In this study, the effect of the chemical nature of confinement on the folding and thermal stability of the telomere G-quadruplex (G4) has been investigated by studying the folding pattern of different telomeric derived DNA sequences with varying numbers and arrangements of thymine loop nucleobases in the presence of anionic and cationic nanosized water pools. The findings suggest that both anionic and cationic water pools fold the telomere sequences into a G4 of the same topology. However, the thermal stability of the folded G4 in the cationic water pool is significantly lower than that in the anionic case. The overall data indicate that the topology of the folded G4 is insensitive to the nature of the confinement; however, the thermal stability of the folded telomeric G4 depends significantly on the chemical nature of the confinement. It is plausible that the interfacial water inside the cationic water pools has a different orientation and hydrogen bonding than in the case of anionic water pools, which may cause the different thermal stabilities of the G4 on these two water pools. These findings may be important in understanding the folding and stability of telomere G4 inside a confined cellular system.

The unfolding of telomeric G-quadruplexes (G4s) is a key step in telomere elongation and regulation. Within cells, the highly crowded intracellular milieu significantly influences the structural stability and dynamics of G4s; however, the molecular mechanism governing their unfolding under such conditions remains poorly understood. In this study, we have investigated the thermal unfolding of various human telomeric G4 sequences in KCl, both in the absence and presence of molecular crowders, using temperature-dependent circular dichroism (CD) spectroscopy combined with singular value decomposition, multivariate curve resolution alternating least-squares (MCR-ALS), and well-tempered metadynamics simulations. In KCl alone, telomeric G4s exhibit a two-state unfolding mechanism, where the hybrid-type topology directly converts into the unfolded random-coil state. In contrast, under crowded conditions, particularly in the presence of hydrophobic crowders, the unfolding follows a three-state pathway involving a distinct intermediate. The hybrid structure initially transitions to a parallel-type topology at elevated temperatures before fully unfolding. This stabilization of the parallel topology arises from preferential interactions between hydrophobic crowders and the exposed loop nucleobases of the parallel G4 form. On the other hand, hydrophilic crowders exert minimal influence on the unfolding pathway, which remains similar to that observed in KCl solution. Overall, these findings provide molecular-level insights into the unfolding process of telomeric G4 DNA in crowded cell-like environments and may be useful in understanding the complex telomere elongation process.

Abstract Hydroxychloroquine (HCQ), a clinically relevant quinoline derivative, impairs both collective and individual cell migration by disrupting actin and vimentin cytoskeletal dynamics during wound healing. In scratch assays with HeLa cells, HCQ treatment significantly reduces wound closure rates and inhibits bursts of coordinated migration, as well as single-cell motility. Quantitative imaging reveals that HCQ diminishes actin filament density at the wound edge and induces reorganization of the vimentin network, resulting in smaller nuclei and compromised structural connectivity. Particle tracking micro-rheology demonstrates that HCQ softens the cytoplasm and decreases cellular mechanical heterogeneity. In vitro spectroscopic studies show that HCQ binds cooperatively to actin with micromolar affinity, perturbing its secondary structure, reducing filament polymerisation rates, and impairs interactions between actin and actin binding proteins (ABPs). HCQ also significantly suppresses lamellipodial protrusive activity, indicating a link between cytoskeletal remodelling and impaired cell migration. Collectively, these findings establish that HCQ disrupts essential mechanisms for directed migration by modulating the cytoskeleton and cell mechanics. This multifaceted impairment may underlie therapeutic potential of HCQ as an anticancer agent by restricting cellular invasive capacity and remodelling required for tumour progression.

