Carbazole Derivatives Binding to Bcl-2 Promoter Sequence G-quadruplex.

In this study, we used ultraviolet-visible (UV-Vis), fluorescence, and circular dichroism (CD) techniques, as well as molecular modeling, to probe the interactions between carbazole derivatives and the G-quadruplex structure formed in the promoter region of gene Bcl-2. This gene is a rational target for anticancer therapy due to its high expression in a variety of tumors as well as resistance to chemotherapy-induced apoptosis. We employed a sequence with a specific dual G-to-T mutation that may form a mixed-type hybrid G-quadruplex structure in the Bcl-2 P1 promoter region. The three tested carbazole compounds differing in substitution on the nitrogen atom of carbazole interact with the Bcl-2 G-quadruplex by the same binding mode with the very comparable binding affinities in the order of 105 M-1. During absorption and fluorescence measurements, large changes in the ligand spectra were observed at higher G4 concentrations. The spectrophotometric titration results showed a two-step complex formation between the ligands and the G-quadruplex in the form of initial hypochromicity followed by hyperchromicity with a bathochromic shift. The strong fluorescence enhancement of ligands was observed after binding to the DNA. All of the used analytical techniques, as well as molecular modeling, suggested the π-π interaction between carbazole ligands and a guanine tetrad of the Bcl-2 G-quadruplex. Molecular modeling has shown differences in the interaction between each of the ligands and the tested G-quadruplex, which potentially had an impact on the binding strength.

A Label-Free Fluorescent Amplification Strategy for High-Sensitive Detection of Pseudomonas aeruginosa based on Protective-EXPAR (p-EXPAR) and Catalytic Hairpin Assembly.

This study presents a fluorescent mechanism for two-step amplification by combining two widely used techniques, exponential amplification reaction (EXPAR) and catalytic hairpin assembly (CHA). Pseudomonas aeruginosa (P. aeruginosa) engaged in competition with the complementary DNA in order to attach to the aptamer that had been fixed on the magnetic beads. The unbound complementary strand in the liquid above was utilized as a trigger sequence to initiate the protective-EXPAR (p-EXPAR) process, resulting in the generation of a substantial quantity of short single-stranded DNA (ssDNA). The amplified ssDNA can initiate the second CHA amplification process, resulting in the generation of many double-stranded DNA (dsDNA) products. The CHA reaction was initiated by the target/trigger DNA, resulting in the release of G-quadruplex sequences. These sequences have the ability to bond with the fluorescent amyloid dye thioflavin T (ThT), generating fluorescence signals. The method employed in this study demonstrated a detection limit of 16 CFU/ml and exhibited a strong linear correlation within the concentration range of 50 CFU/ml to 105 CFU/ml. This method of signal amplification has been effectively utilized to create a fluorescent sensing platform without the need for labels, enabling the detection of P. aeruginosa with high sensitivity.

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

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

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

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

Discovery of A G-Quadruplex Unwinder That Unleashes the Translation of G-Quadruplex-Containing mRNA without Inducing DNA Damage.

To explore the mechanisms and therapeutic strategies for G-quadruplex (G4) mediated diseases, it is crucial to manipulate and intervene in intracellular G4 structures using small molecular tools. While hundreds of G4 stabilizers have been developed, there is a significant gap in the availability of G4 unwinding agents. Here, we propose a strategy to disrupt G-quadruplexes by forming G-C hydrogen bonds with chemically modified cytidine trimers. We validated a good G4 unwinder, the 2'-F cytidine trimer (2'-F C3). 2'-F C3 does not inhibit cell growth nor cause severe DNA damage at a concentration below 10 µM. Moreover, 2'-F C3 does not affect gene transcription nor RNA splicing, while it significantly enhances the translation of G4-containing mRNA and upregulates RNA splicing, RNA processing and cell cycle pathways. The discovery of this G4 unwinder provides a functional tool for the chemical modulation of G4s in living cells.

