Search Results (50)

Lipid-based nanoparticles are widely explored as non-viral vectors for nucleic acid delivery, where the molecular structure of cationic lipids strongly determines their performance. Five-membered heterocyclic linkers were explored as a new structural motif in cationic amphiphilic lipids for the development of promising gene delivery candidates. Novel lipids incorporating pyrrole, furan, and thiophene linkers were synthesized alongside structurally related aliphatic analogues, enabling systematic evaluation of how linker type influences physicochemical behavior and self-assembly properties. Self-assembly behavior in aqueous media was characterized by dynamic light scattering, and pDNA encapsulation efficiency was measured using the Quant-iT Pico-Green method. The resulting liposomes exhibited hydrodynamic diameters ranging from 92 to 1317 nm, while corresponding lipoplexes ranged from 302 to 1159 nm. Amphiphiles containing heterocyclic linkers demonstrated high pDNA encapsulation (>80% at optimal N/P ratios), whereas aliphatic analogues showed significantly reduced performance. These results demonstrate that linker structure strongly influences both self-assembly and nucleic acid binding properties. By evaluating structure–activity relationships, five-membered heterocycles are proposed as promising structural elements for the rational development of lipid-based gene delivery candidates. Full article

The global rise in antimicrobial resistance (AMR) demands innovative strategies beyond traditional antibiotics. Peptide Nucleic Acids (PNAs), synthetic DNA analogues with peptide-like backbones, act as thermically, chemically, and enzymatically stable sequence-specific agents capable of silencing essential bacterial genes. Through antisense mechanisms, PNAs bind bacterial mRNA or rRNA, blocking translation or ribosome assembly and thereby inducing species-specific growth inhibition. Their programmable design enables precise targeting of multidrug-resistant pathogens while sparing commensal microbiota. Recent advances, including γ-modified backbones, cationic substitutions, and delivery platforms such as cell-penetrating peptides (CPPs), dendron conjugates, and nanoparticles, have improved solubility, stability, and cellular uptake. Studies show promising in vitro and, albeit less frequently, in vivo efficacy against both Gram-positive and Gram-negative bacteria, often with synergistic activity when combined with conventional antibiotics. Although challenges remain in delivery and large-scale production, PNAs represent a promising class of antimicrobials to combat AMR through targeted gene inhibition. Full article

Peptide nucleic acids (PNAs) are synthetic analogues of DNA/RNA characterized by the absence of negative phosphate groups, which confer a low sensitivity to ionic strength for hybridization with respect to the canonical counterpart. PNAs are a suitable probe for miRNAs, as well as endogenous molecules of single-strand non-coding RNA whose dysregulation is often linked to several diseases. The interaction forces between PNA and microRNA-155 (miR-155), a multifunctional microRNA overexpressed in a variety of tumors, were investigated by Atomic Force Spectroscopy (AFS) in fluid under different conditions. We found that the unbinding forces acquired at the ionic strength of 150 mM for a rather wide range of loading rates (ΔF/Δt) can be described using the Bell–Evans model. This allows us to extract information on the kinetics and thermodynamic properties of the miR-155/PNA duplex. Additionally, we probed the unbinding forces and the target recognition times between miR-155 and PNA in the 50–300 mM ionic strength range. Our results indicate that both of these parameters are practically independent from the ionic strength in the analyzed range. The results provide information that is useful for a wider use of PNA in biosensors for diagnostics and therapeutics, even in situ. Full article

Thrombin binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers that efficiently binds to thrombin, resulting in anticoagulant effects. TBA also possesses promising antiproliferative properties. As with most therapeutic oligonucleotides, chemical modifications are critical for therapeutic applications, particularly to improve thermodynamic stability, resistance in biological environment, and target affinity. To evaluate the effects of nucleobase and/or sugar moiety chemical modifications, five TBA analogues have been designed and synthesized considering that the chair-like G4 structure is crucial for biological activity. Their structural and biological properties have been investigated by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), native polyacrylamide gel electrophoresis (PAGE) techniques, and PT and MTT assays. The analogue TBAB contains 8-bromo-2′-deoxyguanosine (B) in G-syn glycosidic positions, while TBAL and TBAM contain locked nucleic acid guanosine (L) or 2′-O-methylguanosine (M) in G-anti positions, respectively. Instead, both the two types of modifications have been introduced in TBABL and TBABM with the aim of obtaining synergistic effects. In fact, both derivatives include B in syn positions, exhibiting in turn L and M in the anti ones. The most appealing results have been obtained for TBABM, which revealed an interesting cytotoxic activity against breast and prostate cancer cell lines, while in the case of TBAB, extraordinary thermal stability (T_m approximately 30 °C higher than that of TBA) and an anticoagulant activity higher than original aptamer were observed, as expected. These data indicate TBAB as the best TBA anticoagulant analogue here investigated and TBABM as a promising antiproliferative derivative. Full article