Poly(ADP-ribose) polymerases (PARPs) have emerged as pivotal therapeutic targets due to their essential function in DNA single-strand break repair, particularly in cancers with BRCA1/2 mutations. Although PARP inhibitors show promising clinical responses, their long-term efficacy is often compromised by the development of drug resistance, which limits sustained therapeutic success. To address this, identifying molecules capable of modulating additional cancer-relevant pathways has become an emerging strategy. G-quadruplexes (G4s), enriched in oncogenic promoter regions, have gained considerable attention as therapeutic targets. In this study, we investigated the ability of two PARP inhibitors, PARP inhibitor VIII (PI VIII) and PARP inhibitor XI (PI XI), to bind and stabilize G4 structures using biophysical assays and molecular docking. Our results reveal that PI VIII exhibits a markedly stronger affinity for G4 structures compared to PI XI, with a preference for the G4 motif present in the c-myc oncogene, which is frequently overexpressed in cancer cells. Moreover, PI VIII demonstrates enhanced binding and stabilization of the c-myc G4 relative to duplex DNA. The observed stabilizing effect involves electrostatic interactions, hydrogen bonding, and π-π stacking. These findings identify PI VIII as a promising dual-mode anticancer candidate capable of simultaneously inhibiting PARP activity and stabilizing oncogenic G-quadruplexes.

The structural interconversion between the G-quadruplex (G4) and duplex DNA is central to many biological processes, yet the factors governing this transition remain incompletely understood. Here, we systematically examine the roles of guanine (G)-tract length and loop architecture in modulating the kinetics of the G4-to-duplex transition using circular dichroism (CD), NMR spectroscopy, gel electrophoresis and time-dependent FRET measurements. Our results show that increasing the number of guanines in the G-tract significantly enhances the G4 stability and reduces the propensity for duplex formation. Sequences with shorter G-tracts undergo rapid conversion to duplex upon the addition of complementary strands, whereas those with longer G-tracts exhibit slower kinetics or remain predominantly in the G4 state. Binding studies reveal that the affinity of the complementary strand decreases markedly with an increasing G-tract length, establishing an inverse correlation between G4 stability and duplex formation. Kinetic analyses further indicate the presence of multiple G4 conformational populations that interconvert at distinct rates, reflecting the structural heterogeneity in the G4 ensemble. In contrast, variations in loop length exert a relatively modest and nonmonotonic effect on binding affinity but significantly influence the kinetics of the transition. Increasing the loop length reduces structural inhomogeneity and enhances the contribution of the fast-converting population. Overall, these findings demonstrate that the G-tract length is the dominant factor controlling G4 stability and duplex conversion, whereas the loop length and composition fine-tune the kinetic pathways by modulating the underlying conformational landscape. This study provides molecular-level insight into the role of the G-tract and loop nucleobases of the G4 in the structural transition from the G4 to the duplex.

Dodine, a widely used fungicide, exhibits amphiphilic behavior that drives its accumulation at aqueous interfaces, where its structural organization can influence interactions with biomolecules and the overall surface activity. In agricultural systems, salts are commonly present and are expected to modulate the interfacial behavior of dodine. Here, we investigate the effect of varying salt concentrations on the interfacial enrichment and structural arrangement of different doses of dodine at the air/water interface by using vibrational sum frequency generation (VSFG) spectroscopy and surface pressure measurements. Dodine adopts disordered, ordered, and well-packed states at the aqueous interface depending on its concentration. The addition of monovalent salt to the subphase enhances the accumulation of dodine at the interface and modulates its packing, likely due to the greater stabilization of ion pairs between dodine cations and salt anions at the interface compared with the bulk solution. Divalent salts induce similar effects at lower concentrations than monovalent salts, highlighting the importance of ionic charge in this process. The VSFG and surface pressure data indicate that salt anions, depending on their charge and the initial ordering of dodine, can drive dodine molecules to the interface, transforming the air/dodine/water interface from a disordered state to an ordered and ultimately well-packed arrangement. The salt-anion-induced recovery of dodine packing at the aqueous interface may provide valuable insights into the interaction of different biomolecules with this fungicide at the aqueous interface.

