INTRODUCTION:G-quadruplexes have emerged as a versatile class of oligonucleotides that combine molecular recognition, intrinsic anticancer activity and drug carrier functions, offering new opportunities to improve tumor selectivity and overcome limitations of conventional chemotherapy. AREAS COVERED:This review summarizes structural and biological features that make G-quadruplexes attractive platforms for anticancer drug delivery, including cancer-selective uptake, high stability and scaffold versatility. We discuss G-quadruplexes with intrinsic antiproliferative activity, covalent G-quadruplex-drug conjugates including small molecules and nucleoside antimetabolites and G-quadruplexes that non-covalently host G-quadruplex ligands. We further examine G-quadruplex-decorated nanoparticles and DNA nanostructures, as well as, higher-order G-quadruplex architectures that enable multivalent receptor targeting. A literature search was conducted using the Web of Science database to identify peer-reviewed publications related to 'G-quadruplex.' The search covered the period from 2000 to the present (as of 12 January 2026). EXPERT OPINION:G-quadruplexes represent a platform with unique capacity to unify targeting and therapy, offering a promising alternative to antibody-based systems. However, issues such as structural polymorphism, pharmacokinetics, and controlled drug release must be addressed through rational design and chemical modification to fully translate these delivery systems into clinically relevant anticancer therapeutics.

Poly(lactic-co-glycolic acid) (PLGA)-based polymeric nanoparticles (PLGA NPs) have proven to be effective as potential drug delivery systems. The presence of carboxylate groups on their surface facilitates the development of multifunctional NPs enhancing therapeutic efficacy through synergetic effects. Our study describes the preparation of PLGA NPs using oil-in-water polymeric nano-emulsions, generated via a phase inversion composition low-energy emulsification method. Rosmarinic acid (RA), a phytochemical with neuroprotective effects, and an antisense oligonucleotide (ASO) were selected respectively as a phytochemical to be entrapped and as a ligand to decorate the surface of PLGA nanoparticles respectively, aiming to enhance ASO delivery to neuronal cells. Physicochemical characterization confirmed that RA and ASO incorporation preserved colloidal stability, with no adverse effect on particle size, surface charge, or morphology. In vitro-controlled release experiments showed a cumulative RA release of ca. 12% over 24 h governed by a semi-Fickian diffusion mechanism after adjusting to different equation models. Importantly, RA entrapment displayed measurable radical scavenging capacity, leading to a EC50 of 76 ± 0.9 μg·mL-1. Cell culture experiments confirmed biocompatibility in both a non-cancer cell line (HEK293) and neuroblastoma cell model (SH-SY5Y). Uptake studies revealed efficient internalization of PLGA NPs by SH-SY5Y cells and primary murine neurons, promoting gene silencing of luciferase expression (53.7 ± 7.9%). Together, these results show a modular PLGA nanoplatform that enables the simultaneous incorporation of an antioxidant payload and a covalently grafted antisense oligonucleotide, allowing independent assessment of redox modulation and gene silencing in neuronal models.

By combining in silico, biophysical, and in vitro experiments, we decipher the topology, physical, and potential biological properties of hybrid-parallel nucleic acids triplexes, an elusive structure at the basis of life. We found that hybrid triplex topology follows a stability order: r(Py)-d(Pu)·r(Py) > r(Py)-d(Pu)·d(Py) > d(Py)-d(Pu)·d(Py) > d(Py)-d(Pu)·r(Py). The r(Py)-d(Pu)·d(Py) triplex is expected to be preferred in the cell as it avoids the need to open the duplex reducing the torsional stress required for triplex formation in the r(Py)-d(Pu)·r(Py) topology. Upon a massive collection of melting data, we have created the first predictor for hybrid triplex stability. Leveraging this predictor, we conducted a comprehensive scan to assess the likelihood of the human genome and transcriptome to engage in triplex formation. Our findings unveil a remarkable inclination—of both the human genome and transcriptome—to generate hybrid triplex formation, particularly within untranslated (UTRs) and regulatory regions, thereby corroborating the existence of a triplex-mediated regulatory mechanism. Furthermore, we found a correlation between nucleosome linkers and Triplex-forming sequence (TFS) which agree with a putative role of triplexes in arranging chromatin structure.