Recent advances in genetics and nucleic acid chemistry have created fundamentally new tools, both for practical applications in therapy and diagnostics and for fundamental genome editing tasks. Nucleic acid-based therapeutic agents offer a distinct advantage of selectively targeting the underlying cause of the disease. Nevertheless, despite the success achieved thus far, there remain unresolved issues regarding the improvement of the pharmacokinetic properties of therapeutic nucleic acids while preserving their biological activity. In order to address these challenges, there is a growing focus on the study of safe and effective delivery methods utilising modified nucleic acid analogues and their lipid bioconjugates. The present review article provides an overview of the current state of the art in the use of chemically modified nucleic acid derivatives for therapeutic applications, with a particular focus on oligonucleotides conjugated to lipid moieties. A systematic analysis has been conducted to investigate the ability of amphiphilic oligonucleotides to self-assemble into micelle-like structures, as well as the influence of non-covalent interactions of such derivatives with serum albumin on their biodistribution and therapeutic effects. Full article

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N⁴-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK. Full article

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides. Full article

Our study was motivated by the urgent need to develop or improve antivirals for effective therapy targeting RNA viruses. We hypothesized that analogues of favipiravir (FVP), an inhibitor of RNA-dependent RNA polymerase (RdRp), could provide more effective nucleic acid recognition and binding processes while reducing side effects such as cardiotoxicity, hepatotoxicity, teratogenicity, and embryotoxicity. We proposed a set of FVP analogues together with their forms of triphosphate as new SARS-CoV-2 RdRp inhibitors. The main aim of our study was to investigate changes in the mechanism and binding capacity resulting from these modifications. Using three different approaches, QTAIM, QSPR, and MD, the differences in the reactivity, toxicity, binding efficiency, and ability to be incorporated by RdRp were assessed. Two new quantum chemical reactivity descriptors, the relative electro-donating and electro-accepting power, were defined and successfully applied. Moreover, a new quantitative method for comparing binding modes was developed based on mathematical metrics and an atypical radar plot. These methods provide deep insight into the set of desirable properties responsible for inhibiting RdRp, allowing ligands to be conveniently screened. The proposed modification of the FVP structure seems to improve its binding ability and enhance the productive mode of binding. In particular, two of the FVP analogues (the trifluoro- and cyano-) bind very strongly to the RNA template, RNA primer, cofactors, and RdRp, and thus may constitute a very good alternative to FVP. Full article

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2′-deoxyguanosine (G^Br) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G^LNA) in the analogue TBABL, 2’-O-methylguanosine (G^OMe) in TBABM, and 2’-F-riboguanosine (G^F) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (T_m approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer. Full article

Favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide, FPV), an active pharmaceutical component of the drug discovered and registered in March 2014 in Japan under the name Avigan, with an indication for pandemic influenza, has been studied. The study of this compound was prompted by the idea that effective processes of recognition and binding of FPV to the nucleic acid are affected predominantly by the propensity to form intra- and intermolecular interactions. Three nuclear quadrupole resonance experimental techniques, namely ¹H-¹⁴N cross-relaxation, multiple frequency sweeps, and two-frequency irradiation, followed by solid-state computational modelling (density functional theory supplemented by the quantum theory of atoms in molecules, 3D Hirshfeld Surfaces, and reduced density gradient) approaches were applied. The complete NQR spectrum consisting of nine lines indicating the presence of three chemically inequivalent nitrogen sites in the FPV molecule was detected, and the assignment of lines to particular sites was performed. The description of the nearest vicinity of all three nitrogen atoms was used to characterize the nature of the intermolecular interactions from the perspective of the local single atoms and to draw some conclusions on the nature of the interactions required for effective recognition and binding. The propensity to form the electrostatic N−H···O, N−H···N, and C−H···O intermolecular hydrogen bonds competitive with two intramolecular hydrogen bonds, strong O−H···O and very weak N−H···N, closing the 5-member ring and stiffening the structure, as well as π···π and F···F dispersive interactions, were analysed in detail. The hypothesis regarding the similarity of the interaction pattern in the solid and the RNA template was verified. It was discovered that the -NH₂ group in the crystal participates in intermolecular hydrogen bonds N–H···N and N–H···O, in the precatalytic state only in N–H···O, while in the active state in N–H···N and N–H···O hydrogen bonds, which is of importance to link FVP to the RNA template. Our study elucidates the binding modes of FVP (in crystal, precatalytic, and active forms) in detail and should guide the design of more potent analogues targeting SARS-CoV-2. Strong direct binding of FVP-RTP to both the active site and cofactor discovered by us suggests a possible alternative, allosteric mechanism of FVP action, which may explain the scattering of the results of clinical trials or the synergistic effect observed in combined treatment against SARS-CoV-2. Full article