The folding of the guanine repetitive region in the telomere unit into G-quadruplex (G4) by drugs has been suggested as an alternative approach for cancer therapy. Hydroxychloroquine (HCQ) and chloroquine (CQ) are two important drugs in the trial stage for cancer. Both drugs can induce the folding of telomere-guanine-rich sequences into G4 even in the absence of salt. However, the guanine repetitive telomeric sequences are always flanked by other nucleobases at both the terminal (5' or 3') that can affect the drug-induced folding pathways and stability of the G4 significantly. Hence, in this study, the HCQ and CQ drug-induced folding of the guanine repetitive telomeric sequences into G4 and its stability by varying the chemical nature, number, and positions of the flanking nucleobases has been explored using several biophysical techniques and docking studies. It has been found that the drug-induced folding of telomere with single flanking nucleobases is similar to that without flanking nucleobases irrespective of the chemical nature and position of the flanking nucleobase. However, the propensity of the folding and the stability of the telomeric G4 induced by drugs decrease significantly with the increase of the flanking nucleobases more than one of any chemical nature and position. The data suggest that the number of flanking nucleobases rather than their chemical nature and location is a critical factor in the folding of the telomere into G4 induced by both drugs. Further, it has been observed that both drugs mainly interact with the G-tract and thymine of the loop region rather than the flanking nucleobases of the telomeric sequences without or with one flanking nucleobase. In contrast, the flanking nucleobases also participate in the interaction with the HCQ and CQ along with the core guanine repeat telomeric unit in the case of the telomeric sequences with more than one flanking nucleobases. The participation of the flanking nucleobases in the interaction with the HCQ and CQ affects the hydrogen bonding of the positively charged side chain of drugs with G quartet and loop nucleobases of telomere along with the with π···π and C-H···π weak interactions between the quinoline part of the drugs with the core telomeric guanine repeat unit which affects the folding pattern of the telomere sequences with more than one flanking nucleobases into G4.

G-quadruplexes (G4) have been proposed as an alternative target for cancer therapy, as the folding of DNA sequences into stabilized G4 in the cancer microenvironment affects key biological functions. The antimalarial drugs, hydroxychloroquine (HCQ) and chloroquine (CQ), are in the clinical trial stage for cancer therapy and have been found to fold DNA sequences into the stabilized G4 even in the absence of KCl salt. In this study, the role of loop nucleobases in terms of chemical nature, number, and location in the HCQ-/CQ-induced folding of DNA sequences into G4 in the absence of KCl has been investigated systematically. The data indicate that both drugs selectively induce the folding of DNA sequences into G-quadruplexes (G4) that contain thymine loop nucleobases. The folding tendency of DNA sequences into stabilized G4 decreases with the increase in the thymine loop nucleobases. Moreover, DNA sequences with fewer thymine loop nucleobases tend to fold into stable G4 when the thymine residues are present at the terminal positions, whereas sequences with more thymine loop nucleobases show higher G4 folding propensity when these bases are located at the central loop. These findings are important in understanding the anticancer effect of antimalarial drugs.

Multielectron, multiproton CO2 reduction selectively to C-1 products is an important area of research. In nature, metalloenzymes use second-sphere interactions like hydrogen bonding and electrostatic interactions to control the rate and selectivity to these multielectron and multiproton reactions, e.g., NO2-, SO2, H+, etc. Recent developments have suggested that hydrogen bonding as well as electrostatic interactions in molecular catalysts, like iron porphyrins, results in selective 2e(-)/2H(+) reduction of CO2, as well, to CO or HCOOH and suppresses competitive reduction of protons to H-2, which occurs at similar reduction potentials. However, until date, there is no direct and systematic investigation of these two different second-sphere effects on multielectron, multiproton transformation like CO2 reduction. A series of iron porphyrins is synthesized where second-sphere hydrogen bonding and electrostatic interactions are installed in the ortho position of a mesophenyl group in an iron tetraphenyl framework. The results show that both hydrogen bonding and electrostatic interactions can facilitate the selective reduction of CO2 to CO by iron porphyrins using H2O as a proton source. In iron porphyrins with hydrogen bonding interactions, the selectivity for CO increases with an increase in H2O concentrations. However, in the iron porphyrins with electrostatic interactions, the selectivity for CO decreases, and the iron porphyrin becomes more selective for H-2 evolution instead at higher H2O concentrations. Excited-state lifetime measurements and molecular dynamics simulations of porphyrins suggest that the solvation of the cationic groups in the periphery of the porphyrin by H2O leads to an increased concentration of water near the metal center, which promotes H-2 evolution over CO2 reduction.

