNA polymerase progression along the duplex, thereby triggering DNA damage and ultimately jeopardizing genomic stability. Their stabilization by ad hoc ligands is currently being explored as a putative anticancer strategy since it might represent an efficient way to inflict toxic DNA damage specifically to rapidly dividing cancer cells. The relevance of this strategy is only emerging for three-way DNA junctions (TWJs) and, to date, no molecule has been recognized as a reference TWJ ligand, featuring both high affinity and selectivity. Herein, we characterize such reference ligands through a combination of in vitro techniques comprising affinity and selectivity assays (competitive FRET-melting and TWJ Screen assays), functional tests (qPCR and Taq stop assays) and structural analyses (molecular dynamics and NMR investigations). We identify novel azacryptands TrisNP-amphi and TrisNP-ana as the most promising ligands, interacting with TWJs with high affinity and selectivity. These ligands represent new molecular tools to investigate the cellular roles of TWJs and explore how they can be exploited in innovative anticancer therapies.

Unlocking the potential of protein-derived peptides to target G-quadruplex DNA: from recognition to anticancer activity.

Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.

Investigating the preferential interaction between imatinib mesylate and VEGF G-quadruplex DNA as therapeutic strategies for cancer treatment: Biophysical and molecular modelling approaches

G-Quadruplex DNA (GQ–DNA) is one of the most important non-canonical nucleic acid structures. GQ–DNA forming sequences are present in different crucial genomic regions and are abundant in promoter regions of several oncogenes. Therefore, GQ–DNA is an important target for anticancer drugs and hence binding interactions between GQ–DNA and small molecule ligands are of great importance. Since GQ–DNA is a highly polymorphic structure, it is important to identify ligand molecules which preferentially target a particular quadruplex sequence. In this present study, we have used a FDA approved drug called imatinib mesylate (ligand) which is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia, gastrointestinal stromal tumours. Different spectroscopic techniques as well as molecular docking investigations and molecular simulations have been used to explore the interaction between imatinib mesylate with VEGF GQ DNA structures along with duplex DNA, C-Myc, H-Telo GQ DNA. We found that imatinib mesylate shows preferential interaction towards VEGF GQ DNA compared to C-Myc, H-Telo GQ and duplex DNA. Imatinib mesylate seems to be an efficient ligand for VEGF GQ DNA, suggesting that it might be used to regulate the expression of genes in cancerous cells.

Mitochondrial RelA empowers mtDNA G-quadruplex formation for hypoxia adaptation in cancer cells

Mitochondrial DNA (mtDNA) G-quadruplexes (G4s) have important regulatory roles in energy metabolism, yet their specific functions and underlying regulatory mechanisms have not been delineated. Using a chemical-genetic screening strategy, we demonstrated that the JAK/STAT3 pathway is the primary regulatory mechanism governing mtDNA G4 dynamics in hypoxic cancer cells. Further proteomic analysis showed that activation of the JAK/STAT3 pathway facilitates the translocation of RelA, a member of the NF-κB family, to the mitochondria, where RelA binds to mtDNA G4s and promotes their folding, resulting in increased mtDNA instability, inhibited mtDNA transcription, and subsequent mitochondrial dysfunction. This binding event disrupts the equilibrium of energy metabolism, catalyzing a metabolic shift favoring glycolysis. Collectively, the results provide insights into a strategy employed by cancer cells to adapt to hypoxia through metabolic reprogramming.

Systematic Evaluation of Benchmark G4 Probes and G4 Clinical Drugs using three Biophysical Methods: A Guideline to Evaluate Rapidly G4-Binding Affinity.

G-quadruplex DNA structures (G4) are proven to interfere with most genetic and epigenetic processes. Small molecules binding these structures (G4 ligands) are invaluable tools to probe G4-biology and address G4-druggability in various diseases (cancer, viral infections). However, the large number of reported G4 ligands (>1000) could lead to confusion while selecting one for a given application. Herein we conducted a systematic affinity ranking of 11 popular G4 ligands vs 5 classical G4 sequences using FRET-melting, G4-FID assays and SPR. Interestingly SPR data globally align with the rankings obtained from the two semi-quantitative assays despite discrepancies due to limits and characteristics of each assay. In the whole, PhenDC3 emerges as the most potent binder irrespective of the G4 sequence. Immediately below PDS, PDC-360A, BRACO19, TMPyP4 and RHPS4 feature strong to medium binding again with poor G4 topology discrimination. More strikingly, the G4 drugs Quarfloxin, CX5461 and c-PDS exhibit weak affinity with all G4s studied. Finally, NMM and Cu-ttpy showed heterogeneous behaviors due, in part, to their physicochemical particularities poorly compatible with screening conditions. The remarkable properties of PhenDC3 led us to propose its use for benchmarking FRET-melting and G4-FID assays for rapid G4-affinity evaluation of newly developed ligands.

Biomimetic Hydrogen-Bonded G ⋅ C ⋅ G ⋅ C Quadruplex within a Tetraphenylethene-Based Octacationic Spirobicycle in Water.

