DNA G4s Research Articles

Dominant and genome-wide formation of DNA:RNA hybrid G-quadruplexes in living yeast cells

Guanine-rich DNA forms G-quadruplexes (G4s) that play a critical role in essential cellular processes. Previous studies have mostly focused on intramolecular G4s composed of four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has not been strictly confirmed in the genome of living eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and two other independent G4-specific detections reveal that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of potential G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s readily form and even dominate at G4 sites that are theoretically capable of forming the intramolecular DNA G4s (dG4s) by themselves. Compared to dG4s, hG4s exhibit broader kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing it to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also establishes a sensitive and currently the only method for detecting the structural form of G4s in living cells.

The first embryonic landscape of G-quadruplexes related to myogenesis

BackgroundDNA G-quadruplexes (G4s) represent a distinctive class of non-canonical DNA secondary structures. Despite their recognition as potential therapeutic targets in some cancers, the developmental role of G4 structures remains enigmatic. Mammalian embryonic myogenesis studies are hindered by limitations, prompting the use of chicken embryo-derived myoblasts as a model to explore G4 dynamics. This study aims to reveal the embryonic G4s landscape and elucidate the underlying mechanisms for candidate G4s that influence embryonic myogenesis.ResultsThis investigation unveils a significant reduction in G4s abundance during myogenesis. G4s stabilizer pyridostatin impedes embryonic myogenesis, emphasizing the regulatory role of G4s in this process. G4 Cut&Tag sequencing and RNA-seq analyses identify potential G4s and DEGs influencing embryonic myogenesis. Integration of G4 and DEG candidates identifies 32 G4s located in promoter regions capable of modulating gene transcription. WGBS elucidates DNA methylation dynamics during embryonic myogenesis. Coordinating transcriptome data with DNA G4s and DNA methylation profiles constructs a G4-DMR-DEG network, revealing nine interaction pairs. Notably, the NFATC2 promoter region sequence is confirmed to form a G4 structure, reducing promoter mCpG content and upregulating NFATC2 transcriptional activity.ConclusionsThis comprehensive study unravels the first embryonic genomic G4s landscape, highlighting the regulatory role of NFATC2 G4 in orchestrating transcriptional activity through promoter DNA methylation during myogenesis.

Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5′ single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5′ most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

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.

Near-infrared fluorescent probe for sensitive detection and imaging of DNA G4s in living cells

G-quadruplexs (G4s), four-stranded nucleic acid secondary structures, which formed by guanine-rich sequences play a vital role in human biological systems. Studies have shown that the formation of G4s is closely related to tumor development and apoptosis, which is considered as a new target for the development of anti-tumor drugs. Therefore, it is important to develop novel probes for G4s imaging. In this article, we engineered a near-infrared fluorescent probe (TOH) which can be activated by DNA G4s in living cells and tumor. TOH exhibits high selectivity to the structure of DNA G4s with the limit of detection for DNA G4s (Mito-0.5–2) is calculated to be 0.43 nM. Imaging studies of different cell lines revealed that the brighter fluorescence in cancer cell lines than in normal, indicating that DNA G4s maybe highly express in tumor cell lines. Simultaneously, TOH is also introduced into live tumor tissue imaging and found that the fluorescence intensity of tumor is the brightest relative to normal tissue, further validating the high expression of DNA G4s structures in tumor tissue. These features demonstrate TOH not only have the ability to image DNA G4 structures in real time, but also may have tumor diagnostic capabilities.

A NIR fluorescent probe with large stokes shift for sensitive detection and imaging of G4s in live cells

G4s play a very important role not only in human biological systems but are associated with tumor formation, which is considered as a novel target for the exploitation of anti-tumor drugs. Therefore, it is essential to develop near-infrared (NIR) fluorescent probes to study G4s. In this article, we designed a large stokes shift probe (AOC) which can be activated NIR fluorescent by G4s in living cells and tumor tissue. In vitro experiments shown that AOC exhibited no fluorescence emission in aqueous solution, while significantly enhanced at 768 nm in the presence of the G4s. The optimal excitation wavelength of the probe is about 640 nm, indicating that the probe AOC has a large stokes shift (128 nm). Meanwhile, AOC is dynamically responsive to the concentrations of DNA G4s (22AG, Mito-160) and exhibits high selectivity toward DNA G4s with desirable stability in the physiological pH conditions. Imaging studies of different cell lines have shown that cancer cell lines fluoresced brightly than normal cell lines, implication that the expression of G4s may be higher in tumor cell lines. We further performed fluorescence imaging of tumor tissues using AOC, which showed that tumor tissues had the brightest fluorescence intensity relative to normal organ tissues. These experimental results further suggest that the NIR fluorescent probe may have tumor diagnostic properties with great potential for application.

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.

