Non-canonical DNA Research Articles

DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death.

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs). The role of DDX41 in hematopoiesis and how its germline and somatic mutations contribute to MNs remain unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for the development of other hematopoietic lineages. Using stage-specific Cre models for erythropoiesis, we reveal that Ddx41 knockout in early erythropoiesis is embryonically lethal, while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4), noncanonical DNA structures that tend to accumulate in the early stages of erythroid precursors. We show that DDX41 co-localizes with G4 on the erythroid genome. DDX41 directly binds to and dissolves G4, which is significantly compromised in MN-associated DDX41 mutants. Accumulation of G4 by DDX41 deficiency induces erythroid genome instability, defects in ribosomal biogenesis, and upregulation of p53. However, p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel, genome instability also activates the cGas-Sting pathway, which is detrimental to survival since cGas-deficient and hematopoietic-specific Ddx41 knockout mice are viable without detectable hematologic phenotypes, although these mice continue to show erythroid ribosomal defects and upregulation of p53. These findings are further supported by data from a DDX41 mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 dissolver, essential for erythroid genome stability and suppressing the cGAS-STING pathway.

Platinum(II)-Salphen Complexes as DNA Binders and Photosensitisers.

Current anticancer therapies suffer from issues such as off-target side effects and the emergence of drug resistance; therefore, the discovery of alternative therapeutic approaches is vital. These can include the development of drugs with different modes of action, and the exploration of new biomolecular targets. For the former, there has been increasing interest in drugs that are activated by an external stimulus (e. g. light irradiation) to generate cytotoxic chemicals such as reactive oxygen species (ROS). For the latter, significant efforts are being directed to explore non-canonical DNA and RNA structures (e. g. guanine-quadruplexes), as alternative biomolecular targets. Herein we report the synthesis of a library of 21 new platinum(II)-Salphen complexes (square planar platinum(II) complexes coordinated to tetradentate O,N,N,O-Schiff base ligands), and the investigation, for all complexes, of their photophysical and photochemical properties, their interactions with duplex and quadruplex DNA, and their cytotoxicity against HeLa cancer cells both in the dark and upon light irradiation. Thanks to the intrinsic phosphorescence of the platinum(II) complexes, confocal microscopy was used for six of the complexes to determine their cellular permeability and localisation in two cancer cell lines (HeLa and U2OS). Altogether, these studies have allowed us to identify two lead platinum(II) complexes with high guanine-quadruplex DNA affinity and selectivity, good cell permeability and nuclear localisation, and high cytotoxicity against HeLa cancer cells upon irradiation with no detected cytotoxicity in the dark.

G-quadruplex DNA and RNA in cellular senescence.

Normal cells divide, are damaged, and are repaired across their lifetime. As cells age, they enter cellular senescence, characterized by a permanent state of cell-cycle arrest triggered by various stressors. The molecular mechanisms that regulate senescent phenotypes have been actively investigated over the last several decades; however, one area that has been neglected is how G-quadruplex (G4) DNA and RNA (G4-DNA and G4-RNA) mediate senescence. These non-canonical four-stranded DNA and RNA structures regulate most normative DNA and RNA-dependent processes, such as transcription, replication, and translation, as well as pathogenic mechanisms, including genomic instability and abnormal stress granule function. This review also highlights the contribution of G4s to sex differences in age-associated diseases and emphasizes potential translational approaches to target senescence and anti-aging mechanisms through G4 manipulation.

Sequence Context in DNA i-Motifs Can Nurture Very Stable and Persistent Kinetic Traps.

I-motifs are non-canonical DNA structures with recognized biological significance and a proven utility in material engineering. Consequently, understanding and control of i-motif properties is essential to sustain progress across both disciplines. In this work, we systematically investigate how proximity to the most common form of DNA, a double-stranded duplex, influences the thermodynamic and kinetic properties of adjacent i-motifs. We demonstrate that double-stranded stems in i-motif loops promote kinetic trapping of very stable and persistent partially folded conformations. Further, we investigate pathways toward rational control over a folding topology makeup.

Chalcogen bonds enable efficient photoreduction of sulfur-containing heterocycles.