Nucleoside analogues (NAs) are a family of compounds which include a variety of purine and pyrimidine derivatives, widely used as anticancer and antiviral agents. For their ability to compete with physiological nucleosides, NAs act as antimetabolites exerting their activity by interfering with the synthesis of nucleic acids. Much progress in the comprehension of their molecular mechanisms has been made, including providing new strategies for potentiating anticancer/antiviral activity. Among these strategies, new platinum-NAs showing a good potential to improve the therapeutic indices of NAs have been synthesized and studied. This short review aims to describe the properties and future perspectives of platinum-NAs, proposing these complexes as a new class of antimetabolites. Full article

The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N⁴-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications Full article

Willardiine is a nonprotein amino acid containing uracil, and thus classified as nucleobase amino acid or nucleoamino acid, that together with isowillardiine forms the family of uracilylalanines isolated more than six decades ago in higher plants. Willardiine acts as a partial agonist of ionotropic glutamate receptors and more in particular it agonizes the non-N-methyl-D-aspartate (non-NMDA) receptors of L-glutamate: ie. the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate receptors. Several analogues and derivatives of willardiine have been synthesised in the laboratory in the last decades and these compounds show different binding affinities for the non-NMDA receptors. More in detail, the willardiine analogues have been employed not only in the investigation of the structure of AMPA and kainate receptors, but also to evaluate the effects of receptor activation in the various brain regions. Remarkably, there are a number of neurological diseases determined by alterations in glutamate signaling, and thus, ligands for AMPA and kainate receptors deserve attention as potential neurodrugs. In fact, similar to willardiine its analogues often act as agonists of AMPA and kainate receptors. A particular importance should be recognized to willardiine and its thymine-based analogue AlaT also in the peptide chemistry field. In fact, besides the naturally-occurring short nucleopeptides isolated from plant sources, there are different examples in which this class of nucleoamino acids was investigated for nucleopeptide development. The applications are various ranging from the realization of nucleopeptide/DNA chimeras for diagnostic applications, and nucleoamino acid derivatization of proteins for facilitating protein-nucleic acid interaction, to nucleopeptide-nucleopeptide molecular recognition for nanotechnological applications. All the above aspects on both chemistry and biotechnological applications of willardine/willardine-analogues and nucleopeptide will be reviewed in this work. Full article

G-quadruplexes are secondary structures originating from nucleic acid regions rich in guanines, which are well known for their involvement in gene transcription and regulation and DNA damage repair. In recent studies from our group, kynurenic acid (KYNA) derivative 1 was synthesized and found to share the structural features typical of G-quadruplex binders. Herein, structural modifications were conducted on this scaffold in order to assist the binding with a G-quadruplex, by introducing charged hydrophilic groups. The antiproliferative activity of the new analogues was evaluated on an IGROV-1 human ovarian cancer cell line, and the most active compound, compound 9, was analyzed with NMR spectrometry in order to investigate its binding mode with DNA. The results indicated that a weak, non-specific interaction was set with duplex nucleotides; on the other hand, titration in the presence of a G-quadruplex from human telomere d(TTAGGGT)₄ showed a stable, although not strong, interaction at the 3′-end of the nucleotidic sequence, efficiently assisted by salt bridges between the quaternary nitrogen and the external phosphate groups. Overall, this work can be considered a platform for the development of a new class of potential G-quadruplex stabilizing molecules, confirming the crucial role of a planar system and the ability of charged nitrogen-containing groups to facilitate the binding to G-quadruplex grooves and loops. Full article

