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.

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.

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.

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.

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.

A Cost-Effective Hemin-Based Artificial Enzyme Allows for Practical Applications.

Nanomaterials excel in mimicking the structure and function of natural enzymes while being far more interesting in terms of structural stability, functional versatility, recyclability, and large-scale preparation. Herein, the story assembles hemin, histidine analogs, and G-quadruplex DNA in a catalytically competent supramolecular assembly referred to as assembly-activated hemin enzyme (AA-heminzyme). The catalytic properties of AA-heminzyme are investigated both in silico (by molecular docking and quantum chemical calculations) and in vitro (notably through a systematic comparison with its natural counterpart horseradish peroxidase, HRP). It is found that this artificial system is not only as efficient as HRP to oxidize various substrates (with a turnover number kcat of 115 s-1) but also more practically convenient (displaying better thermal stability, recoverability, and editability) and more economically viable, with a catalytic cost amounting to <10% of that of HRP. The strategic interest of AA-heminzyme is further demonstrated for both industrial wastewater remediation and biomarker detection (notably glutathione, for which the cost is decreased by 98% as compared to commercial kits).

Development of a multi-targeted chemotherapeutic approach based on G-quadruplex stabilisation and carbonic anhydrase inhibition.

A novel class of compounds designed to hit two anti-tumour targets, G-quadruplex structures and human carbonic anhydrases (hCAs) IX and XII is proposed. The induction/stabilisation of G-quadruplex structures by small molecules has emerged as an anticancer strategy, disrupting telomere maintenance and reducing oncogene expression. hCAs IX and XII are well-established anti-tumour targets, upregulated in many hypoxic tumours and contributing to metastasis. The ligands reported feature a berberine G-quadruplex stabiliser scaffold connected to a moiety inhibiting hCAs IX and XII. In vitro experiments showed that our compounds selectively stabilise G-quadruplex structures and inhibit hCAs IX and XII. The crystal structure of a telomeric G-quadruplex in complex with one of these ligands was obtained, shedding light on the ligand/target interaction mode. The most promising ligands showed significant cytotoxicity against CA IX-positive HeLa cancer cells in hypoxia, and the ability to stabilise G-quadruplexes within tumour cells.

Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target.

Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

Toward the Establishment of a Harmonized Physicochemical Profiling Platform for Therapeutic Oligonucleotides: A Case Study for Aptamers Where the Higher-Order Structure Influences Physical Properties.

Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.

Michael addition-activated alkylation of G-quadruplex DNA with methylamine-protected vinyl-quinazolinone derivatives.

The role of G-quadruplex (G4) in cellular processes can be investigated by the covalent modification of G4-DNA using alkylating reagents. Controllable alkylating reagents activated by external stimuli can react elegantly and selectively. Herein, we report a chemical activation system that can significantly boost the reaction rate of methylamine-protected vinyl-quinazolinone (VQ) derivative for the alkylation of G4-DNA. The two screened activators can transform low-reactive VQ-NHR' to highly reactive intermediates following the Michael addition mechanism. This approach expands the toolbox of activable G4 alkylating reagents.

Novel aerogels based on supramolecular G-quadruplex assembly with intrinsic flame retardancy and thermal insulation.

The construction of supramolecular aerogels still faces great challenges. Herein, we present a novel bio-based supramolecular aerogel derived from G-Quadruplex self-assembly of guanosine (G), boric acid (B) and sodium alginate (SA) and the obtained GBS aerogels exhibit superior flame-retardant and thermal insulating properties. The entire process involves environmentally friendly aqueous solvents and freeze-drying. Benefiting from the supramolecular self-assembly and interpenetrating dual network structures, GBS aerogels exhibit unique structures and sufficient self-supporting capabilities. The resulting GBS aerogels exhibit overall low densities (36.5-52.4 mg/cm3), and high porosities (>95 %). Moreover, GBS aerogels also illustrate excellent flame retardant and thermal insulating properties. With an oxygen index of 47.0-51.1 %, it can easily achieve a V-0 rating and low heat, smoke release during combustion. This work demonstrates the preparation of intrinsic flame-retardant aerogels derived from supramolecular self-assembly and dual cross-linking strategies, and is expected to provide an idea for the realization and application of novel supramolecular aerogel materials.