Providing viral load numbers of infection events aids in the identification of disease severity and in the effective overall patient management. Gold-standard polymerase chain reaction (PCR) techniques make this possible but cannot be applied at the point of need and in low-resource settings. Here, we report on the development of a compact analytical platform that can detect a conserved sequence of the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in 40 min in nasopharyngeal swab samples without the need for any previous purification or gene amplification steps. It combines electrochemical and paper fluidic approaches together with a sandwich hybridization assay performed on magnetic nanoparticles (MNPs) modified with a tailor-designed capture DNA hairpin. The device proves to quantitatively detect viral RNA in a retrospective study carried out with nasopharyngeal swab samples. A sensitivity of 100% and a specificity of 93% were estimated by the receiver operating characteristic (ROC) analysis. However, although molar concentration values of the target RNA sequence are provided, these estimates do not fully correlate with the viral load numbers estimated by RT-qPCR over the whole Ct sample range. Empirical studies have been carried out that have provided clear insights into this hurdle and simple solutions to overcome it, without depriving the device of the features required for potential use in a point-of-care (PoC) environment.

The formation of nucleic acid triple helices ("triplexes") is an area of great interest due to their potential role in the natural and artificial regulation of gene expression or for use in analytical, diagnostic, or synthetic methods. During the coronavirus pandemic, a large search for novel methods for the detection of SARS-CoV-2 was undertaken. Based on triplex affinity capture and using polypurine reverse-Hoogsteen hairpins, a method known as Triplex Enhanced Nucleic Acid Detection Assay (TENADA) was developed for the rapid detection of SARS-CoV-2 without the need for polymerase chain reaction (PCR) amplification. In this work, to expand the targeting scope of this method, we explored triplex-forming bis-pyrimidine clamps targeting a polypurine sequence in the ORF1a region of SARS-CoV-2. To enhance parallel triplex stability, 2'-sugar and 5-methylpyrimidine modifications were incorporated into both strands of the clamps, and their effect on the triplexes formed was assessed via NMR and other biophysical methods. The results revealed distinct stabilizing effects of the modifications, influenced by their size, sugar puckering, and capacity to form short contacts with neighboring residues. The dual ability of clamps to simultaneously form Watson-Crick and Hoogsteen hydrogen bonds offers a novel perspective on the effect of modifications on triplex stability, previously unexplored with triplex-forming oligonucleotides (TFOs). Finally, the bis-pyrimidine clamps that formed the most stable parallel triplexes were applied in a thermal lateral flow (TLF) sensing device, demonstrating their potential as biosensing probes. These clamps effectively detected the synthetic DNA target with limits of detection (LoDs) ranging from 0.05 to 0.001 nM. Understanding the best modification strategies and their impact on the triplex structure will advance the development of clamps as biosensing and therapeutic agents.

In this work, strategies for the detection of pyrimidine-rich DNA target sequences based on the formation of duplex and antiparallel triplex structures are studied. The presence of the target is detected from the changes in fluorescence of silver nanoclusters stabilized by the corresponding complementary DNA probes. In all cases, the formation of intermolecular structures has been assessed by means of melting experiments and multivariate analysis. In the case studied, it has been observed that the formation of antiparallel triplex structures produces changes in fluorescence properties that could be more useful for analytical purposes than those observed when only duplex structures are formed. In particular, the use of silver nanoclusters confined within a loop rich in cytosinetype bases in the antiparallel triplex structure resulting from the interaction of probe and analyte has been shown to produce an increase in red fluorescence. This latter probe has been shown to be selective against target sequences that have mismatches that could affect the formation of stable duplex structures, while it has been shown to tolerate a small number of purine mismatches that could affect the stability of the resulting antiparallel triplex structure. As a final remark, it should be noted that this methodology could also be used in the development of analytical procedures that allow the detection of antiparallel triplex structures, which are difficult to observe with other spectroscopic methods.