Considering that practically all reactions that involve nucleotides also involve metal ions, it is evident that the coordination chemistry of nucleotides and their derivatives is an essential corner stone of biological inorganic chemistry. Nucleotides are either directly or indirectly involved in all processes occurring in Nature. It is therefore no surprise that the constituents of nucleotides have been chemically altered—that is, at the nucleobase residue, the sugar moiety, and also at the phosphate group, often with the aim of discovering medically useful compounds. Among such derivatives are acyclic nucleoside phosphonates (ANPs), where the sugar moiety has been replaced by an aliphatic chain (often also containing an ether oxygen atom) and the phosphate group has been replaced by a phosphonate carrying a carbon–phosphorus bond to make the compounds less hydrolysis-sensitive. Several of these ANPs show antiviral activity, and some of them are nowadays used as drugs. The antiviral activity results from the incorporation of the ANPs into the growing nucleic acid chain—i.e., polymerases accept the ANPs as substrates, leading to chain termination because of the missing 3′-hydroxyl group. We have tried in this review to describe the coordination chemistry (mainly) of the adenine nucleotides AMP and ATP and whenever possible to compare it with that of the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA²⁻ = adenine(N9)-CH₂-CH₂-O-CH₂-${\mathrm{PO}}_3^2$) [or its diphosphate (PMEApp⁴⁻)] as a representative of the ANPs. Why is PMEApp⁴⁻ a better substrate for polymerases than ATP⁴⁻? There are three reasons: (i) PMEA²⁻ with its anti-like conformation (like AMP²⁻) fits well into the active site of the enzyme. (ii) The phosphonate group has an enhanced metal ion affinity because of its increased basicity. (iii) The ether oxygen forms a 5-membered chelate with the neighboring phosphonate and favors thus coordination at the P_α group. Research on ANPs containing a purine residue revealed that the kind and position of the substituent at C2 or C6 has a significant influence on the biological activity. For example, the shift of the (C6)NH₂ group in PMEA to the C2 position leads to 9-[2-(phosphonomethoxy)ethyl]-2-aminopurine (PME2AP), an isomer with only a moderate antiviral activity. Removal of (C6)NH₂ favors N7 coordination, e.g., of Cu²⁺, whereas the ether O atom binding of Cu²⁺ in PMEA facilitates N3 coordination via adjacent 5- and 7-membered chelates, giving rise to a Cu(PMEA)_cl/O/N3 isomer. If the metal ions (M²⁺) are M(α,β)-M(γ)-coordinated at a triphosphate chain, transphosphorylation occurs (kinases, etc.), whereas metal ion binding in a M(α)-M(β,γ)-type fashion is relevant for polymerases. It may be noted that with diphosphorylated PMEA, (PMEApp⁴⁻), the M(α)-M(β,γ) binding is favored because of the formation of the 5-membered chelate involving the ether O atom (see above). The self-association tendency of purines leads to the formation of dimeric [M₂(ATP)]₂(OH)⁻ stacks, which occur in low concentration and where one half of the molecule undergoes the dephosphorylation reaction and the other half stabilizes the structure—i.e., acts as the “enzyme” by bridging the two ATPs. In accord herewith, one may enhance the reaction rate by adding AMP²⁻ to the [Cu₂(ATP)]₂(OH)⁻ solution, as this leads to the formation of mixed stacked Cu₃(ATP)(AMP)(OH)⁻ species, in which AMP²⁻ takes over the structuring role, while the other “half” of the molecule undergoes dephosphorylation. It may be added that Cu₃(ATP)(PMEA) or better Cu₃(ATP)(PMEA)(OH)⁻ is even a more reactive species than Cu₃(ATP)(AMP)(OH)⁻. – The matrix-assisted self-association and its significance for cell organelles with high ATP concentrations is summarized and discussed, as is, e.g., the effect of tryptophanate (Trp⁻), which leads to the formation of intramolecular stacks in M(ATP)(Trp)³⁻ complexes (formation degree about 75%). Furthermore, it is well-known that in the active-site cavities of enzymes the dielectric constant, compared with bulk water, is reduced; therefore, we have summarized and discussed the effect of a change in solvent polarity on the stability and structure of binary and ternary complexes: Opposite effects on charged O sites and neutral N sites are observed, and this leads to interesting insights. Full article