Chalcogen bonding interactions have attracted significant attention in a broad chemistry community, with a particular focus on their ability to stabilise the key transition states in various organic synthetic routes. In this work, we demonstrate that they can also be harnessed in selective photoredox reactions, which cannot be otherwise achieved with alternative approaches to photoreduction. We demonstrate this concept through the photoreduction of the sulfur-containing DNA nucleoside precursor thioanhydrouridine to 2'-deoxy-thiouridine, revealing the previously unrecognized role of bisulfide in this process. Based on quantum chemical simulations, we identify a stable chalcogen-bonding complex of the hydrosulfide anion and thionhydrouridine (HS-…S contacts), which enables directional photoinduced electron transfer, resulting in the formation of non-canonical DNA nucleoside. We also disprove the possibility that photoreduction of thioanhydronucleosides could be initiated by hydrated electrons generated from irradiated bisulfide anions which do not interact with the chromophore. Finally, we show that selective photoreduction mediated by chalcogen bonds can only occur for chromophores, which exhibit sufficiently long excited-state lifetimes in the locally-excited states to undergo transition to the productive charge transfer state. These findings can be further used in the design of similar photoredox reactions which can employ the potential of chalcogen bonding interactions.

Insights into the Molecular Structure, Stability, and Biological Significance of Non-Canonical DNA Forms, with a Focus on G-Quadruplexes and i-Motifs

This article provides a comprehensive examination of non-canonical DNA structures, particularly focusing on G-quadruplexes (G4s) and i-motifs. G-quadruplexes, four-stranded structures formed by guanine-rich sequences, are stabilized by Hoogsteen hydrogen bonds and monovalent cations like potassium. These structures exhibit diverse topologies and are implicated in critical genomic regions such as telomeres and promoter regions of oncogenes, playing significant roles in gene expression regulation, genome stability, and cellular aging. I-motifs, formed by cytosine-rich sequences under acidic conditions and stabilized by hemiprotonated cytosine–cytosine (C:C+) base pairs, also contribute to gene regulation despite being less prevalent than G4s. This review highlights the factors influencing the stability and dynamics of these structures, including sequence composition, ionic conditions, and environmental pH. Molecular dynamics simulations and high-resolution structural techniques have been pivotal in advancing our understanding of their folding and unfolding mechanisms. Additionally, the article discusses the therapeutic potential of small molecules designed to selectively bind and stabilize G4s and i-motifs, with promising implications for cancer treatment. Furthermore, the structural properties of these DNA forms are explored for applications in nanotechnology and molecular devices. Despite significant progress, challenges remain in observing these structures in vivo and fully elucidating their biological functions. The review underscores the importance of continued research to uncover new insights into the genomic roles of G4s and i-motifs and their potential applications in medicine and technology. This ongoing research promises exciting developments in both basic science and applied fields, emphasizing the relevance and future prospects of these intriguing DNA structures.

Human hnRNPA1 reorganizes telomere-bound replication protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection.

Calcium-Dependent Chemiluminescence Catalyzed by a Truncated c-MYC Promoter G-Triplex DNA.

The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5'-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs.

Complex Biophysical and Computational Analyses of G-Quadruplex Ligands: The Porphyrin Stacks Back.

G-quadruplexes (G4s), as non-canonical DNA structures, attract a great deal of research interest in the molecular biology as well as in the material science fields. The use of small molecules as ligands for G-quadruplexes has emerged as a tool to regulate gene expression and telomeres maintenance. Meso-tetrakis-(N-methyl-4-pyridyl)porphyrin (TMPyP4) was shown as one of the first ligands for G-quadruplexes and it is still widely used. We report an investigation comprising molecular docking and dynamics, synthesis and multiple spectroscopic and spectrometric determinations on simple cationic porphyrins and their interaction with different DNA sequences. The study allowed to synthesize a few compounds that have shown to interact with DNA; the detailed characterization has shown that the presence of amide groups at the periphery improves selectivity for parallel G4s binding over other structures. Taking into account the ease of synthesis, 5,10,15,20-tetrakis-(1-acetamido-4-pyridyl)porphyrin bromide could be considered a better alternative to TMPyP4 in studies involving G4 binding.

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.

Hindered intermolecular stacking of anti-parallel telomeric G-quadruplexes.