Probing G-quadruplex-ligand binding using DNA intrinsic fluorescence.

G-quadruplexes (G4s) are helical four-stranded nucleic acid structures that can form in guanine-rich sequences, which are mostly found in functional cellular regions, such as telomeres, promoters, and DNA replication origins. Great efforts are being made to target these structures towards the development of specific small molecule G4 binders for novel anti-cancer, neurological, and viral therapies. Here, we introduce an optical assay based on quenching of the intrinsic fluorescence of DNA G-quadruplexes for assessing and comparing the G4 binding affinity of various small molecule ligands in solutions. We show that the approach allows direct quantification of ligand binding to distinctive G4 topologies. We believe that this method will facilitate quick and reliable evaluation of small molecule G4 ligands and support their development.

Aptamer-Drug conjugates for a targeted and synergistic anticancer Response: Exploiting T30923-5-fluoro-2'-deoxyuridine (INT-FdU) derivatives.

One of the most appealing approaches for cancer treatment is targeted therapy, which is based on the use of drugs able to target cancer cells without affecting normal ones. This strategy lets to overcome the major limitation of conventional chemotherapy, namely the lack of specificity of anticancer drugs, which often leads to severe side effects, decreasing the therapy effectiveness. Delivery of cell-killing substances to tumor cells is one-way targeted drug therapy can work. Generally, monoclonal antibodies are combined with chemotherapeutic drugs, allowing cellular uptake through the binding to their targets on the surface of cancer cells. Aptamer-drug conjugates represent a promising alternative solution to antibodies to minimize off-target effects, considering the remarkable selective binding capabilities of aptamers. In this study, to enhance the therapeutic efficacy of the antineoplastic agent 5-fluoro-2'-deoxyuridine (FdU) in various cancer cells, we focused on the development of a novel conjugate using the antiproliferative aptamer T30923 (INT) as a drug vehicle. Three derivatives composed of T30923 conjugated with a different number of FdU units were synthesized, and their structural and biological properties were thoroughly characterized, highlighting their potential for targeted and synergistic anticancer responses.

Enzyme-free and rapid colorimetric analysis of osteopontin via triple-helix aptamer probe coupled with catalytic hairpin assembly reaction.

BACKGROUND: Osteopontin (OPN) is closely associated with tumorigenesis, growth, invasion, and immune escape and it serves as a plasma biomarker for hepatocellular carcinoma (HCC). Nevertheless, the accurate and rapid detection of low-abundance OPN still poses significant challenges. Currently, the majority of protein detection methods rely heavily on large precision instruments or involve complex procedures. Therefore, developing a simple, enzyme-free, rapid colorimetric analysis method with high sensitivity is imperative. RESULTS: In this study, we have developed a portable colorimetric biosensor by integrating the triple-helix aptamer probe (THAP) and catalytic hairpin assembly (CHA) strategy, named as T-CHA. After binding to the OPN, the trigger probe can be released from THAP, then initiates the CHA reaction and outputs the signal through the formation of a G-quadruplex/Hemin DNAzyme with horseradish peroxidase-like activity. Consequently, this colorimetric sensor achieves visual free-labeled detection without additional fluorophore modification and allows for accurate quantification by measuring the optical density of the solution at 650 nm. Under optimal conditions, the logarithmic values of various OPN concentrations exhibit satisfactory linearity in the range of 5 pg mL-1 to 5 ng mL-1, with a detection limit of 2.04 pg mL-1. Compared with the widely used ELISA strategy, the proposed T-CHA strategy is rapid (∼105 min), highly sensitive, and cost-effective. SIGNIFICANCE: The T-CHA strategy, leveraging the low background leakage of THAP and the high catalytic efficiency of CHA, has been successfully applied to the detection of OPN in plasma, demonstrating significant promise for the early diagnosis of HCC in point-of-care testing. Given the programmability of DNA and the universality of T-CHA, it can be readily modified for analyzing other useful tumor biomarkers.

Balancing act: BRCA2's elaborate management of telomere replication through control of G-quadruplex dynamicity.

