G-quadruplex DNA Research Articles

Mechanistic Insights into Oxygen-Independent DNA Photodegradation by Pheophorbide a: Implications for Future Photodynamic Therapy Applications.

This study explores the unique properties of Pheophorbidea(Pheda) in the photodegradation of G-quadruplex DNA under anoxic conditions, emphasizing its potential for photodynamic therapy (PDT) in hypoxic tumor environments. We used electron spin resonance (ESR), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopies to assess the radical generation and DNA interaction capabilities of Phedacompared to Pyropheophorbidea(Pyroa) under both oxygenated and anoxic conditions. Our results reveal that Phedaeffectively degrades G-quadruplex DNA in the absence of oxygen, whereas Pyroadoes not. Under anoxic conditions, Phedagenerates unique carbon-centered radicals, as indicated by distinctive ESR signals, which are not observed in aerobic environments or with Pyroa. These radicals facilitate DNA decomposition without relying on reactive oxygen species (ROS). Pheda's ability to degrade DNA independently of oxygen underscores its potential as a versatile and effective PDT agent, especially for hypoxic tumors where traditional photosensitizers are ineffective. This study paves the way for the development of new PDT agents that can operate under different oxygen levels. Pheda's radical generation and oxygen-independent DNA degradation mechanisms make it a promising candidate for future PDT applications.

G-quadruplex stabilizer CX-5461 effectively combines with radiotherapy to target ATRX-deficient malignant glioma.

Inactivation of α-thalassaemia/mental retardation X-linked (ATRX) represents a defining molecular feature in large subsets of malignant glioma. ATRX deficiency gives rise to abnormal G-quadruplex (G4) DNA secondary structures, enhancing replication stress and genomic instability. Building on earlier work, we evaluated the extent to which pharmacological G4 stabilization selectively enhances DNA damage and cell death in ATRX-deficient preclinical glioma models. Using the G4 stabilizer CX-5461, we treated patient-derived glioma stem cells (GSCs) in vitro and GSC flank and intracranial murine xenografts in vivo to evaluate efficacy as both a single agent and in combination with ionizing radiation (IR), the latter a central element of current treatment standards. CX-5461 promoted dose-sensitive lethality in ATRX-deficient GSCs relative to ATRX-intact controls. Mechanistic studies revealed that CX-5461 disrupted histone variant H3.3 deposition, enhanced replication stress and DNA damage, activated p53-independent apoptosis, and induced G2/M arrest to a greater extent in ATRX-deficient GSCs than in ATRX-intact counterparts. These data were corroborated in vivo, where CX-5461/IR treatment profoundly delayed tumor growth and prolonged survival in mice bearing ATRX-deficient flank xenografts. Histopathological analyses revealed decreased proliferation, increased apoptosis, and significant G4 induction, replication stress, and DNA damage in CX-5461-treated tumors, both alone and in combination with IR. Finally, despite suboptimal blood-brain-barrier penetration, systemic CX-5461 treatment induced tangible pharmacodynamic effects in ATRX-deficient intracranial GSC models. In totality, our work substantively demonstrates efficacy and defines mechanisms of action for G4 stabilization as a novel therapeutic strategy targeting ATRX-deficient malignant glioma, laying the groundwork for clinical translation.

Neurotransmitters and neural hormone-based probes for quadruplex DNA sequences associated with neurodegenerative diseases

The potential of neurotransmitters and neural hormones as possible G-quadruplex DNA binders was analyzed using fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), DNA melting analysis, and molecular docking. G-quadruplex sequences, (GGC)3 and G4C2, with roles in Fragile X syndrome and amyotrophic lateral sclerosis (ALS), respectively, were selected, and their interactions with melatonin, serotonin, and gamma-aminobutyric acid (GABA), were studied. Both melatonin and serotonin demonstrated strong interactions with the DNA sequences with hydrogen bonding being the primary mode of interaction, with some non-intercalative interactions involving the π systems. GABA demonstrated much weaker interactions and may not be a suitable candidate as a probe for low concentrations of G-quadruplex DNA.