Telomeric G-quadruplexes (G4s) are non-canonical DNA structures composed of TTAGGG repeats. They are extensively studied both as biomolecules key for genome stability and as promising building blocks and functional elements in synthetic biology and nanotechnology. This is why it is extremely important to understand how the interaction between G4s is affected by their topology. We used small-angle x-ray scattering to investigate the end-to-end stacking of antiparallel telomeric G-quadruplexes formed by the sequence AG3(T2AG3)3. To represent the experimental data, we developed a highly efficient coarse-grained fitting tool, which successfully described the samples as an equilibrium mixture of monomeric and dimeric G4 species. Our findings indicate that the antiparallel topology prevents the formation of long multimeric structures under self-crowding conditions, unlike the hybrid/parallel structures formed by the same DNA sequence. This result supports the idea that the stacking of monomeric G-quadruplexes is strongly affected by the presence of diagonal loops.

Synthesis and biological properties of small molecules — ligands of non-canonical DNA and RNA structures

Nucleic acids are important targets for many anticancer drugs. Apart from the canonical B-DNA double helix, DNA forms a number of non-canonical structures (G-quadruplexes, i-motifs, hairpins, triplexes, <i>etc</i>.), which play an important role in the regulation of biological processes. Binding to non-canonical DNA structures occurs mainly by π–π-stacking. Therefore, aromatic and heteroaromatic compounds such as fused polyaromatic compounds (acridines, anthraquinones, carbazoles), porphyrins, benzothiazoles, benzimidazoles, pyridines, and quinolines, as well as their complexes are used as ligands for secondary structures. These ligands should possess not only high selectivity for non-canonical structures over double-stranded DNA, but also relatively high solubility and ability to penetrate through cell membranes. This review summarizes the achievements of 2020–2024 in the synthesis and biological studies of (hetero)arenes (acridines, anthraquinones, benzazoles, xanthones, porphyrins) and coordination compounds that have exhibited anticancer activity as a result of binding to non-canonical DNA or RNA structures. Ligands of various types of non-canonical nucleic acid structures (G-quadruplexes, i-motifs, triplexes, hairpins) are considered and their cytotoxic activity and structure–property relationships are compared <br> The bibliography includes 166 references.

G-quadruplex forming regions in GCK and TM6SF2 are targets for differential DNA methylation in metabolic disease and hepatocellular carcinoma patients

The alarming increase in global rates of metabolic diseases (MetDs) and their association with cancer risk renders them a considerable burden on our society. The interplay of environmental and genetic factors in causing MetDs may be reflected in DNA methylation patterns, particularly at non-canonical (non-B) DNA structures, such as G-quadruplexes (G4s) or R-loops. To gain insight into the mechanisms of MetD progression, we focused on DNA methylation and functional analyses on intragenic regions of two MetD risk genes, the glucokinase (GCK) exon 7 and the transmembrane 6 superfamily 2 (TM6SF2) intron 2-exon 3 boundary, which harbor non-B DNA motifs for G4s and R-loops.Pyrosequencing of 148 blood samples from a nested cohort study revealed significant differential methylation in GCK and TM6SF2 in MetD patients versus healthy controls. Furthermore, these regions harbor hypervariable and differentially methylated CpGs also in hepatocellular carcinoma versus normal tissue samples from The Cancer Genome Atlas (TCGA). Permanganate/S1 nuclease footprinting with direct adapter ligation (PDAL-Seq), native polyacrylamide DNA gel electrophoresis and circular dichroism (CD) spectroscopy revealed the formation of G4 structures in these regions and demonstrated that their topology and stability is affected by DNA methylation. Detailed analyses including histone marks, chromatin conformation capture data, and luciferase reporter assays, highlighted the cell-type specific regulatory function of the target regions. Based on our analyses, we hypothesize that changes in DNA methylation lead to topological changes, especially in GCK exon 7, and cause the activation of alternative regulatory elements or potentially play a role in alternative splicing.Our analyses provide a new view on the mechanisms underlying the progression of MetDs and their link to hepatocellular carcinomas, unveiling non-B DNA structures as important key players already in early disease stages.

