G4 DNA Structures Research Articles

The age-related decline of helicase function-how G-quadruplex structures promote genome instability.

The intricate mechanisms underlying transcription-dependent genome instability involve G-quadruplexes (G4) and R-loops. This perspective elucidates the potential link between these structures and genome instability in aging. The co-occurrence of G4 DNA and RNA-DNA hybrid structures (G-loop) underscores a complex interplay in genome regulation and instability. Here, we hypothesize that the age-related decline of sirtuin function leads to an increase in acetylated helicases that bind to G4 DNA and RNA-DNA hybrid structures, but are less efficient in resolving them. We propose that acetylated, less active, helicases induce persistent G-loop structures, promoting transcription-dependent genome instability in aging.

Disaggregation-driven far-red BODPIY dye for selective G4 DNA structures detection

Push-pull solvatochromic dyes undergoing intramolecular charge transfer (ICT) have emerged as useful tools for imaging target biomolecules directly in living cells and monitoring interactions of biomolecules by changing the signal of their fluorescence. However, the currently reported solvatochromic fluorescent dyes frequently suffer from the problem of inherit background noise, and cause unspecific fluorescence that masks the specific signal from biomolecules. Herein, based on the combined the effect of aggregation-caused quenching (ACQ), a D-π-A structural far-red BODIPY dye BSK was designed and developed as a G-quadruplex (G4) DNA structures probe. In organic solvents, the spectra results underlined the excellent characteristics of BSK, such as high polarity sensitivity and strong solvatochromic effect due to the electron donor–acceptor system. In aqueous solution, BSK underwent aggregation to form nonfluorescent amorphous aggregates in which the dye kept low background fluorescence in a solution. Upon binding of BSK with c-MYC G4 DNA, an intense fluorescence was trigged by the synergistic properties of disaggregation-induced emission and inhibited twisted intramolecular charge transfer, with a low detection limit of 16 nM. In addition, BSK exhibited a selective ability to target G4 DNA structures and effectively differentiated from duplex and single-strand DNAs. Moreover, BSK has the potential to be used in visualizing the cellular nucleus in cells.

Potential protein kinase inhibitors that target G-quadruplex DNA structures in the human telomeric regions.

Telomeric regions contain Guanine-rich sequences arranged in a planar manner and connected by Hoogsteen hydrogen bonds that can fold into G-quadruplex (G4) DNA structures, and can be stabilized by monovalent metal cations. The presence of G4 DNA holds significance in cancer-related processes, especially due to their regulatory potential at transcriptional and translational levels of oncogene and tumor suppressor genes. The objective of this current research is to explore the evolving realm of FDA-approved protein kinase inhibitors, with a specific emphasis on their capacity to stabilize the G4 DNA structures formed at the human telomeric regions. This involves investigating the possibility of repurposing FDA-approved protein kinase inhibitors as a novel approach for targeting multiple cancer types. In this context, we have selected 16 telomeric G4 DNA structures as targets and 71 FDA-approved small-molecule protein kinase inhibitors as ligands. To investigate their binding affinities, molecular docking of human telomeric G4 DNA with nuclear protein kinase inhibitors and their corresponding co-crystalized ligands were performed. We found that Ponatinib and Lapatinib interact with all the selected G4 targets, the binding free energy calculations, and molecular dynamic simulations confirm their binding efficacy and stability. Thus, it is hypothesized that Ponatinib and Lapatinib may stabilize human telomeric G4 DNA in addition to their ability to inhibit BCR-ABL and the other members of the EGFR family. As a result, we also hypothesize that the stabilization of G4 DNA might represent an additional underlying mechanism contributing to their efficacy in exerting anti-cancer effects.

Targeting of G-quadruplex DNA with 99mTc(I)/Re(I) Tricarbonyl Complexes Carrying Pyridostatin Derivatives.