A Rationally Designed Azobenzene Photoswitch for DNA G-Quadruplex Regulation in Live Cells.

G-quadruplex (G4) DNA structures are increasingly acknowledged as promising targets in cancer research, and the development of G4-specific stabilizing compounds may lay a fundamental foundation in precision medicine for cancer treatment. Here, we propose a light-responsive G4-binder for precise modulation of drug activation, providing dynamic and spatiotemporal control over G4-associated biological processes contributing to cancer cell death. We developed a specialized fluorinated azobenzene (AB) switch equipped with a quinoline unit and a positively charged carboxamide side chain, Q-Azo4F-C, designed for targeted binding to G4 structures within cells. Biophysical studies, combined with molecular dynamics simulations, provide insights into the unique coordination modes of the photoswitchable ligand in its trans and cis configurations when interacting with G4s. The observed variations in complexation processes between the two isomeric states in different cancer cell lines manifest in more than 25-fold reversible cytotoxic activity. Immunostaining conducted with the structure-specific G4 antibody (BG4), establishes a direct correlation between cytotoxicity and the varying extent of G4 induction regulated by the two isoforms. Finally, we demonstrate the photo-driven reversible regulation of G4 structures in lung cancer cells by Q-Azo4F-C. Our findings highlight the potential of light-responsive G4-binders in advancing precision cancer therapy through dynamic control of G4-mediated pathways.

Construction of G-quadruplex/thioflavin T fluorescent probe for label-free detection of malachite green

Malachite green (MG), owing to its extreme toxicity, is strictly prohibited as a drug in aquaculture across numerous countries. To facilitate an economical and user-friendly strategy for MG detection, we rigorously screened c-MYC22 from a wide range of G-quadruplex (G4) DNAs, aiming to craft a fluorescent probe in tandem with the water-soluble fluorescent dye thioflavin T (ThT). This endeavor culminated in the development of a label-free, rapid and sensitive method for MG detection. The fluorescence of the G4/ThT complex dims significantly when MG interacts with G4, allowing for the quantitative analysis of MG. Our method boasts linear detection ranges between 50–1000 µg/L and 400–6000 µg/L, with an impressive limit of detection as low as 6.82 µg/L and a short detection time of 15 min. The fluorescent probe demonstrates excellent specificity, rendering it ideal for MG detection in real water samples. Furthermore, this method stands out for its simplicity and efficiency, the probe used does not need to be modified, and the detection takes only one step with a short detection time. These features make it a strong contender for commercialization as a method of MG detection, presenting significant potential for further advancement.

Factors Affecting Liquid-Liquid Phase Separation of RGG Peptides with DNA G-Quadruplex.

Liquid-liquid phase separation (LLPS), mediated by G-quadruplexes (G4 s) and intrinsically disordered proteins, particularly those containing RGG domains, plays a critical role in cellular processes and diseases. However, the molecular mechanism and the role of individual amino acid residues of the protein in LLPS with G4 (G4-LLPS) are still unknown. Here, we systematically designed peptides and investigated the roles of arginine residues in G4-LLPS. It was found that the FMRP-derived RGG peptide induced LLPS with G4-forming Myc-DNA, whereas a point-mutated peptide, in which all arginine residues were replaced with lysine, was unable to undergo LLPS, indicating the importance of arginine residues. Moreover, systematically truncated peptides showed that at least five positive net charges of peptide are required to induce G4-LLPS. Furthermore, quantitative investigation demonstrated that the higher binding affinity of peptides with G4 led to a higher LLPS ability, whereas threshold of the binding affinity for undergoing LLPS was identified. These insights elucidate the pivotal role of arginine in G4-LLPS and the specific requirement for multiple arginine residues, contributing to a deeper understanding of the complex interplay between intrinsically disordered proteins and nucleic acids.

G-Quadruplex Recognition by Tetraalkylammonium Ions: A New Paradigm for Discrimination between Parallel and Antiparallel G-Quadruplexes.