In this study, the effect of the chemical nature of the confinement on the folding and thermal stability of the telomere G-quadruplex (G4) has been investigated by studying the folding pattern of different telomere DNA sequences with varying numbers and arrangements of thymine loop nucleobases in the presence of anionic and cationic nanosized water pools. The findings suggest that both anionic and cationic water pools fold the telomere sequences into G4 of the same topology. However, the thermal stability of the folded G4 in the cationic water pool is significantly lower than that of the anionic case. The overall data indicate that the topology of the folded G4 is insensitive to the nature of the confinement, however, the thermal stability of the folded telomeric G4 depends significantly on the chemical nature of the confinement. It is plausible that the interfacial water inside the cationic water pools has a different orientation and hydrogen bonding than the case of anionic water pools, which may cause the different thermal stability of the G4 on these two water pools. These findings may be important in understanding the folding and stability of telomere G4 inside the confined cellular system. ### Competing Interest Statement The authors have declared no competing interest.

This work presents the rational design and successful synthesis of three ionic tetraphenylbuta-1,3-diene (TPB) derivatives (TPB-Py-butyl, TPB-Py-SO3, and TPB-Py-NMe3) with short alkyl chains, containing different terminal groups. While the aromatic core remains unchanged, the overall charge of the moiety is modulated by altering the terminal ionic group attached to the alkyl chains. These derivatives exhibit excellent water solubility, allowing us to investigate the detailed photophysical studies in aqueous environments. Temperature-dependent photoluminescence (PL) studies indicate that these moieties display highly reversible emission in water with good linearity and fatigue resistance. Additionally, their ionic natures are leveraged to evaluate their interactions with and stabilization of DNA G-quadruplex structures, including c-myc, c-kit, bcl2, KRAS, and VGEF. Among the three ionic TPBs, the tetra-cationic TPB-Py-NMe3 derivative has revealed the highest stabilization efficiency, particularly towards c-myc. Cytotoxicity assessments using the MTT assay have confirmed that these TPBs exhibit low cytotoxicity while displaying anticancer activity. The combination of their reversible optical properties, DNA binding capabilities, and biocompatibility highlights their potential applications in biosensing, bioimaging, and cancer therapeutics.

G-quadruplexes (G4s) in the telomere region are important targets for cancer therapy. Molecules that can fold and stabilize the telomere DNA sequences, even in the absence of salt, can be an exciting prospect for therapy purposes. Anti-inflammatory drugs hydroxychloroquine (HCQ) and chloroquine (CQ) have shown promising effects in cancer therapy and also in the different levels of trial stages. In this study, we have investigated the structure and stability of several natural and mutated telomeric sequences with anti-inflammatory drugs and their analogues in the absence of salts using the biophysical and docking methods to understand the role of the quartet and loop nucleobases of DNA along with the functional group of drugs responsible for triggering the folding of telomeric DNA sequences into G4. The findings indicate that the hydrogen bonding between the charged side chain with the guanine repeating unit associated with the quartet and the thymine in the terminal loops of telomere DNA is the main driving force for the folding of telomere DNA sequences into G4 induced by anti-inflammatory drugs. The data indicate that the adenine nucleobase in the loop of the telomere does not play any role in its folding process induced by HCQ and CQ.