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

G-quadruplex DNA sequences present in the promoter and telomere regions of the genomic sequence are considered therapeutic targets for the treatment of cancer. Curcumin, derived from Curcuma longa, has been known as a quadruplex binder and has a potential role in the apoptosis of cancer cells. Here, we have reported the Schiff base ligand of curcumin synthesized through the condensation of the amino acid L-tryptophan and the knoevenagel derivative of curcumin (4-nitrobenzylidene curcumin (NBC)) as a potential G-quadruplex binder. Thus, spectroscopic and biophysical studies reveal a higher binding affinity of the ligand Sb-NBC towards the promoter and telomere G-quadruplex sequence as compared to the parent NBC. The ligand Sb-NBC highly stabilizes the parallel and hybrid G-quadruplex topologies to 10.5 0C- 6.4 0C. Interestingly, the ligands also exhibit selective cytotoxicity toward cancer cells over normal cells. Taken together, this work provides evidence of the possibility of applying curcumin Schiff base in cancer therapy to regulate oncogene expression in cancer cells.

Gas-sensing riboceptors.

Understanding how cells sense gases or gaseous solutes is a fundamental question in biology and is pivotal for the evolution of molecular and organismal life. In numerous organisms, gases can diffuse into cells, be transported, generated, and sensed. Controlling gases in the cellular environment is essential to prevent cellular and molecular damage due to interactions with gas-dependent free radicals. Consequently, the mechanisms governing acute gas sensing are evolutionarily conserved and have been experimentally elucidated in various organisms. However, the scientific literature on direct gas sensing is largely based on hemoprotein-based gasoreceptors (or sensors). As RNA-based G-quadruplex (G4) structures can also bind to heme, I propose that some ribozymes can act as gas-sensing riboceptors (ribonucleic acid receptors). Additionally, I present a few other ideas for non-heme metal ion- or metal cluster-based gas-sensing riboceptors. Studying riboceptors can help understand the evolutionary origins of cellular and gasocrine signaling.

G-quadruplex structures in FGFR3 promoter negatively regulate its gene expression and DNA replication.

FGFR3 activating mutations and abnormal expression are linked to tumor development. However, the current state of research on FGFR3 gene expression regulation is relatively insufficient. In this study, we have reported that the FGFR3 promoter's positive strand contains several G-tracts and most likely forms a G-quadruplex (G4) structure. Circular dichroism investigations revealed that oligonucleotides from this region exhibit G-quadruplex-like molar ellipticity. We further validated the G4 structure of the FGFR3 promoter using biochemical and cellular molecular biology techniques. The G-quadruplex mutation enhanced the transcriptional activity of the FGFR3 promoter and DNA replication, suggesting that the G4 structure inhibits its expression. Furthermore, we conducted a preliminary screen for helicases associated with FGFR3 expression and explored their regulatory effects on FGFR3 gene transcription. Subsequently, we investigated the effect of curcumin on the stability of the G4 structure of the FGFR3 promoter and its regulatory effect on FGFR3 expression.

GQIcombi application to subdue glioma via differentiation therapy.

Current therapy protocols fail to cure high-grade gliomas and prevent recurrence. Therefore, novel approaches need to be developed. A re-programing of glioma cell fate is an alternative attractive way to stop tumor growth. The two-step protocol applies the antiproliferative GQ bi-(AID-1-T) and small molecule inducers with BDNF to trigger neural differentiation into terminally differentiated cells, and it is very effective on GB cell cultures. This original approach is a successful example of the "differentiation therapy". To demonstrate a versatility of this approach, in this publication we have extended a palette of cell cultures to gliomas of II, III and IV Grades, and proved an applicability of that version of differential therapy for a variety of tumor cells. We have justified a sequential mode of adding of GQIcombi components to the glioma cells. We have shown a significant retardation of tumor growth after a direct injection of GQIcombi into the tumor in rat brain, model 101/8. Thus, the proposed strategy of influencing on cancer cell growth is applicable to be further translated for therapy use.

Triple modal aptasensor arrays driven by CHA-mediated DNAzyme for signal-amplified atrazine pesticide accumulation monitoring in agricultural crops.