G-quadruplexes are an important class of noncanonical secondary structures of DNA that exist in the cell and are involved in the regulation of principal genomic events. Any regulatory role of a G-quadruplex in the genome is coupled with the attendant interconversions between the G-quadruplex and duplex states. Much effort has been invested in the quest for agents that can recognize individual G-quadruplexes and shift the associated duplex-G-quadruplex equilibria toward the G-quadruplex state. In this communication, we demonstrate that, notwithstanding their simplicity, tetraalkylammonium ions, [H(CH2)n]4N+, recognize and strongly stabilize the parallel c-MYC G-quadruplex, while not binding to the antiparallel human telomeric G-quadruplex or duplex DNA. The affinity of TAA+ ions for the c-MYC G-quadruplex correlates with the length of the aliphatic side chains. Our CD spectral data suggest that the binding of tetraalkylammonium ions is external, not resulting in structural changes in the host G-quadruplex. The observed discriminatory power identifies tetraalkylammonium salts as a starting point for developing topology-selective G-quadruplex-binding agents.

A Novel Approach for Colorimetric Detection of Glyphosate in Food Based on a Split Aptamer Nanostructure and DNAzyme Activity.

Glyphosate has become the most widely used herbicide worldwide in recent years. There are many concerns about toxicity and mutagenicity from long-term use of glyphosate in humans and animals. Therefore, the methods that can help in easy and quick detection of this chemical compound in food and water are critical. In this work, a biosensor was fabricated by combining the enzymatic properties of a specific DNA G-quadruplex and selectivity of a split aptamer to detect glyphosate in foods and water in a quick and simple colorimetric manner. The color change in this method is based on the oxidation of TMB by the G-quadruplex enzyme and the function of aptamer to trap glyphosate, which is visible to the naked eye in the presence and absence of the herbicide. The biosensor showed its high performance in various real samples of water and foods and provided a detection limit of 1.37 nM (R² = 0.9899) with a linear response range of 100 to 400 nM of glyphosate. This biosensor can provide an innovative, cheap and fast approach for the detection and monitoring of glyphosate in various foods and water.

Rationally Designed G-Quadruplex Selective "Turn-On" NIR Fluorescent Probe with Large Stokes Shift for Nucleic Acid Research-Based Applications.

Guanine-rich DNA/RNA sequences can form Hoogsteen bonds to adopt noncanonical secondary structures called G-quadruplexes, and these have been associated with diverse cellular processes. There has been considerable research interest in the design of G4-interacting ligands for cellular probing of the G4 structure and understanding its associated biological function. Most of the fluorescent G4 ligands either do not have significant selectivity over other nucleic acid structures, have high Stokes shift, or are not in the near-infrared (NIR) region, which limits its cellular visualization. The current work involves the rational design and synthesis of NIR fluorescent probes comprising a (Z)-1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium scaffold. Among the designed molecules, 4a exhibited far-red fluorescence (λmax = 680 nm) with large Stokes shift (∼182 nm) upon selective binding to human telomeric G-quadruplexes. The dye 4a does not disturb the conformation and stability of G-quadruplexes, thereby making it suitable for nucleic acid research based applications. Interestingly, 4a showed remarkable selectivity over single- and double-stranded structures in contrast to a commercially available quadruplex binding probe, Thiazole orange (TO). The molecular docking studies indicate that 4a binds at the groove region of the telomeric DNA G-quadruplex through π-π stacking interactions with the quinoline and amine-substituted phenyl ring and with the phosphate backbone through anion-π interactions with the benzothiazole ring. The designed molecule 4a has interesting photophysical properties, cell permeability, and biocompatibility with minimal cytotoxicity. Fluorescence imaging studies in live HeLa cells showed that probe 4a binds to the transient population of the DNA G-quadruplex in the nucleus and RNA quadruplexes in the cytoplasm. In brief, G-quadruplex NIR fluorescent probe 4a with a higher signal/noise ratio has significant potential for cellular imaging studies and thus opens avenues to decipher the biological pathways for better understanding of G-quadruplex biology.