Disruption of a DNA G-quadruplex causes a gain-of-function SCL45A1 variant relevant to developmental disorders.

SLC45A1 encodes a glucose transporter protein highly expressed in the brain. Mutations in SLC45A1 may lead to neurological diseases and developmental disorders, but its exact role is poorly understood. DNA G-quadruplexes (DNA G4s) are stable structures formed by four guanine bases and play a role in gene regulation and genomic stability. Changes in DNA G4s may affect brain development and function. The mechanism linking alterations in DNA G-quadruplex structures to SLC45A1 pathogenicity remains unknown. In this study, we identify a functional DNA G-quadruplex and its key binding site on SLC45A1 (NM_001080397.3: exon 2: c.449 G>A: p.R150K). This variant results in the upregulation of mRNA and protein expression, which may lead to intellectual developmental disorder with neuropsychiatric features. Mechanistically, the mutation is found to disrupt DNA G-quadruplex structures on SLC45A1, leading to transcriptional enhancement and a gain-of-function mutation, which further causes increased expression and function of the SLC45A1 protein. The identification of the functional DNA G-quadruplex and its effects on DNA G4s may provide new insights into the genetic basis of SLC45A1 pathogenicity and highlight the importance of DNA G4s of SLC45A1 in regulating gene expression and brain development.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 μM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Synthesis and Biological Evaluation of Benzo [4,5]- and Naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone Derivatives.

Azacarbazoles have attracted significant interest due to their valuable properties, such as anti-pathogenic and antitumor activity. In this study, a series of structurally related tricyclic benzo[4,5]- and tertacyclic naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone derivatives with one or two positively charged tethers were synthesized and evaluated for anti-proliferative activity. Lead tetracyclic derivative 5b with two amino-bearing arms inhibited the metabolic activity of A549 lung adenocarcinoma cells with a CC50 value of 3.6 μM, with remarkable selectivity (SI = 17.3) over VA13 immortalized fibroblasts. Cell-cycle assays revealed that 5b triggers G2/M arrest without signs of apoptosis. A study of its interaction with various DNA G4s and duplexes followed by dual luciferase and intercalator displacement assays suggests that intercalation, rather than the modulation of G4-regulated oncogene expression, might contribute to the observed activity. Finally, a water-soluble salt of 5b was shown to cause no acute toxic effects, changes in mice behavior, or any decrease in body weight after a 72 h treatment at concentrations up to 20 mg/kg. Thus, 5b is a promising candidate for studies in vivo; however, further investigations are needed to elucidate its molecular target(s).

Discovery of a minimalistic DIE-based fluorescent probe for detection of parallel G-quadruplex forms

G-quadruplexes (G4s) are considered to be involved in some key biological processes, leading to the development of a large number of G4 fluorescent probes, which offer possibilities to study G4 dynamics as well as their biological roles. However, the structures of G4s show high polymorphism, which can be classified into parallel, hybrid and antiparallel forms, and the probes targeting a certain topology are limited. In this study, we have developed a minimalistic fluorescent probe by exploiting the disaggregation-induced emission (DIE) principle. The further studies demonstrated that this probe exhibited promising selectivity toward parallel DNA and RNA G4 forms in vitro. Moreover, it was found that this probe could be applied to map the RNA G4s that always form into parallel topologies in live cells, which distinguished it from other reported DIE-based probes that often targeted the mitochondrial or nuclear DNA G4s. To the best of our knowledge, this was the first DIE-based fluorescent probe for mapping cellular RNA G4s.

Promoter R-Loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing.

R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) data sets, we found that many splicing factors, including U2AF1 whose recognition of the 3' splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We also revealed that U2AF1 binds directly to R-loops and DNA G4 structures at a low-nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1's interaction with 3' splicing site and leads to augmented distribution of RNA polymerase II (RNAPII) to promoters over gene bodies, thereby modulating cotranscriptional pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interactions of U2AF1 with these nucleic acid structures, and established a biochemical rationale for U2AF1's occupancy in gene promoters. We also unveiled that interaction with R-loops promotes U2AF1's phase separation, and our work suggests that U2AF1 modulates pre-mRNA splicing by regulating RNAPII's partition in transcription initiation versus elongation.

An overview on nucleic-acid G-quadruplex prediction: from rule-based methods to deep neural networks.