Developing sensors with high selectivity and sensitivity is of great significance for pesticide analysis in environmental assessment. Herein, a versatile three-way sensor array was designed for the detection of the pesticide atrazine, based on the integration of catalytic hairpin assembly (CHA) amplification and three-mode signal transducers. With atrazine, CHA was triggered to generate abundant G-quadruplex. The produced G-quadruplex hybrid could assemble with thioflavin T (TFT) or hemin to mimic enzyme and induce the fluorescence enhancement by TFT, or the colorimetric increase by the oxidized chromogenic substrate and the naked-eye color change by inhibiting the L-cysteine-mediated aggregation of gold nanoparticles. A distinctive three-mode array was successfully constructed with convenience, on-site accessibility and high sensitivity for enzyme-free practical analysis of atrazine. It is also effective and reliable for analyzing real samples including paddy water, paddy soil and polished rice. The detection limits for atrazine were as low as 7.4 pg/mL by colorimetric observation and 0.25 pg/mL by fluorescent detection. Furthermore, the array was exploited to monitor the residue, distribution and bioaccumulation of atrazine in maize and rice for food security and environmental assessment. Hence, this work presented a versatile example for sensitive and on-site all-in-one pesticide analysis arrays with multiple signal report modes.

Interaction analysis of RNA G-quadruplex with ligands and in situ imaging application.

RNA G4, as an integral branch of G4 structure, possesses distinct interactions with ligands compared to the common DNA G4, thus the investigation of RNA G4/ligand interactions might be considered as a fresh breakthrough to improve the biosensing performance of G4/ligand system. In this study, we comparatively explored the structural and functional mechanisms of RNA G4 and DNA G4 in the interaction with ligands, hemin and thioflavin T (ThT), utilizing the classical PS2.M sequence as a model. We found that although the catalytic performance of RNA G4/hemin system was lower than DNA G4/hemin, RNA G4/ThT fluorescence system exhibited a significant improvement (2∼3-fold) compared to DNA G4/ThT, and adenine modification could further enhance the signaling. Further, by exploring the interaction between RNA G4 and ThT, we deemed that RNA G4 and ThT were stacked in a bimolecular mode compared to single-molecule binding of DNA G4/ThT, thus more strongly limiting the structural spin in ThT excited state. Further, RNA G4/ThT displayed higher environmental tolerance and lower ion dependence than DNA G4/ThT. Finally, we employed RNA G4/ThT as a highly sensitive label-free fluorescent signal output system for in situ imaging of isoforms BCR-ABL e13a2 and e14a2. Overall, this study successfully screened a high-performance RNA G4 biosensing system through systematic RNA G4/ligands interaction studies, which was expected to provide a promising reference for subsequent G4/ligand research.

[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-ß-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.

Characterization of G-quadruplexes in the Helicobacter pylori genome and assessment of therapeutic potential of G4 ligands.

Helicobacter pylori, a leading human pathogen associated with duodenal ulcer and gastric cancer, presents a significant threat to human health due to increasing antibiotic resistance rates. This study investigates G-quadruplexes (G4s), which are non-canonical secondary structures form in G-rich regions within the H. pylori genome. Extensive research on G4s in eukaryotes has revealed their role in epigenetically regulating cellular processes like gene transcription, DNA replication, and oncogene expression. However, understanding of G4-mediated gene regulation in other organisms, especially bacterial pathogens, remains limited. Although G4 motifs have been extensively studied in a few bacterial species such as Mycobacterium, Streptococci, and Helicobacter, research on G4 motifs in other bacterial species is still sparse. Like in other organisms such as archaea, mammals, and viruses, G4s in H. pylori display a non-random distribution primarily situated within open reading frames of various protein-coding genes. The occurrence of G4s in functional regions of the genome and their conservation across different species indicates that their placement is not random, suggesting an evolutionary pressure to maintain these sequences at specific genomic sites. Moreover, G-quadruplexes show enrichment in specific gene classes, suggesting their potential involvement in regulating the expression of genes related to cell wall/membrane/envelope biogenesis, amino acid transport, and metabolism. This indicates a probable regulatory role for G4s in controlling the expression of genes essential for H. pylori survival and virulence. Biophysical techniques such as Circular Dichroism spectroscopy and Nuclear Magnetic Resonance were used to characterize G4 motifs within selected H. pylori genes. The study revealed that G-quadruplex ligand inhibited the growth of H. pylori, with minimal inhibitory concentrations in the low micromolar range. This suggests that targeting G4 structures could offer a promising approach for developing novel anti-H. pylori drugs.

