Arnau Domínguez

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.

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.

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.

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. We developed a novel prodrug, an oligonucleotide, comprising 10 units of Floxuridine (5-fluoro-2’-deoxyuridine) (FdU) nucleoside antimetabolites, to enhance half-life and reduce rapid metabolism. Encapsulated in soluble colloidal silica nanoparticles, this compound is protected and directed towards 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 was 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. Our study paves the way for innovative targeted therapies, promising significant advancements in treatment strategies and improved outcomes for patients with metastatic cancers. Key Points: Triple-strategy for treating melanoma: FdU ₁₀ prodrug, silica nanoparticle and targeted delivery. Oligonucleotide prodrug (Floxuridine units) enhances half-life and reduces metabolism. Soluble silica nanoparticles protect therapeutic FdU ₁₀ from nucleases and decorated with protein ligands are directed to tumor neovasculature endothelial cells. Significant 50% tumor reduction in preclinical melanoma models after systemic administration with targeted therapies.

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.