Search

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide (cex-NDI) was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes. Aiming at evaluating how the fluorescence behaviour of this compound is associated with specific binding modes to the different DNA targets, cex-NDI was here studied in its interaction with hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex models by biophysical techniques, molecular docking, and biological assays. cex-NDI showed different binding modes associated with different amounts of stacking interactions with the three DNA targets. The preferential binding sites were the groove, outer quartet, or intercalative site of the hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex, respectively. Interestingly, our data show that the fluorescence intensity of DNA-bound cex-NDI correlates with the amount of stacking interactions formed by the ligand with each DNA target, thus providing the rationale behind the conformation-sensitive properties of cex-NDI and supporting its use as a fluorescent probe of G-quadruplex structures. Notably, biological assays proved that cex-NDI mainly localizes in the G-quadruplex-rich nuclei of cancer cells.

In the search for new therapeutic strategies to contrast SARS-CoV-2, we here studied the interaction of polydatin (PD) and resveratrol (RESV)—two natural stilbene polyphenols with manifold, well known biological activities—with Spike, the viral protein essential for virus entry into host cells, and ACE2, the angiotensin-converting enzyme present on the surface of multiple cell types (including respiratory epithelial cells) which is the main host receptor for Spike binding. Molecular Docking simulations evidenced that both compounds can bind Spike, ACE2 and the ACE2:Spike complex with good affinity, although the interaction of PD appears stronger than that of RESV on all the investigated targets. Preliminary biochemical assays revealed a significant inhibitory activity of the ACE2:Spike recognition with a dose-response effect only in the case of PD.

Exploiting a variant of SELEX called “Ligand-Guided Selection” (LI-GS), we recently identified two novel truncated G-rich aptamers, called R1.2 and R1.3, specific for membrane-bound IgM (mIgM), the hallmark of B cells. Herein, the conformational behaviour of these aptamers has been analysed by multiple biophysical methods. In order to investigate their functional secondary structures, these studies have been carried out in pseudo-physiological buffers mimicking different cellular environments. Both aptamers proved to be highly polymorphic, folding into stable, unimolecular G-quadruplex structures in K+-rich buffers. In turn, in buffered solutions containing Na+/Mg2+ ions, R1.2 and R1.3 formed mainly duplex structures. Remarkably, these aptamers were able to effectively bind mIgM on B-cell lymphoma exclusively in the presence of potassium ions. These findings demonstrate the key role of G-quadruplex folding in the molecular recognition and efficient binding of R1.2 and R1.3 to mIgM expressed in lymphoma and leukemia cells, providing a precious rational basis for the design of effective aptamer-based biosensors potentially useful for the detection of cancer-relevant biomarkers.

A focused library of analogs of a lead-like G-quadruplex (G4) targeting compound (4), sharing a furobenzoxazine naphthoquinone core and differing for the pendant groups on the N-atom of the oxazine ring, has been here analyzed with the aim of developing more potent and selective ligands. These molecules have been tested vs. topologically different G4s by the G4-CPG assay, an affinity chromatography-based method for screening putative G4 ligands. The obtained results showed that all these compounds were able to bind several G4 structures, both telomeric and extra-telomeric, thus behaving as multi-target ligands, and two of them fully discriminated G4 vs. duplex DNA. Biological assays proved that almost all the compounds produced effective DNA damage, showing marked antiproliferative effects on tumor cells in the low μM range. Combined analysis of the G4-CPG binding assays and biological data led us to focus on compound S4-5, proved to be less cytotoxic than the parent compound 4 on normal cells. An in-depth biophysical characterization of the binding of S4-5 to different G4s showed that the here identified ligand has higher affinity for the G4s and higher ability to discriminate G4 vs. duplex DNA than 4. Molecular docking studies, in agreement with the NMR data, suggest that S4-5 interacts with the accessible grooves of the target G4 structures, giving clues for its increased G4 vs. duplex selectivity. A focused library of analogs of a lead-like G-quadruplex (G4) targeting compound (4), sharing a furobenzoxazine naphthoquinone core and differing for the pendant groups on the N-atom of the oxazine ring, has been here analyzed with the aim of developing more potent and selective ligands. These molecules have been tested vs. topologically different G4s by the G4-CPG assay, an affinity chromatography-based method for screening putative G4 ligands. The obtained results showed that all these compounds were able to bind several G4 structures, both telomeric and extra-telomeric, thus behaving as multi-target ligands, and two of them fully discriminated G4 vs. duplex DNA. Biological assays proved that almost all the compounds produced effective DNA damage, showing marked antiproliferative effects on tumor cells in the low μM range. Combined analysis of the G4-CPG binding assays and biological data led us to focus on compound S4-5, proved to be less cytotoxic than the parent compound 4 on normal cells. An in-depth biophysical characterization of the binding of S4-5 to different G4s showed that the here identified ligand has higher affinity for the G4s and higher ability to discriminate G4 vs. duplex DNA than 4. Molecular docking studies, in agreement with the NMR data, suggest that S4-5 interacts with the accessible grooves of the target G4 structures, giving clues for its increased G4 vs. duplex selectivity.