The main goal of this work was to elucidate the potential relevance of (radio)metal chelates of 99mTc and Re targeting G-quadruplex structures for the design of new tools for cancer theranostics. 99mTc provides the complexes with the ability to perform single-photon-emission computed tomography imaging studies, while the Re complexes should act as anticancer agents upon interaction with specific G4 DNA or RNA structures present in tumor tissues. Towards this goal, we have developed isostructural 99mTc(I) and Re(I) tricarbonyl complexes anchored by a pyrazolyl-diamine (Pz) chelator carrying a pendant pyridostatin (PDS) fragment as the G4-binding motif. The interaction of the PDF-Pz-Re (8) complex with different G4-forming oligonucleotides was studied by circular dichroism, fluorescence spectroscopy and FRET-melting assays. The results showed that the Re complex retained the ability to bind and stabilize G4-structures from different DNA or RNA sequences, namely those present on the SRC proto-oncogene and telomeric RNA (TERRA sequence). PDF-Pz-Re (8) showed low to moderate cytotoxicity in PC3 and MCF-7 cancer cell lines, as typically observed for G4-binders. Biodistribution studies of the congener PDF-Pz-99mTc (12) in normal mice showed that the complex undergoes a fast blood clearance with a predominant hepatobiliary excretion, pointing also for a high in vitro stability.

G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

In-gel staining methods of G4 DNA and RNA structures.

G-quadruplexes (G4) are functionally important nucleic acid structures, involved in many cellular pathways. They are often dynamically regulated in cells, which makes detecting them in vivo challenging and dependent on sophisticated technical equipment. Therefore, in vitro studies are commonly performed as a first step to confirm a candidate sequence folds into a G4. Several methods have been developed, each with its individual pros and cons. A highly accessible and quick approach, without the need for specialized equipment, is the detection of G4s in native gels using light-up probes. These molecules become fluorescent after specifically binding to G4s. Several different classes have been discovered, emitting light in various colors, and some possess specificity for certain G4 topologies, which makes them highly versatile tools for G4 visualization. Here, we will explore the general procedure using the light-up probe NMM on RNA G4s and discuss advantages and limitations of this method.

Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.

UV-induced G4 DNA structures recruit ZRF1 which prevents UV-induced senescence

Senescence has two roles in oncology: it is known as a potent tumor-suppressive mechanism, which also supports tissue regeneration and repair, it is also known to contribute to reduced patient resilience, which might lead to cancer recurrence and resistance after therapy. Senescence can be activated in a DNA damage-dependent and -independent manner. It is not clear which type of genomic lesions induces senescence, but it is known that UV irradiation can activate cellular senescence in photoaged skin. Proteins that support the repair of DNA damage are linked to senescence but how they contribute to senescence after UV irradiation is still unknown. Here, we unraveled a mechanism showing that upon UV irradiation multiple G-quadruplex (G4) DNA structures accumulate in cell nuclei, which leads to the recruitment of ZRF1 to these G4 sites. ZRF1 binding to G4s ensures genome stability. The absence of ZRF1 triggers an accumulation of G4 structures, improper UV lesion repair, and entry into senescence. On the molecular level loss of ZRF1 as well as high G4 levels lead to the upregulation of DDB2, a protein associated with the UV-damage repair pathway, which drives cells into senescence.

Syntheses, Characterization, and DNA Binding Studies of a Series of Copper(II), Nickel(II), Platinum(II) and Zinc(II) Complexes Derived from Schiff Base Ligands

AbstractThree series of metal salophen complexes derived from Zn2+, Cu2+, Pt2+ and Ni2+ have been synthesized and their interaction with quadruplex DNA has been evaluated. The compounds differ on the number of ethyl piperidine substituents. They have been characterized by 1H NMR, IR and UV‐visible spectroscopies and by HR‐mass spectrometry. Their luminescent properties have been also evaluated and we can observe that, as expected, Zn2+ and Pt2+ complexes are those displaying more interesting luminescence with an emission band red‐shifted with respect to the corresponding uncoordinated ligand. DNA interactions with G4 and duplex DNA were evaluated by FRET melting assays (for the Zn2+, Cu2+ and Ni2+ complexes) and by emission titrations (for one Pt2+ complex) which indicated that the disubstituted compounds 2‐Ni and 2‐Pt are the only ones that display good affinity for G4 DNA structures.