G-quadruplexes as pivotal components of cis-regulatory elements in the human genome

BackgroundCis-regulatory elements (CREs) are crucial for regulating gene expression, and G-quadruplexes (G4s), as prototypal non-canonical DNA structures, may play a role in this regulation. However, the relationship between G4s and CREs, especially with non-promoter-like functional elements, requires further systematic investigation. We aimed to investigate the associations between G4s and human cCREs (candidate CREs) inferred from the Encyclopedia of DNA Elements (ENCODE) data.ResultsWe found that G4s are prominently enriched in most types of cCREs, especially those with promoter-like signatures (PLS). The co-occurrence of CTCF signals with H3K4me3 or H3K27ac signals strengthens the association between cCREs and G4s. Genetic variants in G4s, particularly within their G-runs, exhibit higher regulatory potential and deleterious effects compared to cCREs. The G-runs within G4s near transcriptional start sites (TSSs) are more evolutionarily constrained compared to G-runs in cCREs, while those far from the TSS are relatively less conserved. The presence of G4s is often linked to a more favorable local chromatin environment for the activation and execution of regulatory function of cCREs, potentially attributable to the formation of G4 secondary structures. Finally, we discovered that G4-associated cCREs exhibit widespread activation in a variety of cancers.ConclusionsOur study suggests that G4s are integral components of human cis-regulatory elements, extending beyond their potential role in promoters. The G4 primary sequences are associated with the localization of CREs, while the G4 structures are linked to the activation of these elements. Therefore, we propose defining G4s as pivotal regulatory elements in the human genome.

An i-motif-regulated enhancer, eRNA and adjacent lncRNA affect Lhb expression through distinct mechanisms in a sex-specific context

Genome-wide studies have demonstrated regulatory roles for diverse non-coding elements, but their precise and interrelated functions have often remained enigmatic. Addressing the need for mechanistic insight, we studied their roles in expression of Lhb which encodes the pituitary gonadotropic hormone that controls reproduction. We identified a bi-directional enhancer in gonadotrope-specific open chromatin, whose functional eRNA (eRNA2) supports permissive chromatin at the Lhb locus. The central untranscribed region of the enhancer contains an iMotif (iM), and is bound by Hmgb2 which stabilizes the iM and directs transcription specifically towards the functional eRNA2. A distinct downstream lncRNA, associated with an inducible G-quadruplex (G4) and iM, also facilitates Lhb expression, following its splicing in situ. GnRH activates Lhb transcription and increased levels of all three RNAs, eRNA2 showing the highest response, while estradiol, which inhibits Lhb, repressed levels of eRNA2 and the lncRNA. The levels of these regulatory RNAs and Lhb mRNA correlate highly in female mice, though strikingly not in males, suggesting a female-specific function. Our findings, which shed new light on the workings of non-coding elements and non-canonical DNA structures, reveal novel mechanisms regulating transcription which have implications not only in the central control of reproduction but also for other inducible genes.

CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci.

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs.

Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Accelerated Screening of Alternative DNA Base‐Organic Molecule‐Base Architectures via Integrated Theory and Experiment

AbstractOrganic molecule‐mediated noncanonical DNA self‐assembly expands the standard DNA base‐pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base‐pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub‐stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule‐incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA‐molecule architectures beyond Watson–Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA‐based delivery system and probe design for sensitive detection of certain molecules.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Two rhenium compounds with benzimidazole ligands: synthesis and DNA interactions.

Two rhenium compounds: cis-tetrachlorotetrabenzimidazoldirhenium(iii) chloride - I and tetrabenzimidazoldioxorhenium(v) - II have been synthesized and characterized. X-ray data are presented for the new complex II. I and II show strong emission that has been used to investigate their interaction with several non-canonical DNA structures. Both compounds have a quenching effect on the fluorescence intensity upon addition of the investigated oligonucleotides; I was more selective for binding G4-than II. Association constant values obtained for I and II with G-quadruplexes reached 106 M-1, which suggests a strong interaction between both complexes and these sequences. FRET-melting assays show that I and II have a rather high level of stabilization of ckit1 and ckit2 quadruplexes. I is toxic against macrophages RAW267.7 only in high concentrations, while complex II shows no toxicity against these cells. I and II accumulate inside cells in different degrees. Molecular dynamic simulation studies have provided insights into the binding modes of II with ckit1 and ckit2 G-quadruplexes. The results obtained show the DNA binding activity of the rhenium complexes and their ability to be players in the anti-cancer fight since they can bind to non-canonical DNA forms in oncogene promoters, accumulate in some cancer cells, and influence the cancer cells microenvironment.