In biological systems, nucleotide quadruplexes (such as G-quadruplexes) in DNA and RNA that are held together by multiple hydrogen bonds play a crucial functional role. The biomimetic formation of these hydrogen-bonded quadruplexes captured by artificial systems in water poses a significant challenge but can offer valuable insights into these complex functional structures. Herein, we report the formation of biomimetic hydrogen-bonded G ⋅ C ⋅ G ⋅ C quadruplex captured by a tetraphenylethene (TPE) based octacationic spirobicycle (1). The spirobicyclic compound possesses a three-dimensional (3D) crossing dual-cavity structure, which enables the encapsulation of four d(GpC) dinucleotide molecules, thereby realizing 1 : 4 host-guest complexation in water. The X-ray structure reveals that four d(GpC) molecules further form a two-layer G ⋅ C ⋅ G ⋅ C quadruplex with Watson-Crick hydrogen bonds, which are stabilized within the dual hydrophobic cavities of 1 through the cooperative non-covalent interactions of hydrogen bonds, CH⋅⋅⋅π interactions, and hydrophobic effect. Due to the dynamically-rotational propeller chirality of TPE units, 1 with adaptive chirality can further serve as a chiroptical sensor to exhibit opposite Cotton effects with mirror-image CD spectra for the pH-dependent hydrogen-bonded assemblies of d(GpC) including the Watson-Crick G ⋅ C ⋅ G ⋅ C (pH 9.22) and Hoogsteen G ⋅ C+ ⋅ G ⋅ C+ (pH 5.74) quartets through the host-guest chirality transfer in water.

Nucleic Acid-Binding Dyes as Versatile Photocatalysts for Atom-Transfer Radical Polymerization.

Nucleic acid-binding dyes (NuABDs) are fluorogenic probes that light up after binding to nucleic acids. Taking advantage of their fluorogenicity, NuABDs have been widely utilized in the fields of nanotechnology and biotechnology for diagnostic and analytical applications. We demonstrate the potential of NuABDs together with an appropriate nucleic acid scaffold as an intriguing photocatalyst for precisely controlled atom-transfer radical polymerization (ATRP). Additionally, we systematically investigated the thermodynamic and electrochemical properties of the dyes, providing insights into the mechanism that drives the photopolymerization. The versatility of the NuABD-based platform was also demonstrated through successful polymerizations using several NuABDs in conjunction with diverse nucleic acid scaffolds, such as G-quadruplex DNA or DNA nanoflowers. This study not only extends the horizons of controlled photopolymerization but also broadens opportunities for nucleic acid-based materials and technologies, including nucleic acid-polymer biohybrids and stimuli-responsive ATRP platforms.

Pathogenic SNPs Affect Both RNA and DNA G-Quadruplexes' Responses to Ligands.

Single nucleotide polymorphisms (SNPs) are common genetic variations that are present in over 1% of the population and can significantly modify the structures of both DNA and RNA. G-quadruplex structures (G4) are formed by the superposition of tetrads of guanines. To date, the impact of SNPs on both G4 ligands' binding efficacies and specificities has not been investigated. Here, using a bioinformatically predicted G4 and SNPs found in the α-synuclein gene as a proof-of-concept, it was demonstrated that SNPs can modulate both DNA and RNA G4s' responses to ligands. Specifically, six widely recognized ligands (Phen-DC3, PDS, 360A, RHPS4, BRACO19, and TMPyP4) were shown to differentially affect both the structure and the polymerase stalling of the different SNPs. This work highlights the importance of choosing the appropriate G4 ligand when dealing with an SNP identified in a G-rich gene.

Comparative Analysis of 1,9-Dimethyl-Methylene Blue and Toluidine Blue Interactions with ct-DNA, G-Quadruplex DNA, and ssDNA

This study presents a comprehensive investigation into the interactions of two distinct synthetic dyes—1,9-Dimethyl-Methylene Blue and Toluidine Blue —with different DNA structures, namely calf thymus DNA (ct-DNA), G-quadruplex DNA, and single stranded DNA (ssDNA). The objective of this research is to elucidate the molecular affinities of these dyes for specific DNA structures and explore their potential applications in molecular biology and diagnostics. The experimental approach involved detailed UV-visible spectroscopic analyses, to probe the binding affinities of each dye with ct-DNA, G-quadruplex DNA, and ssDNA. The study aimed to assess the selectivity of these dyes towards the unique structural features of each DNA entity. The binding stoichiometry is defined from Job’s method. The selectivity of the dyes towards DNA also investigated with competitive dialysis experiments. The binding stoichiometry were 1:1 for 1,9-Dimethyl-Methylene Blue and Toluidine Blue and G-quadruplex DNA or ssDNA. Besides, results indicate that 1,9-Dimethyl-Methylene Blue and Toluidine Blue exhibit a pronounced affinity for G-quadruplex DNA, and ct-DNA. While single-stranded DNA is a fundamental component of DNA replication and transcription, our dyes exhibit lower affinity for this structure. The selectivity is advantageous, as it allows for the discrimination between single-stranded and structured DNA regions. The potential applications in studying DNA dynamics and unwinding processes are vast.

Molecular Docking Study of Binding of Perylene Di-imide to a Bio Molecular Human Telomeric G-quadruplex

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands stabilizing these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells , telomerase adds multiple copies of the 5’-GGTTAG-3’ motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, which are sensitive to the nature of the cations present, to the flanking sequences, and probably also to concentration, as confirmed by the different conformations deposited in the Protein Data Bank (PDB). The existence of different polymorphism in the DNA quadruplex and the absence of a uniquely precise binding site give rise to check docking approach . As target we have selected six different experimental models of the human telomeric sequence d[AG3(T2AG3)3] based on three G-tetrads and as ligands the perylene di-imide . We checked out molecular docking simulation of binding of perylene di-imide to a slected G-quadruplex using dock 6.9 to examine whether or not to reproduced the loop binding mode of perylene di-imide. The simulation gave the two highest rank docking pose of perylene di-imide and the binding mode were external stacking on the terminal guanine tetrade and the groove binding.