Parallel DNA G-Quadruplex Induced and Stabilized by Curaxin CBL0137

G-quadruplex (G4) is one of the higher-order DNA structures in guanine-rich sequences which are widely distributed across the genome. Due to their presence in oncogenic promoters and telomeres, G4 DNA structures become the novel targets in anticancer drug designs. Curaxin CBL0137, as an important candidate anticancer drug, can effectively inhibit the growth of multiple cancers. Although there is evidence that anticancer activity of curaxin is associated with its ability to bind DNA and to change the DNA topology, its therapeutic target and the underlying anti-cancer mechanism are still unclear. Here we show, for the first time, that curaxin CBL0137 induces G4 folding from anti-parallel to parallel structures, by single-molecule fluorescence resonance energy transfer technique. More importantly, we find that curaxin CBL0137 promotes G4 folding as well as stabilizes the folded G4 structures with long loops, giving a novel insight into effects of curaxin CBL0137 on DNA structures. Our work provides new ideas for the therapeutic mechanism of curaxin CBL0137 and for designs of new G4-targeting anticancer drugs.

Electrochemical detection of Oxaliplatin induced DNA damage in G-quadruplex structures

Oxaliplatin (OXP) is a platinum-based chemotherapeutic agent that induces DNA damage by forming intra- and interstrand crosslinks, mainly at the N7s of adenine (A) and guanine (G) bases. In addition to double-stranded DNA, G-rich G-quadruplex (G4)-forming sequences can also be targeted by OXP. However, high doses of OXP can lead to drug resistance and cause serious adverse effects during treatment. To better understand the targeting of G4 structures by OXP, their interactions as well as the molecular mechanisms underlying OXP resistance and adverse effects, there is a need for a rapid, quantitative, and cost-effective method to detect OXP and the damage it causes. In this study, we successfully fabricated a graphite electrode biosensor modified with gold nanoparticles (AuNPs) to investigate the interactions between OXP and the G4-forming promoter region (Pu22) of Vascular endothelial growth factor (VEGF). The overexpression of VEGF is known to be associated with tumor progression and the stabilization of VEGF G4 by small molecules is shown to suppresses VEGF transcription in different cancer cell lines. Differential pulse voltammetry (DPV) was used to investigate the interactions between OXP and Pu22-G4 DNA by monitoring the decrease in the oxidation signal of guanine with increasing OXP concentration. Under the optimized conditions (37 °C, 1:2 v/v AuNPs/water as electrode surface modifier, and 180 min incubation time) the developed probe showed a linear dynamic range of 1.0–10.0 μM with a detection limit of 0.88 μM and limit of quantification of 2.92 μM. Fluorescence spectroscopy was also used to support the electrochemical studies. We observed a decrease in the fluorescence emission of Thioflavin T in the presence of Pu22 upon addition of OXP. To our knowledge, this is the first electrochemical sensor developed to study OXP-induced damage to G4 DNA structures. Our findings provide new insights into the interactions between VEGF G4 and OXP, which could aid in targeting VEGF G4 structures and the development of new strategies to overcome OXP resistance.

A red light-triggered chemical tool for sequence-specific alkylation of G-quadruplex and I-motif DNA.

The importance of non-canonical DNA structures such as G-quadruplexes (G4) and intercalating-motifs (iMs) in the fine regulation of a variety of cellular processes has been recently demonstrated. As the crucial roles of these structures are being unravelled, it is becoming more and more important to develop tools that allow targeting these structures with the highest possible specificity. While targeting methodologies have been reported for G4s, this is not the case for iMs, as evidenced by the limited number of specific ligands able to bind the latter and the total absence of selective alkylating agents for their covalent targeting. Furthermore, strategies for the sequence-specific covalent targeting of G4s and iMs have not been reported thus far. Herein, we describe a simple methodology to achieve sequence-specific covalent targeting of G4 and iM DNA structures based on the combination of (i) a peptide nucleic acid (PNA) recognizing a specific sequence of interest, (ii) a pro-reactive moiety enabling a controlled alkylation reaction, and (iii) a G4 or iM ligand orienting the alkylating warhead to the reactive residues. This multi-component system allows for the targeting of specific G4 or iM sequences of interest in the presence of competing DNA sequences and under biologically relevant conditions.

New Xanthone Derivatives as Potent G-Quadruplex Binders for Developing Anti-Cancer Therapeutics.