Single-Molecule adenosine detection and chiral selectivity by DNA aptamer conformational changes using α-Hemolysin nanopore

Nucleic acid aptamers, which are short single-stranded RNA or DNA molecules, undergo conformational changes upon recognizing small molecules. However, their structural characterization is challenging due to the flexibility of single-stranded oligonucleotides. Nanopores provide a sensitive method for single-molecule analysis of molecular conformational changes as they transport through the pore. In this study, we employed α-hemolysin (α-HL) nanopore with high spatiotemporal resolution to investigate the conformational changes of DNA aptamer (ADO-23) upon binding to adenosine (Ado) molecules. Unlike the events generated by the aptamer alone, the DNA-Ado complex produced two distinct new characteristic events, indicating the formation of two unique secondary structures. By analyzing current events, the structures formed by the DNA-Ado complex were speculated to be hairpin DNA and G-quadruplex (G4) DNA. Furthermore, we demonstrated the potential of the aptamer as a chiral selector within the nanopore. The results showed that the aptamer specifically bound to D-Ado and not to L-Ado. Finally, we realized specific and sensitive detection of small molecule Ado by α-HL pore, with a detection limit as low as 10.5 nM, and successfully applied it to the detection of real human serum samples. The nanopore platform provides a powerful tool for studying small molecule-biomolecule conformational changes, and we constructed sensors for detecting adenosine through the aptamer. This work also offers a promising new approach to the recognition of enantiomers at the single-molecule level.

Discovery of Arene Ruthenium(II) Complexes as Potential VEGF Inhibitors for Glioblastoma Metastasis Suppression.

Developing drugs for treating glioblastoma has been a significant challenge. Herein, a series of arene ruthenium(II) complexes have been synthesized and investigated as potential candidates to suppress the proliferation and metastasis of glioblastoma. It is found that para-substituent-modified molecules, especially 6, exhibit higher antitumor activity than ortho-substituents. Further studies show that 6 can trigger tumor cell autophagy by regulating the PI3K/AKT/mTOR pathway. Moreover, it is also found that 6 can induce DNA damage in glioblastoma cells through binding and stabilizing VEGF G-quadruplex DNA. Furthermore, it is confirmed that 6 can inhibit the proliferation and metastasis of U87-MG glioblastoma cell in situ xenograft in the zebrafish model. Hence, arene ruthenium(II) complexes can be developed as promising therapeutic agents for glioblastoma treatment in the future.

Computational modeling to understand the interaction of TMPyP4 with a G-quadruplex

The potential of small molecules to bind to G-quadruplex-forming sequences in oncogene promoter regions, thereby regulating their structural equilibrium, has been explored as a promising strategy for cancer chemotherapy. The model drug 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4) has been shown to have an affinity toward G-quadruplex DNA. However, the precise sites and modes of TMPyP4 binding to G-quadruplex DNA remain a subject of debate. In this study, we focus on identifying potential binding sites on a mutant c-MYC sequence known to fold into a single 1:2:1 loop isomer quadruplex. Our findings provide insights into the 4:1 stoichiometry reported for TMPyP4 binding to this G-quadruplex. Binding enthalpy and free energy calculations show that intercalation of a TMPyP4 molecule between the quadruplexes is thermodynamically favorable. Our calculations suggest that two of the binding sites are located at the top and bottom of the quadruplex, respectively, while the remaining two are likely intercalations.

Visualization of Mitochondrial DNA G-Quadruplexes with Isaindigotone Derived Near-Infrared Fluorogenic Probe.