I-motifs are non-canonical, cytosine-rich DNA structures stabilized by hemiprotonated C•C+ base pairs, whose formation is highly pH-dependent. While certain chemical modifications can enhance i-motif stability, modifications at the sugar moiety often disrupt essential inter-strand contacts. In this study, we examine the structural and thermodynamic impact of incorporating 2′-fluoro-ribocytidine (2′F-riboC) into i-motif-forming sequences derived from d(TCCCCC). Using a combination of UV, 1H NMR, and 19F NMR spectroscopy, we demonstrate that full substitution with 2′F-riboC strongly destabilizes i-motif, whereas partial substitutions (one or two substitutions per strand) support well-folded structures at acidic pH (pH 5). High-resolution NMR structures reveal well-defined i-motif architectures with conserved C•C+ pairing and characteristic interstrand NOEs. Sugar conformational analysis reveals a predominant North pucker for cytosines, which directs the fluorine substituent toward the minor groove of the i-motif. 19F NMR further confirms slow exchange between folded and unfolded species, enabling the simultaneous detection of both under identical experimental conditions and, consequently, highlighting the utility of fluorine at the 2′ sugar position as a spectroscopic probe. These findings provide insights into fluorine-mediated modulation of i-motif stability and further extend the utility of 19F NMR in nucleic acid research.

Chemical modifications of nucleic acids are widely used to tune stability and functionality in therapeutic and nanotechnological applications. Among these, fluorinated cytidine derivatives such as 2'-fluoro-arabinocytidine (2'F-araC) and 2'-fluoro-ribocytidine (2'F-riboC) have been shown to influence i-motif structures differently, with 2'F-araC strongly stabilizing and 2'F-riboC exerting a mildly deleterious effect. In this study, we investigate the impact of gemcitabine (2'-deoxy-2',2'-difluorocytidine, dFdC) on i-motif stability. dFdC exhibits small effects in single or double substituted sequences, but a pronounced stabilization when multiple consecutive residues are incorporated. Thermal and pH-dependent analyses demonstrate that sequences containing fully substituted dFdC maintain i-motif folding at neutral pH and show enhanced thermal stability. Structural insights suggest that this stabilization arises from a combination of factors, such as hyperconjugative interactions, hydrogen bonding, and dipole alignment, while the adaptable sugar conformation mitigates destabilizing minor groove contacts observed in other more rigid modifications, such as 2'-F-riboC. Cooperative interactions among adjacent dFdC residues and potential changes in hydration may play a key factor in reinforcing stability. These results highlight the unique capacity of dFdC to enhance i-motif robustness and suggest that strategically placed difluoro substitutions can be exploited to design i-motifs with improved stability, expanding their potential in biotechnology and therapeutic applications.

Malignant melanoma presents a significant challenge in oncology due to its aggressive nature and high metastatic potential. Conventional systemic treatments often fail to effectively reach tumor sites, limiting their therapeutic impact. This study introduces a groundbreaking triple-strategy approach for treating malignant melanoma. A novel prodrug, an oligonucleotide, comprising 10 units of Floxuridine (5-fluoro-2'-deoxyuridine) (FdU) nucleoside antimetabolites are developed, to enhance half-life and reduce rapid metabolism. Encapsulated in soluble colloidal silica nanoparticles, this compound is protected and directed toward tumor neovasculature precursor endothelial cell receptors, ensuring local delivery. The strategy focuses on releasing the prodrug in the tumor microenvironment, aiming to eradicate both melanoma cells and their supportive structures. Efficacy is demonstrated in cell culture studies and preclinical models of malignant melanoma, showing a remarkable 50% reduction in tumor size after just three intravenous treatments. These findings underscore the transformative potential of targeting endothelial cell membrane proteins for drug delivery. This study paves the way for innovative targeted therapies, promising significant advancements in treatment strategies and improves outcomes for patients with metastatic cancers.