Xanthone is an important scaffold for various medicinally relevant compounds. However, it has received scant attention in the design of agents that are cytotoxic to cancer cells via targeting the stabilization of G-quadruplex (G4) nucleic acids. Specific G4 DNA recognition against double-stranded (ds) DNA is receiving epoch-making interest for the development of G4-mediated anticancer agents. Toward this goal, we have synthesized xanthone-based derivatives with various functionalized side-arm substituents that exhibited significant selectivity for G4 DNA as compared to dsDNA. The specific interaction has been demonstrated by performing various biophysical experiments. Based on the computational study as well as the competitive ligand binding assay, it is inferred that the potent compounds exhibit an end-stacking mode of binding with G4 DNA. Additionally, compound-induced conformational changes in the flanking nucleotides form the binding pocket for effective interaction. Selective action of the compounds on cancer cells suggests their effectiveness as potent anti-cancer agents. This study promotes the importance of structure-based screening approaches to get molecular insights for new scaffolds toward desired specific recognition of non-canonical G4 DNA structures.

An environmentally insensitive fluorescent probe for G4 DNA detection: Design, synthesis, and mechanism studies

G4 DNA structure highly localized to functionally important sites within the human genome, has been identified as a biomarker for regulation of multiple biological processes. Identification G4-responsive fluorescence probes has broad application prospects for addressing G4 biological functions, as well as developing of new families of anticancer drugs. However, some currently designed G4 DNA probes may suffer from serious solvent-dependent effect, and cause unspecific fluorescence that masks the specific signal from G4 DNA. Herein, with a bulky imidazole-cored molecular rotor fusing in D-A building block of carbazole-pyridinium, we constructed a new probe ACPS. This new probe with desirable environmentally insensitive property exhibited a “fluorescence-off” state in various polarity solvents. In the presence of G4 DNA, the intra-molecular rotations would be restricted, triggering intense fluorescence enhancement. Especially, probe ACPS bound to G4 DNA structures with superior selectivity, exhibiting much weaker fluorescence response in the presence of non-G4 DNA structures. This probe was also able to realize fluorescence visualization in cell imaging. Collectively, the probe design strategy eliminates the background fluorescence caused by uncontrollable environmental polarity change, thereby achieving high-fidelity sensing G4 DNA structures in complicated systems.

The effect of side chain variations on quinazoline-pyrimidine G-quadruplex DNA ligands

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

Unique development of a new dual application probe for selective detection of antiparallel G-quadruplex sequences.

G-Quadruplex (G4) structures play vital roles in many biological processes; consequently, they have been implicated in various human diseases like cancer, Alzheimer's disease etc. The selective detection of G4 DNA structures is of great interest for understanding their roles and biological functions. Hence, development of multifunctional fluorescent probes is indeed essential. In this investigation, we have synthesized a quinolinium based dual application probe (QnMF) that presents molecular rotor properties. This dual application molecular rotor is able to detect selectively antiparallel G4 sequences (22AG in 100 mM NaCl) through a turn-on response over other G4 topologies. The QnMF also contains a distinct fluorine-19 that undergoes a significant chemical shift in response to microenvironmental changes around the molecule when bound to G4 structures. The probe QnMF exhibits significantly brighter fluorescence emissions in glycerol (ε × ϕ = 2800 cm-1 M-1) and relatively less brighter fluorescence emissions in methanol (ε × ϕ = 40.5 cm-1 M-1). The restricted rotation inherent property of the QnMF molecular rotor is responsible for brighter fluorescence and leads to enhancement in the fluorescence upon binding to the G4 structure. Overall, the probe's dual detection method makes it useful for monitoring the G4 structures that are abundant and plays a vital role in living organisms.

G-quadruplex-mediated specific recognition, stabilization and transcriptional repression of bcl-2 by small molecule

The presence of the G-quadruplex (G4) structure in the promoter region of the human bcl-2 oncogenes makes it a promising target for developing anti-cancer therapeutics. Bcl-2 inhibits apoptosis, and its frequent overexpression in cancer cells contributes to tumor initiation, progression, and resistance to therapy. Small molecules that can specifically bind to bcl-2 G4 with high affinity and selectivity are remaining elusive. Here, we report that small molecule 1,3-bis-) furane-2yl-methylidene-amino) guanidine (BiGh) binds to bcl-2 G4 DNA structure with very high affinity and selectivity over other genomic G4 DNA structures and duplex DNA. BiGh stabilizes folded parallel conformation of bcl-2 G4 via non-covalent and electrostatic interactions and increases the thermal stabilization up to 15 °C. The ligand significantly suppresses the bcl-2 transcription in HeLa cells by a G4-dependent mechanism and induces cell cycle arrest which promotes apoptosis. The in silico ADME profiling confirms the potential ‘drug-likeness’ of BiGh. Our results showed that BiGh stabilizes the bcl-2 G-quadruplex motif, downregulates the bcl-2 gene transcription as well as translation process in cervical cancer cells, and exhibits potential anti-cancer activity. This work provides a potential platform for the development of lead compound(s) as G4 stabilizers with drug-like properties of BiGh for cancer therapeutics.