Suppression of CTC1 inhibits hepatocellular carcinoma cell growth and enhances RHPS4 cytotoxicity.

BACKGROUND: Although DNA repair mechanisms function to maintain genomic integrity, in cancer cells these mechanisms may negatively affect treatment efficiency. The strategy of targeting cancer cells via inhibiting DNA damage repair has been successfully used in breast and ovarian cancer using PARP inhibitors. Unfortunately, such strategies have not yet yielded results in liver cancer. Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a treatment-resistant malignancy. Despite the development of guided therapies, treatment regimens for advanced HCC patients still fall short of the current need and significant problems such as cancer relapse with resistance still exist. In this paper, we targeted telomeric replication protein CTC1, which is responsible for telomere maintenance. METHODS: CTC expression was analyzed using tumor and matched-tissue RNA-sequencing data from TCGA and GTEx. In HCC cell lines, q-RT-PCR and Western blotting were used to detect CTC1 expression. The knock-down of CTC1 was achieved using lentiviral plasmids. The effects of CTC1 silencing on HCC cells were analyzed by flow cytometry, MTT, spheroid and colony formation assays. RESULTS: CTC1 is significantly downregulated in HCC tumor samples. However, CTC1 protein levels were higher in sorafenib-resistant cell lines compared to the parental groups. CTC1 inhibition reduced cell proliferation in sorafenib-resistant HCC cell lines and diminished their spheroid and colony forming capacities. Moreover, these cells were more sensitive to single and combined drug treatment with G4 stabilizer RHPS4 and sorafenib. CONCLUSION: Our results suggest that targeting CTC1 might be a viable option for combinational therapies designed for sorafenib resistant HCC patients.

Effect of molecular crowders on ligand binding kinetics with G-quadruplex DNA probed by fluorescence correlation spectroscopy.

Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect-a nearly three-fold decrease-observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.

Strategy for modeling higher-order G-quadruplex structures recalcitrant to NMR determination.

Guanine-rich nucleic acids can form intramolecularly folded four-stranded structures known as G-quadruplexes (G4s). Traditionally, G4 research has focused on short, highly modified DNA or RNA sequences that form well-defined homogeneous compact structures. However, the existence of longer sequences with multiple G4 repeats, from proto-oncogene promoters to telomeres, suggests the potential for more complex higher-order structures with multiple G4 units that might offer selective drug-targeting sites for therapeutic development. These larger structures present significant challenges for structural characterization by traditional high-resolution methods like multi-dimensional NMR and X-ray crystallography due to their molecular complexity. To address this current challenge, we have developed an integrated structural biology (ISB) platform, combining experimental and computational methods to determine self-consistent molecular models of higher-order G4s (xG4s). Here we outline our ISB method using two recent examples from our lab, an extended c-Myc promoter and long human telomere G4 repeats, that highlights the utility and generality of our approach to characterizing biologically relevant xG4s.

Design and synthesis of dual functional NBD-fluorophore-incorporated naphthalene diimide derivatives as G-quadruplex ligands.

Nitrobenzoxadiazole (NBD)-incorporated naphthalene diimide derivatives were designed and synthesized as candidate of antitumor agents with cytotoxicity against human pancreatic cancer cell MIA PaCa-2. Among these, compound 1NND and 3NND exhibited fluorescent "turn-off" property toward human telomeric G-quadruplex (G4), which allows the direct measurement of dissociation constant (Kd) of ligands against G4 by fluorescence titration method. Notably, the compound 1NND not only exhibited great cytotoxic activity against MIA PaCa-2 with a half maximal inhibitory concentration (IC50) of 77.9 nM, but also exhibited high affinity against G4 with Kd of 1.72 µM. Furthermore, the target binding properties were investigated by circular dichroism (CD) spectra and further studied by molecular docking methods.