Precision targeting is a hot topic in cancer nanomedicine, as conventional chemotherapies cause systemic toxicities, creating an urgent need for more selective treatments. Although antibody-drug conjugates (ADCs) are the current gold standard in targeted therapy, their clinical performance remains limited. As an alternative, we previously developed a multivalent protein nanocarrier (T22-GFP-H6) displaying the CXCR4-targeting peptide T22, which offers super-selective tumor accumulation driven by CXCR4 overexpression. This innovative nanovehicle showed favorable biodistribution for targeted delivery of antitumor drugs, including monomethyl auristatin E (MMAE), in a first-generation stochastic nanoconjugate format. However, unlike ADCs, where conjugation strategy is known to influence pharmacokinetics and efficacy, these parameters remain largely unexplored in non-antibody multivalent nanocarriers. Here, we evaluated the impact of precise payload accommodation using two site-specific strategies that attach a single MMAE molecule at distinct structural sites, and we compared them with first-generation nanoconjugates. The conjugation strategy substantially affected the biodistribution and antitumor efficacy, with a solvent-exposed cysteine-conjugation distal to the targeting ligand proving most effective. At equimolar nanocarrier dosing, this construct achieved tumor control similar to the stochastic conjugate in a disseminated hematologic malignancy despite an approximately 4-fold lower MMAE load (drug-to-protein ratio, DPR = 1 vs DPR ≈ 4). Moreover, at equimolar MMAE dosing, it clearly outperformed both the stochastic conjugate and the alternative site-directed design. These findings align with trends in advanced ADCs and provide practical design rules for rational, site-specific conjugation in next-generation protein-based nanomedicines aimed at enabling dose-sparing in oncology.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

2′-deoxy-5-fluorouridine (Floxuridine, FdU) oligomers are repeated FdU residues linked to each other through phosphate groups exhibiting anticancer properties. One of the major hurdles exhibited by natural oligomers is their reduced stability and limited ability to impart cellular uptake. To overcome these drawbacks, electrostatic and covalent strategies have been used to combine oligomers with nanoparticles (NPs) to enhance stability and cellular internalization. Polymeric NPs, prepared from oil-in-water (O/W) PLGA nano-emulsions (NEs) using low-energy emulsification methods, have shown potential in many biomedical applications owing to their ability to transport drugs across cellular membranes. Carbodiimide-mediated amine coupling reaction and thiol-based Michael-type addition were used to attach FdU oligomers formed by five FdU residues (FdU5) to PLGA NPs. Colloidally stable dispersions of FdU5-decorated PLGA NPs were obtained and characterized in terms of size, surface charge, and morphology in physiological medium. Additionally, synthetic strategies were envisaged to functionalize the surface of FdU5-decorated polymeric NPs with folic acid (FA). Cell culture experiments showed that FdU5-decorated PLGA NPs were able to internalize efficiently inducing cytotoxic effect and inhibiting cell proliferation in tumor cells. These outcomes suggest the feasibility of grafting oligomeric FdU produgs in a covalent fashion into nanoscale platforms as a potential approach for cancer therapy.

A critical step in the manufacturing of oligonucleotides is the selection of ideal solid supports. Herein, we evaluate a hybrid core–shell solid support made of polystyrene and polyethyleneglycol, for the preparation of oligonucleotides using phosphoramidite chemistry. The results were compared with those obtained using conventional solid supports including controlled-pore glass and polystyrene NittoPhase®. The core–shell supports provided good yields when phosphoramidites were dissolved in dichloromethane. Automatic DNA synthesizer was used to prepare several oligonucleotides carrying natural phosphate, phosphorothioate linkages, lipid conjugates and mesyl phosphoramidate dimers in acceptable yields. This study represents the first reported use of core–shell particles for the synthesis of chemically modified oligonucleotide sequences of therapeutic significance.