Nucleic-acid G-quadruplexes (G4s) play vital roles in many cellular processes. Due to their importance, researchers have developed experimental assays to measure nucleic-acid G4s in high throughput. The generated high-throughput datasets gave rise to unique opportunities to develop machine-learning-based methods, and in particular deep neural networks, to predict G4s in any given nucleic-acid sequence and any species. In this paper, we review the success stories of deep-neural-network applications for G4 prediction. We first cover the experimental technologies that generated the most comprehensive nucleic-acid G4 high-throughput datasets in recent years. We then review classic rule-based methods for G4 prediction. We proceed by reviewing the major machine-learning and deep-neural-network applications to nucleic-acid G4 datasets and report a novel comparison between them. Next, we present the interpretability techniques used on the trained neural networks to learn key molecular principles underlying nucleic-acid G4 folding. As a new result, we calculate the overlap between measured DNA and RNA G4s and compare the performance of DNA- and RNA-G4 predictors on RNA- and DNA-G4 datasets, respectively, to demonstrate the potential of transfer learning from DNA G4s to RNA G4s. Last, we conclude with open questions in the field of nucleic-acid G4 prediction and computational modeling.

G-quadruplex DNA contributes to RNA polymerase II-mediated 3D chromatin architecture.

High-order chromatin organization plays an important role in biological processes and disease development. Previous studies revealed a widespread occurrence of guanine quadruplex (G4) structures in the human genome, with enrichment in gene regulatory regions, especially in promoters. However, it remains unclear whether G4 structures contribute to RNA polymerase II (RNAPII)-mediated long-range DNA interactions and transcription activity. In this study, we conducted an intuitive overlapping analysis of previously published RNAPII ChIA-PET (chromatin interaction analysis with paired-end tag) and BG4 ChIP-seq (chromatin immunoprecipitation followed by sequencing using a G4 structure-specific antibody) data. We observed a strong positive correlation between RNAPII-linked DNA loops and G4 structures in chromatin. Additionally, our RNAPII HiChIP-seq (in situ Hi-C followed by ChIP-seq) results showed that treatment of HepG2 cells with pyridostatin (PDS), a small-molecule G4-binding ligand, could diminish RNAPII-linked long-range DNA contacts, with more pronounced diminutions being observed for those contacts involving G4 structure loci. RNA sequencing data revealed that PDS treatment modulates the expression of not only genes with G4 structures in their promoters, but also those with promoters being connected with distal G4sthrough RNAPII-linked long-range DNA interactions. Together, our data substantiate the function of DNA G4s in RNAPII-associated DNA looping and transcription regulation.

Molecular engineering of a wash-free, clover-like G-quadruplex fluorescent probe that exploits a new mechanism of disaggregation-and-binding-induced emission

In this study, we raised a brand-new principle termed disaggregation-and-binding-induced emission (DBIE) for the design of G4 fluorescent probes, which may overcome the shortcomings of the disaggregation-induced emission (DIE) and aggregation-induced emission (AIE) principles. On this basis, a wash-free, clover-like fluorescent probe (TCN3) that could detect either in vitro or cellular DNA G4s was rationally developed. To be noted, this DBIE mechanism may be utilized to design promising fluorescent probes targeting other biotargets.

Phase Separation Modulates the Formation and Stabilities of DNA Guanine Quadruplex.

In the presence of monovalent alkali metal ions, G-rich DNA sequences containing four runs of contiguous guanines can fold into G-quadruplex (G4) structures. Recent studies showed that these structures are located in critical regions of the human genome and assume important functions in many essential DNA metabolic processes, including replication, transcription, and repair. However, not all potential G4-forming sequences are actually folded into G4 structures in cells, where G4 structures are known to be dynamic and modulated by G4-binding proteins as well as helicases. It remains unclear whether there are other factors influencing the formation and stability of G4 structures in cells. Herein, we showed that DNA G4s can undergo phase separation in vitro. In addition, immunofluorescence microscopy and ChIP-seq experiments with the use of BG4, a G4 structure-specific antibody, revealed that disruption of phase separation could result in global destabilization of G4 structures in cells. Together, our work revealed phase separation as a new determinant in modulating the formation and stability of G4 structures in human cells.

Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs

The search for G-quadruplex (G4)-forming sequences across the genome is motivated by their involvement in key cellular processes and their putative roles in dysregulations underlying human genetic diseases. Sequencing-based methods have been developed to assess the prevalence of DNA G4s genome wide, including G4-seq to detect G4s in purified DNA (invitro) using the G4 stabilizer PDS, and G4 chromatin immunoprecipitation sequencing (G4 ChIP-seq) to detect G4s in in situ fixed chromatin (invivo) using the G4-specific antibody BG4. We recently reported on G4-RNA precipitation and sequencing (G4RP-seq) to assess the invivo prevalence of RNA G4 landscapes transcriptome wide using the small molecule BioTASQ. Here, we apply this technique for mapping DNA G4s in plants (rice) and compare the efficiency of this new technique, G4-DNA precipitation and sequencing, G4DP-seq, to that of BG4-DNA-IP-seq that we developed for mapping of DNA G4s in rice using BG4. By doing so, we compare the G4 capture ability of small-sized ligands (BioTASQ and BioCyTASQ) versus the antibody BG4.