DPA-Substituted Carbazole Derivative as a Fluorescent Ligand for G4 DNA.

Herein a conjugated dipicolylamine/carbazole (Car-DPA) molecule was designed and synthesized to enhance the performance for the application as a G4 fluorescent ligand. This ligand has been found to display distinct and specific fluorescence enhancements in the presence of various G4 DNA structures, but limited with ssDNA or dsDNAs. The detail binding characteristics of the ligand with c-MYC G4 DNA were investigated by fluorescence, UV/VIS absorption, CD spectroscopy, and molecular docking. The present study demonstrated that Car-DPA bound to c-MYC G4s with a two-step complex formation, in which the binding mode appeared to be end-stacking. Confocal fluorescence images indicated that ligand Car-DPA could locate in nucleus, which is quite prominent from the cellular internalization studies.

Design mitochondria-specific fluorescent turn-on probes targeting G-quadruplexes for live cell imaging and mitophagy monitoring study

A series of new mitochondrial-selective fluorescent probe was designed and synthesized based on the integration of two planar molecular scaffolds of benzo-indole/indole with a p-substituted styrene moiety. The small-sized probes are flexible and rotatable via an ethylene bridge. The restriction of free rotation upon interaction with targeting biomolecules of G-quadruplexes in mitochondria generates strong emission in visible range (575–615 nm). Cell imaging study showed that the ligands are targeting mitochondria but not nucleus. Competition experiments showed that the ligand BYM is highly selective towards mitochondrial G4-DNA structures against other non-G4 nucleic acid structures including single-/double-stranded DNA and hairpin. The equilibrium binding constant (Keq) of BYM interacting with mitochondrial G4-DNA (mt6363, Keq = 10.8 × 106 M−1) is almost 1000-fold higher than that of mitochondrial double-stranded DNA (Keq = 0.01 × 106 M−1). The probe also showed high sensitivity (LOD = 1.52 nM) and good linear relationship (R2 = 0.9983) with the mitochondrial G4-DNA. The delivery of BYM to mitochondria is not mitochondrial membrane potential dependent but mainly through the permeability transition pore on the mitochondrial inner membrane. In addition, BYM exhibits low cytotoxicity against a number of human cancer and noncancerous cells, indicating that BYM could be a good ligand for bioorthogonal study in live cells by targeting the mitochondrial G4-DNA structures. In the present study, BYM was demonstrated in monitoring the dynamic process of mitochondrial autophagy. The small-sized fluorescent probe possessing high photostability, selectivity and sensitivity targeting mitochondrial G-quadruplexes may able to provide a useful chemical tool for real-time study of mitochondrial functions in live cells.

Stable G-quadruplex DNA structures promote replication-dependent genome instability

G-quadruplex (G4)–prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites. Furthermore, we show that DNA double-strand breaks at the ectopic site are dependent on the nuclease Mus81. Hypermutagenesis during sister chromatid repair extends several kilobases from the G4 site and breaks at the G4 site resulting in microhomology-mediated translocations. To determine whether H3 or G4 structures are responsible for homopurine/homopyrimidine tract instability, we derived constructs and cell lines from the PKD1 repeat, which can only form H3 or G4 structures. Under normal growth conditions, we found that G4 cell lines lost the G4 consensus sequence early during clonal outgrowth, whereas H3 cells showed DNA instability early during outgrowth but only lost reporter gene expression after prolonged growth. Thus, both the H3 and G4 non-B conformation DNAs exhibit genomic instability, but they respond differently to endogenous replication stress. Our results show that the outcomes of replication-dependent double-strand breaks at non–B-DNAs model the instability observed in microhomology-mediated break-induced replication (BIR). Marked variability in the frequency of mutagenesis during BIR suggests possible dynamic heterogeneity in the BIR replisome.