Graphene solution-gated field-effect transistors (gSGFETs) offer high potential for chemical and biochemical sensing applications. Among the current trends to improve this technology, the functionalization processes are gaining relevance for its crucial impact on biosensing performance. Previous efforts are focused on simplifying the attachment procedure from standard multi-step to single-step strategies, but they still suffer from overreaction, and impurity issues and are limited to a particular ligand. Herein, a novel strategy for single-step immobilization of chemically modified aptamers with fluorenylmethyl and acridine moieties, based on a straightforward synthetic route to overcome the aforementioned limitations is presented. This approach is benchmarked versus a standard multi-step strategy using thrombin as detection model. In order to assess the reliability of the functionalization strategies 48-gSGFETs arrays are employed to acquire large datasets with multiple replicas. Graphene surface characterization demonstrates robust and higher efficiency in the chemical coupling of the aptamers with the single-step strategy, while the electrical response evaluation validates the sensing capability, allowing to implement different alternatives for data analysis and reduce the sensing variability. In this work, a new tool capable of overcome the functionalization challenges of graphene surfaces is provided, paving the way toward the standardization of gSGFETs for biosensing purposes. A novel strategy for direct biofunctionalization on graphene surfaces for biosensing applications based on single-step immobilization is presented. This efficient and faster approach is based on derivatized aptamers with fluorenylmethyl and acridine moieties and validated using arrays of 48 graphene SGFETs allowing multiple replicas. The presented straightforward functionalization is benchmarked versus standard multi-step strategy, offering a simpler alternative.image

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 oneway 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.

Quadruplex-Duplex (Q-D) junctions are unique structural motifs garnering increasing interest as drug targets, due to their frequent occurrence in genomic sequences. The viral HIV LTR-III sequence was chosen as a Q-D junction model to study the affinity of the selected compounds BMH-21, namitecan (ST-1968), and doxorubicin (DOXO), all containing a planar polycyclic aromatic moiety, linked to either one short aminoalkyl or an aminoglycosyl group. A multidisciplinary approach that combines NMR spectroscopy, molecular modelling, circular dichroism (CD) and fluorescence spectroscopy was employed. The studied ligands induced moderate but clear stabilization to the Q-D junction by interacting with the interfacial tetrad. DOXO was found to be the best Q-D junction binder. Interestingly, the removal of the aminoglycosyl group significantly changed the pattern of the interactions, indicating that highly polar substituents have a stronger affinity with the exposed regions of the Q-D junction, particularly at the level of the interfacial tetrad.

Although the COVID-19 pandemic was declared no longer a global emergency by the World Health Organization in May 2023, SARS-CoV-2 is still infecting people across the world. Many therapeutic oligonucleotides such as ASOs, siRNAs, or CRISPR-based systems emerged as promising antiviral strategies for the treatment of SARS-CoV-2. In this work, we explored the inhibitory potential on SARS-CoV-2 replication of Polypurine Reverse Hoogsteen Hairpins (PPRHs), CC1-PPRH, and CC3-PPRH, targeting specific polypyrimidine sequences within the replicase and Spike regions, respectively, and previously validated for COVID-19 diagnosis. Both PPRHs are bound to their target sequences in the viral genome with high affinity in the order of nM. In vitro, both PPRHs reduced viral replication by more than 92% when transfected into VERO-E6 cells 24 h prior to infection with SARS-CoV-2. In vivo intranasal administration of CC1-PPRH in K18-hACE2 mice expressing the human ACE receptor protected all the animals from SARS-CoV-2 infection. The properties of PPRHs position them as promising candidates for the development of novel therapeutics against SARS-CoV-2 and other viral infections.

EDITORIAL article Front. Chem., 03 January 2024Sec. Chemical Biology Volume 11 - 2023 | https://doi.org/10.3389/fchem.2023.1355870