Asymmetric triplex metallohelices stabilise DNA G-quadruplexes in promoter oncogene sequences and efficiently reduce their expression in cancer cells

Some metallo-supramolecular helical assemblies with size, shape, charge and amphipathic architectures similar to short cationic α-helical peptides have been shown to target and stabilise DNA G-quadruplexes (G4s) in vitro and downregulate the expression of G4-regulated genes in human cells. To expand the library of metallohelical structures that can act as efficient DNA G4 binders and downregulate genes containing G4-forming sequences in their promoter regions, we investigated the interaction of the two enantiomeric pairs of asymmetric Fe(II) triplex metallohelices with a series of five different DNA G4s formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC, c-KIT, and k-RAS oncogenes. The metallohelices display preferential binding to G4s over duplex DNA in all investigated G4-forming sequences and induced arrest of DNA polymerase on template strands containing G4-forming sequences. Moreover, the investigated metallohelices suppressed the expression of c-MYC and k-RAS genes at mRNA and protein levels in HCT116 human cancer cells, as revealed by RT-qPCR analysis and western blotting.

Abstract 3098: Structure-based design rules for potent quadruplex-binding compounds based on the naphthalene diimide core

Abstract Quadruplex nucleic acids (G4s) are four-stranded arrangements formed by guanine-rich tracts of DNA or RNA, held together by Hoogsteen hydrogen-bonded guanine quartets. G4 sequences are widely though not randomly distributed in the human genome. They are over-represented in telomeres and in the regulatory regions of many cancer-related genes such as KIT, MYC, VEGF, RAS and HIF. G4 structures can be unwound by helicase activity but can be stabilized by the binding of high-affinity small molecules. The compound QN-302, a tetra-substituted naphthalene diimide (ND) derivative, has nanomolar affinity and selectivity for several DNA and RNA G4s over duplex DNA. QN-302 has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It has significant anti-tumor activity in several models for PDAC - in a MIA-PACA2 xenograft, in the genetic KPC model and in an orthotopic BX-PC3 model. QN-302 is currently in an advanced stage of pre-IND development by Qualigen Inc as a novel anti-cancer agent. QN-302 is the latest in several generations of NDs developed by us and designed by computer modelling based on earlier G4 co-crystal structures determined in this laboratory. The database of seven ND-containing crystal structures includes four different NDs with distinct side chain end groups, all with intramolecular telomeric parallel G4s. End-groups include N-methyl piperazine, hydroxyl, morpholino and N-dimethyl. These structures have been systematically compared for example using least-squares overlays of the structures. All the structures have their four substituents positioned each in a G4 groove, although there are significant differences in orientations of the end groups. Surprisingly, the naphthalene diimide cores do not retain the same orientation in each structure, with three distinct orientations being apparent. Examination of ND core overlap with the adjacent G-quartet has revealed variation in the degree of heterocycle-guanine base overlap, which is dependent on the nature and size of each substituent. However, all structures show minimal ND-guanine overlap, with typically just one out of four guanines in a G-quartet being involved. This strongly implies that most of the contribution to ND-G4 affinity comes from the side chains, and especially from the protonated end groups. These and other observations have led us to develop a structure-activity framework for tetra-substituted NDs, which has culminated in their optimization and the design of QN-302, with enhanced cellular potency and G4 affinity compared to previous NDs. Citation Format: Stephen Neidle. Structure-based design rules for potent quadruplex-binding compounds based on the naphthalene diimide core [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3098.

Metal-Organic Compounds as Anticancer Agents: Versatile Building Blocks for Selective Action on G-quadruplexes.

Since the 1980s, cancer research has focused primarily on developing new therapeutic agents targeting DNA alterations rather than understanding cancer as an integrated system composed of several modules. In this sense, G-quadruplex (G4) nucleic acids are a promising target for drug development for cancer therapy since they exist in the chromosomal telomeric sequences and the promoter regions of numerous genes. The G4 structures within telomeric DNA can inhibit telomerase activity and prevent the proliferation and immortalization of cancer cells. Furthermore, such G4 systems within the promoter regions of oncogenes can inhibit the transcription and expression of the oncogene. The rational design of small molecules such as organic ligands and their metal- organic derivative compounds can stabilize G4 structures through different binding modes on several G4 DNA topologies. Metal-based compounds have demonstrated their competitiveness compared to organic molecules to distinguish G4 over the DNA duplex owing to their convenient coordination features, positive charge, and electron density promoted by organic ligand. This article is a comprehensive review of metal compounds G4-binders and their structural features that confer them the ability to recognize G-quartets and stabilize several DNA G4s. This stabilization can be achieved through extended square aromatic surfaces, increased hydrophobicity, different auxiliary ligands, axially coordinated ligands, and the nature of the metal center.