Mitochondrial DNA G-quadruplexes (mtDNA G4s) play potential regulatory roles in mitochondrial functions. Fluorescent probes for imaging mtDNA G4s may provide useful information to unveil their regulating dynamics and functions. However, the existing probes for mtDNA G4s still exhibit short absorption and emission wavelengths and limited sensitivity. Here, we develop a new isaindigotone-derived near-infrared (NIR) fluorogenic probe for imaging mtDNA G4s in live cells and in vivo. Different fluorescent probes are engineered by conjugating the isaindigotone scaffold with varying electron-donating groups. It is shown that the probe ISAP using dimethylaminophenyl as the electron-donating group exhibits near-infrared absorption/emission and a high fluorescence activation fold in response to G4s. Molecular docking simulations reveal that ISAP binds to c-Myc G4 via multiple π-π stacking and hydrogen-bond interaction. Cellular studies show that ISAP exhibits an excellent mitochondrial targeting ability and allows specific imaging of mtDNA G4s. We further employed ISAP to image the dynamics of mtDNA G4s under glycolysis and oxidative stresses in live cells. Its capability to mtDNA G4s in vivo is showcased using a tumor-bearing mice model. This probe may serve as a useful tool to image mtDNA G4s and interrogate their biological roles in living systems.

Differential binding of curcumin with chair and basket type anti-parallel DNA G-quadruplexes

Guanine rich DNA sequences are widespread throughout the genome and they are prone to form G-quadruplex structures both in-vitro and in-vivo. Owing to their regulatory roles in important biological reactions, DNA G-quadruplexes are the prime target for the development of anti-cancer drug molecules. In the present study, we have investigated the interaction of curcumin with anti-parallel chair and basket type DNA G-quadruplexes formed by thrombin binding aptamer (TBA) and human telomeric repeat (HTG) sequences, respectively. The results obtained from spectroscopic techniques demonstrated that curcumin interacts with both the DNA G-quadruplexes through external binding mode and the estimated apparent binding constant value (Kb) was slightly higher for HTG (Kb = (4.38 ± 0.09) × 105 M−1) than that estimated for TBA (Kb = (0.62 ± 0.02) × 105 M−1) DNA G-quadruplex structures. It was also revealed that upon binding with curcumin, TBA and HTG DNA G-quadruplexes were slightly stabilized and destabilized, respectively. Circular dichroism study revealed that upon binding with curcumin the global structure of both the DNA G-quadruplexes was unaltered, whereas the local structure of HTG DNA G-quaruplex was slightly altered. Conjointly all the aforementioned spectroscopic methods emphasize that curcumin has differential interaction with anti-parallel DNA G-quadruplex structures depending on their loop orientation.

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.

Synergistic Anticancer Effects by Enhancing the G-Quadruplex Binding of Nickel(II) Salphen Complexes through Coupling with S-Doped Carbon Nanodots.

In recent decades, researchers have focused on developing less toxic and more precise cancer therapies. Carbon nanodots (CDs) are among the most promising technologies due to their high biocompatibility, tunable fluorescence, and ability to facilitate photothermal and photodynamic therapy. This study explores the synthesis and characterization of two CDs conjugated with Salphen metal complexes, namely, CDs-PEG-M1 and CDs-PEG-M2, through Sonogashira coupling. Their interaction with G-quadruplex DNA structures (G4s), motifs largely involved in cancer development, was evaluated using various spectroscopic techniques. The results indicate that CDs-PEG-M1 exhibits greater effectiveness in stabilizing G4 structures compared to the metal complex alone or nonfunctionalized CDs. This enhanced stabilization suggests that CDs-PEG-M1 could reduce the concentration of the metal complex needed for potential antitumor applications, thereby minimizing side effects on nontarget tissues. When tested on breast cancer models (MDA-MB-231 as a triple-negative model and MCF-7 as a HER-2 positive model) and on a healthy cell line (HDFa), the CDs-PEG-M1 conjugate reduced cell viability in a concentration- and time-dependent manner, showing greater potency and selectivity against cancer cells compared to virgin CDs and the free M1 complex. This synergistic anticancer effect, driven by the interaction with G4 structures and reactive oxygen species production, underscores the potential of CDs-PEG-M1 as a targeted nanotheranostic tool.

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

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

Untargeted CUT&Tag and BG4 CUT&Tag are both enriched at G-quadruplexes and accessible chromatin.