Overcoming Cancer Persister Cells by Stabilizing the ATF4 Promoter G-quadruplex.

Persister cells (PS) selected for anticancer therapy have been recognized as a significant contributor to the development of treatment-resistant malignancies. It is found that imposing glutamine restriction induces the generation of PS, which paradoxically bestows heightened resistance to glutamine restriction treatment by activating the integrated stress response and initiating the general control nonderepressible 2-activating transcription factor 4-alanine, serine, cysteine-preferring transporter 2 (GCN2-ATF4-ASCT2) axis. Central to this phenomenon is the stress-induced ATF4 translational reprogramming. Unfortunately, directly targeting ATF4 protein has proven to be a formidable challenge because of its flat surface. Nonetheless, a G-quadruplex structure located within the promoter region of ATF4 (ATF4-G4) is uncovered and resolved, which functions as a transcriptional regulator and can be targeted by small molecules. The investigation identifies the natural compound coptisine (COP) as a potent binder that interacts with and stabilizes ATF4-G4. For the first time, the high-resolution structure of the COP-ATF4-G4 complex is determined. The formation of this stable complex disrupts the interaction between transcription factor AP-2 alpha (TFAP2A) and ATF4-G4, resulting in a substantial reduction in intracellular ATF4 levels and the eventual death of cancer cells. These seminal findings underscore the potential of targeting the ATF4-G4 structure to yield significant therapeutic advantages within the realm of persister cancer cells induced by glutamine-restricted therapy.

Molecular mechanisms behind BRACO19 and human telomeric G-quadruplex interaction.

Human telomeres (HTs) can form DNA G-quadruplex (G4), an attractive target for anticancer and antiviral drugs. HT-G4s exhibit inherent structural polymorphism, posing challenges for understanding their specific recognition by ligands. Here, we aim to explore the impact of different topologies within a small segment of the HT (Tel22) on its interaction with BRACO19, a rationally designed G4 ligand with high quadruplex affinity, already employed in in-vivo treatments. Our multi-technique approach is based on the combined use of a set of contactless spectroscopic tools. Circular dichroism and UV resonance Raman spectroscopy probe ligand-induced conformational changes in the G4 sequence, while UV-visible absorption, coupled with steady-state fluorescence spectroscopy, provides further insights into the electronic features of the complex, exploiting the photoresponsive properties of BRACO19. Overall, we find that modifying the topology of the unbound Tel22 through cations (K+ or Na+), serves as a critical determinant for ligand interactions and binding modes, thus influencing the HT-G4's assembly capabilities. Furthermore, we show how fluorescence serves as a valuable probe for recognizing cation-driven multimeric structures, which may be present in living organisms, giving rise to pathological forms.

Unfolding rates of 1:1 and 2:1 complex of CX-5461 and c-MYC promoter G-quadruplexes revealed by single-molecule force spectroscopy.

CX-5461, also known as pidnarulex, is a strong G4 stabilizer and has received FDA fast-track designation for BRCA1- and BRCA2- mutated cancers. However, quantitative measurements of the unfolding rates of CX-5461-G4 complexes which are important for the regulation function of G4s, remain lacking. Here, we employ single-molecule magnetic tweezers to measure the unfolding force distributions of c-MYC G4s in the presence of different concentrations of CX-5461. The unfolding force distributions exhibit three discrete levels of unfolding force peaks, corresponding to three binding modes. In combination with a fluorescent quenching assay and molecular docking to previously reported ligand-c-MYC G4 structure, we assigned the ~69 pN peak corresponding to the 1:1 (ligand:G4) complex where CX-5461 binds at the G4's 5'-end. The ~84 pN peak is attributed to the 2:1 complex where CX-5461 occupies both the 5' and 3'. Furthermore, using the Bell-Arrhenius model to fit the unfolding force distributions, we determined the zero-force unfolding rates of 1:1, and 2:1 complexes to be (2.4 ± 0.9) × 10-8 s-1 and (1.4 ± 1.0) × 10-9 s-1 respectively. These findings provide valuable insights for the development of G4-targeted ligands to combat c-MYC-driven cancers.