Immunomodulatory drugs, particularly hydroxychloroquine (HCQ) and chloroquine are in the preclinical investigation for cancer therapy, along with their extensive application in autoimmune and parasitic diseases. A hallmark of cancer cells is the elevated expression of oncogenes that drive tumor progression, often regulated by G-quadruplex (G4) DNA structures located within their upstream promoter regions. This study elucidates that HCQ stabilizes the cellular G4 landscape most efficiently compared to other quinoline-based immunomodulatory drugs within oncogenic DNA, particularly the c-myc oncogene, a pivotal regulator of cancer progression. The drug-induced stabilization of c-myc G4 correlates with significant suppression of its transcriptional activity, culminating in a reduction of invasion and migration of triple-negative breast cancer cells. Mechanistically, the strong electrostatic interaction between the G4 phosphate backbone and the drug's charged side chain, anchors its quinoline group to enhance stacking with loop and quartet regions, stabilizing the G4. The in vivo investigation unveils the HCQ's capacity to potentiate the efficacy of conventional chemotherapeutic agents, representing it as a plausible candidate for adjunctive therapy. This study depicts an unconventional anticancer mechanism of immunomodulator drugs, wherein it exerts preferential transcriptional repression of the c-myc oncogene through G4-dependent stabilization, unveiling a novel strategy in oncological intervention.

Dodine is an important surfactant-based chemical fungicide used widely to kill fungi associated with black spot and foliar diseases on several fruit plants, such as apples, pears, peaches, and strawberries. However, the extensive use of dodine depicts the genotoxic effect, which may cause gene-associated diseases. Dodine can destabilize G-quadruplex (G4) DNA, which is one of the key targets for cancer therapy. Hence, finding an eco-friendly medium that can reduce or reverse the destabilization effect of dodine on G4 is important. This study investigates the efficacy of ionic liquids (ILs) containing a 1,1,3,3-tetramethyl guanidinium (TMG) cation with various anions (chloride, acetate, trifluoroacetate, octanoate, and perfluorooctanoate) in restoring the structure and stability of G4 induced by dodine. Our findings demonstrate that all ILs effectively reverse dodine-induced destabilization of G4, with the required concentration varying based on the lipophilicity of IL's anions. Specifically, higher concentrations of TMG-chloride and TMG-acetate are needed compared to TMG-perfluorooctanoate for the same effect. The IL anions remove dodine from G4 binding sites, while the TMG cation's interaction with G4 mitigates the destabilizing effect of dodine. This study indicates that ILs can be an eco-friendly medium for the storage of dodine to reverse the effect of dodine on G4.

Hydroxychloroquine (HCQ), and chloroquine (CQ) are in the preclinical trial stage for cancer along with their active application in autoimmune diseases and malaria. One of the critical hallmarks of cancer cells is the elevated expression of various oncogenes which promote cancer progression and contribute to poor prognosis. The upstream of the promoter region of these oncogenes often exhibits a G-quadruplex (G4) DNA structure which regulates the gene expression. Hence, targeting G4 structure has emerged as a promising therapeutic strategy for cancer. In this study, the recognition of HCQ and CQ with the G4 structure of different oncogenes and its effect on gene regulation has been explored by a combination of various biophysical and in-vitro and in-vivo biological methods. This study depicts that HCQ and CQ downregulate the c-myc oncogene transcription significantly in a G4-dependent manner compared to other oncogenes. The different biophysical techniques and molecular dynamics simulation studies illustrate that these drug molecules stack predominately at the terminal of the c-myc G4 and the binding of these molecules stabilizes c-myc G4 significantly higher than the G4 structure of other oncogenes. The in-vitro cell data exhibit a notable reduction in both c-myc mRNA and protein levels in a triple-negative breast cancer cell line following HCQ treatment. The pre-clinical breast cancer mouse model in-vivo data also indicate that HCQ reduces tumor growth through the downregulation of the c-myc oncogene. Simultaneously, HCQ also enhances the therapeutic efficacy of standard chemotherapeutic agents to be a potential candidate for combination therapy. This work demonstrates the alternative strategy of anticancer action of widely used drugs by specifically downregulating the c-myc oncogene in a G4-dependent manner.### Competing Interest StatementThe authors have declared no competing interest.