In billion years of evolution, eukaryotes preserved the chromosome ends with arrays of guanine repeats surrounded by thymines and adenines, which can form stacks of four-stranded planar structure known as G-quadruplex (G4). The rationale behind the evolutionary conservation of the G4 structure at the telomere remained elusive. Our recent study has shed light on this matter by revealing that telomere G4 undergoes oscillation between at least two distinct folded conformations. Additionally, tumor suppressor BRCA2 exhibits a unique mode of interaction with telomere G4. To elaborate, BRCA2 directly interacts with G-triplex (G3)-derived intermediates that form during the interconversion of the two different G4 states. In doing so, BRCA2 remodels the G4, facilitating the restart of stalled replication forks. In this review, we succinctly summarize the findings regarding the dynamicity of telomeric G4, emphasize its importance in maintaining telomere replication homeostasis, and the physiological consequences of losing G4 dynamicity at the telomere.

Investigating the preferential interaction between imatinib mesylate and VEGF G-quadruplex DNA as therapeutic strategies for cancer treatment: Biophysical and molecular modelling approaches.

G-Quadruplex DNA (GQ-DNA) is one of the most important non-canonical nucleic acid structures. GQ-DNA forming sequences are present in different crucial genomic regions and are abundant in promoter regions of several oncogenes. Therefore, GQ-DNA is an important target for anticancer drugs and hence binding interactions between GQ-DNA and small molecule ligands are of great importance. Since GQ-DNA is a highly polymorphic structure, it is important to identify ligand molecules which preferentially target a particular quadruplex sequence. In this present study, we have used a FDA approved drug called imatinib mesylate (ligand) which is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia, gastrointestinal stromal tumours. Different spectroscopic techniques as well as molecular docking investigations and molecular simulations have been used to explore the interaction between imatinib mesylate with VEGF GQ DNA structures along with duplex DNA, C-Myc, H-Telo GQ DNA. We found that imatinib mesylate shows preferential interaction towards VEGF GQ DNA compared to C-Myc, H-Telo GQ and duplex DNA. Imatinib mesylate seems to be an efficient ligand for VEGF GQ DNA, suggesting that it might be used to regulate the expression of genes in cancerous cells.

Synthesis and Molecular Dynamic Simulation of Novel Cationic and Non-cationic Pyrimidine Derivatives as Potential G-quadruplex-ligands.

BACKGROUND: Drug resistance has been a problem in cancer chemotherapy, which often causes shortterm effectiveness. Further, the literature indicates that telomere G-quadruplex could be a promising anti-cancer target. OBJECTIVE: We synthesized and characterized two new pyrimidine derivatives as ligands for G-quadruplex DNA. METHODS: The interaction of novel non-cationic and cationic pyrimidine derivatives (3a, b) with G-quadruplex DNA (1k8p and 3qsc) was explored by circular dichroism (CD) and ultraviolet-visible spectroscopy and polyacrylamide gel electrophoresis (PAGE) methods. The antiproliferative activity of desired compounds was evaluated by the MTT assay. Apoptosis induction was assessed by Propidium iodide (P.I.) staining and flow cytometry. Computational molecular modeling (CMM) and molecular dynamics simulation (MD) were studied on the complexes of 1k8p and 3qsc with the compounds. The van der Waals, electrostatic, polar solvation, solventaccessible surface area (SASA), and binding energies were calculated and analyzed. RESULTS: The experimental results confirmed that both compounds 3a and 3b interacted with 1k8p and 3qsc and exerted cytotoxic and proapoptotic effects on cancer cells. The number of hydrogen bonds and the RMSD values increased in the presence of the ligands, indicating stronger binding and suggesting increased structural dynamics. The electrostatic contribution to binding energy was higher for the cationic pyrimidine 3b, indicating more negative binding energies. CONCLUSION: Both experimental and MD results confirmed that 3b was more prone to form a complex with DNA G-quadruplex (1k8p and 3qsc), inhibit cell growth, and induce apoptosis, compared to the non-cationic pyrimidine 3a.

Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Small molecule telomerase inhibitors are also potent inhibitors of telomeric C-strand synthesis.

Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α -primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and they cause telomere shortening in human cells that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.