G-quadruplex DNA structures (G4s) form within single-stranded DNA in nucleosome-free chromatin. As G4s modulate gene expression and genomic stability, genome-wide mapping of G4s has generated strong research interest. Recently, the Cleavage Under Targets and Tagmentation (CUT&Tag) method was performed with the G4-specific BG4 antibody to target Tn5 transposase to G4s. While this method generated a novel high-resolution map of G4s, we unexpectedly observed a strong correlation between the genome-wide signal distribution of BG4 CUT&Tag and accessible chromatin. To examine whether untargeted Tn5 cutting at accessible chromatin contributes to BG4 CUT&Tag signal, we examined the genome-wide distribution of signal from untargeted (i.e. negative control) CUT&Tag datasets. We observed that untargeted CUT&Tag signal distribution was highly similar to both that of accessible chromatin and of BG4 CUT&Tag. We also observed that BG4 CUT&Tag signal increased at mapped G4s, but this increase was accompanied by a concomitant increase in untargeted CUT&Tag at the same loci. Consequently, enrichment of BG4 CUT&Tag over untargeted CUT&Tag was not increased at mapped G4s. These results imply that either the vast majority of accessible chromatin regions contain mappable G4s or that the presence of G4s within accessible chromatin cannot reliably be determined using BG4 CUT&Tag alone.

Pd(II)/1,10-phenanthroline complexes bearing arene ligands: On the role of N- vs O-coordination to tune their cellular activity and binding ability towards DNA and RNA

Three Pd(II)-based complexes of 1,10-phenanthroline and N- or O-coordinating ligands have been synthesised and tested with different relevant biosubstrates like double-stranded DNA, double and triple helix of RNA, DNA G-quadruplexes of different conformations and bovine serum albumin. Here a correlation between N- vs O-coordinating elements and binding mechanism emerged, where the N-coordinating ligands proved to be the most promising. These outcomes were confirmed also in the cellular experiments. The Pd(II) complex with naphthalene-1,8-diamine is the one that is able to be carried by BSA, to strongly bind nucleic acids, to produce reactive oxygen species (ROS) and to show the best cellular performances (poorly toxic towards healthy cells and highly toxic against the cisplatin-resistant cancer cell line). On the opposite, the complex with benzene-1,2-diolate may be sequestered by BSA, weakly binds nucleic acids, does not produce ROS and shows poor cellular activity. The complex with benzene-1,2-diamine stays in between. Other mechanistic details are discussed which show that the biophysical behaviour is the sum of the contribution of aromaticity, charge and N- or O-coordination.

A Comparative Study on the DNA Interactions and Biological Activities of Benzophenanthridine and Protoberberine Alkaloids.

Numerous small molecules exert antitumor effects by interacting with DNA, thereby influencing processes, such as DNA replication, transcription, meiosis, and gene recombination. Benzophenanthridine and protoberberine alkaloids are known to bind DNA and exhibit many pharmacological activities. In this study, we conducted a comparative analysis of the interactions between these two classes of alkaloids with G-quadruplex (G4) DNA and double-stranded DNA (dsDNA). Protoberberine alkaloids showed a greater affinity for binding with G4s than with dsDNA, while benzophenanthridine alkaloids exhibited a significantly stronger binding capacity for dsDNA, especially in regions that are rich in AT base pairs. Benzophenanthridine alkaloids also exhibited much stronger toxicity to various cancer cells. Compared with protoberberine alkaloids, benzophenanthridine alkaloids displayed much stronger activity in inhibiting cellular DNA and RNA synthesis, arresting the cell cycle in the G2/M phase, inducing cell apoptosis, and leading to intracellular DNA damage. Given that dsDNA constitutes the predominant form of DNA within cells for the majority of the cell cycle, the significant antiproliferative activity of benzophenanthridine alkaloids could be attributed, in part, to their higher binding affinity for dsDNA, thereby exerting a more significant impact on cellular proliferation. These findings have valuable implications for understanding the biological activities of isoquinoline alkaloids and their antitumor applications.