The stability of the human telomere G-quadruplex (G4) is directly linked to cancer disease. The human telomere is mostly associated with the flanking nucleobases, which can affect the stability of G4. Hence, in this study, the effect of the flanking nucleobases in the context of their chemical nature, number, and position on the structure and stability of G4 has been investigated in varying concentrations of KCl mimicking the normal and cancer KCl microenvironments. The addition of flanking nucleobases does not alter the G4 topology. However, the presence of merely a single flanking nucleobase destabilizes the telomeric G4. This destabilizing effect is more prominent for thymine than adenine flanking nucleobase, probably due to the formation of the intermolecular G4 topology by thymine. Interestingly, the change in the stability of the telomeric G4 in the presence of thymine flanking nucleobase is sensitive to the concentration of KCl relevant to the normal and cancerous microenvironments, in contrast to adenine. Flanking nucleobases have a greater impact at the 5' end compared to the 3' end, particularly noticeable in KCl concentrations resembling the normal microenvironment rather than the cancerous one. These findings indicate that the effect of the flanking nucleobases on telomeric G4 is different in the KCl salt relevant to normal and cancerous microenvironments. This study may be helpful in attaining molecular-level insight into the role of G4 in telomeric length regulation under normal and cancerous KCl salt conditions.

In this study, the folding of G-quadruplex (G4) from the telomeric DNA sequences having loop nucleobases of different chemical natures, numbers, and arrangements in 10 mM and 100 mM KCl salt conditions mimicking the cancerous and normal KCl salt microenvironments have been investigated. The data suggest that the structure and stability of the G4 are highly dependent on the KCl salt concentration. In general, the conformational flexibility of the folded G4 is higher in KCl salt relevant to cancer than in the normal case for any loop arrangements with the same number of nucleobases. The stability of the G4 decreases with the increase in the number of loop nucleobases for both salt conditions. However, the decrease in the stability of G4 having adenine in the loop region is significantly higher than the case of thymine, particularly more prominent in the KCl salt relevant to the cancer. The topology of the folded G4 and its stability also depend delicately on the permutation of the nucleobases in the loop and the salt concentrations for a particular sequence. The findings indicate that the structure and stability of G4 are noticeably different in KCl salt relevant to physiological and cancer conditions.

Regulating the equilibrium between the duplex form of DNA and G-quadruplex (Gq) and stabilizing the folded Gq are the critical factors for any drug to be effective in cancer therapy due to the direct involvement of Gq in controlling the transcription process. Antimalarial drugs are in the trial stage for different types of cancer diseases; however, the plausible mechanism of action of these drug molecules is not well known. Hence, we investigate the plausible role of antimalarial drugs in the folding and stabilization of Gq-forming DNA sequences from the telomere and promoter gene regions by varying the salt (KCl) concentrations, mimicking the in vitro cancerous and normal cell microenvironments. The study reveals that antimalarial drugs fold and stabilize specifically to telomere Gq-forming sequences in the cancerous microenvironment than the DNA sequences located in the promoter region of the gene. Antimalarial drugs are not only able to fold Gq but also efficiently protect them from unfolding by their complementary strands, hence significantly biasing the equilibrium toward the Gq formation from the duplex. In contrast, in a normal cell microenvironment, K+ controls the folding of telomeres, and the role of antimalarial drugs is not prominent. This study suggests that the action of antimalarial drugs is sensitive to the cancer microenvironment as well as selective to the Gq-forming region.

Understanding the structural change of lysosomal membranes induced by hydroxychloroquine (HCQ) drug is essential as it has been considered as one of the probable mechanisms of its antimalarial action. In this context, vibrational sum frequency generation (VSFG) spectra of the O-H region of water and C-H of the hydrocarbon chain of negatively charged and zwitterionic phospholipids associated with the lysosomal membrane in the absence and presence of different concentrations of HCQ have been measured at the air/water interface. The interfacial water at the negatively charged and zwitterionic lipids gets restructured in the presence of HCQ; however, the mechanism of restructuring is different due to the charge of the head groups of lipids. Interestingly, the presence of HCQ leads to a disorder in the negatively charged lipids, irrespective of their chemical nature, mainly by creating the gauche defect in the hydrocarbon chain of the lipid. In contrast, the ordering of the zwitterionic lipid does not show any appreciable change with the addition of HCQ. The finding on the selectivity of HCQ in affecting the ordering of the lipid depending on its head group charge and restructuring of interfacial water may be useful in understanding the molecular level mechanism of the antimalarial action of HCQ.