First studies on thrombin-inhibiting DNA aptamers were reported in 1992, and since then a large number of anticoagulant aptamers has been discovered. TBA - also named HD1, a 15-mer G-quadruplex (G4)-forming oligonucleotide - is the best characterized thrombin binding aptamer, able to specifically recognize the protein exosite I, thus inhibiting the conversion of soluble fibrinogen into insoluble fibrin strands. Unmodified nucleic acid-based aptamers, in general, and TBA in particular, exhibit limited pharmacokinetic properties and are rapidly degraded in vivo by nucleases. In order to improve the biological performance of aptamers, a widely investigated strategy is the introduction of chemical modifications in their backbone at the level of the nucleobases, sugar moieties or phosphodiester linkages. Besides TBA, also other thrombin binding aptamers, able to adopt a well-defined G4 structure, e.g. mixed duplex/quadruplex sequences, as well as homo- and hetero-bivalent constructs, have been identified and optimized. Considering the growing need of new efficient anticoagulant agents associated with the strong therapeutic potential of these thrombin inhibitors, the research on thrombin binding aptamers is still a very hot and intriguing field. Herein, we comprehensively described the state-of-the-art knowledge on the DNA-based aptamers targeting thrombin, especially focusing on the optimized analogues obtained by chemically modifying the oligonucleotide backbone, and their biological performances in therapeutic applications.

The vascular endothelial growth factor (VEGF) family and its receptors play fundamental roles not only in physiological but also in pathological angiogenesis, characteristic of cancer progression. Aiming at finding putative treatments for several malignancies, various small molecules, antibodies, or protein-based drugs have been evaluated in vitro and in vivo as VEGF inhibitors, providing efficient agents approved for clinical use. Due to the high clinical importance of VEGF, also a great number of anti-VEGF nucleic acid-based aptamers-that is, oligonucleotides able to bind with high affinity and specificity a selected biological target-have been developed as promising agents in anticancer strategies. Notable research efforts have been made in optimization processes of the identified aptamers, searching for increased target affinity and/or bioactivity by exploring structural analogues of the lead compounds. This review is focused on recent studies devoted to the development of DNA-based aptamers designed to target VEGF. Their therapeutic potential as well as their significance in the construction of highly selective biosensors is here discussed.

In the context of developing efficient anticancer therapies aimed at eradicating any sort of tumors, G-quadruplexes represent excellent targets. Small molecules able to interact with G-quadruplexes can interfere with cell pathways specific of tumors and common to all cancers. Naphthalene diimides (NDIs) are among the most promising, putative anticancer G-quadruplex-targeting drugs, due to their ability to simultaneously target multiple G-quadruplexes and their strong, selective in vitro and in vivo anticancer activity. Here, all the available biophysical, biological, and structural data concerning NDIs targeting G-quadruplexes were systematically analyzed. Structure-activity correlations were obtained by analyzing biophysical data of their interactions with G-quadruplex targets and control duplex structures, in parallel to biological data concerning the antiproliferative activity of NDIs on cancer and normal cells. In addition, NDI binding modes to G-quadruplexes were discussed in consideration of the structures and properties of NDIs by in-depth analysis of the available structural models of G-quadruplex/NDI complexes.

To selectively target telomeric G-quadruplex (G4) DNA, monomeric and dimeric naphthalene diimides (NDIs) were investigated as binders of multimeric G4 structures able to discriminate duplex DNA. These NDIs were analysed by the affinity chromatography-based screening G4-CPG (G-quadruplex on Controlled Pore Glass), using the sequence d[AGGG(TTAGGG)7] (tel46), folding into two consecutive G4s, as model of the human telomeric G4 multimer. In parallel, a telomeric G4 monomer (tel26) and a duplex structure (ds27) were used as controls. According to G4-CPG screening, NDI-5 proved to be the best ligand in terms of dimeric G4 vs. duplex DNA selectivity and was analysed by circular dichroism (CD), gel electrophoresis, isothermal titration calorimetry (ITC) and fluorescence spectroscopy in its interactions with tel46. NDI-5 strongly binds and stabilizes tel46 G4, favouring a hybrid folding in K+-containing buffer. Under these conditions, the binding process comprises a first event involving three molecules of NDI-5 and a second one in which other six molecules bind to the DNA. In a metal cation-free system, NDI-5 induces tel46 G4 folding, as indicated by CD and PAGE, favouring an antiparallel structuring. Docking simulations showed that NDI-5 can effectively bind to the pocket between two G4 units, representing a promising ligand for multimeric G4s.

A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands&mdash, according to an affinity chromatography-based screening method named G4-CPG&mdash, were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC50 values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays&rsquo, data.

Nucleic acid aptamers are single-stranded DNA or RNA molecules identified to recognize with high affinity specific targets including proteins, small molecules, ions, whole cells and even entire organisms, such as viruses or bacteria. They can be identified from combinatorial libraries of DNA or RNA oligonucleotides by SELEX technology, an in vitro iterative selection procedure consisting of binding (capture), partitioning and amplification steps. Remarkably, many of the aptamers selected against biologically relevant protein targets are G-rich sequences that can fold into stable G-quadruplex (G4) structures. Aiming at disseminating novel inspiring ideas within the scientific community in the field of G4-structures, the emphasis of this review is placed on: 1) recent advancements in SELEX technology for the efficient and rapid identification of new candidate aptamers (introduction of microfluidic systems and next generation sequencing); 2) recurrence of G4 structures in aptamers selected by SELEX against biologically relevant protein targets; 3) discovery of several G4-forming motifs in important regulatory regions of the human or viral genome bound by endogenous proteins, which per se can result into potential aptamers; 4) an updated overview of G4-based aptamers with therapeutic potential and 5) a discussion on the most attractive G4-based aptamers for diagnostic applications. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio., Highlights • Oligonucleotide aptamers and methods for their selection against specific targets • The recurrence of G-quadruplex (G4) motifs in oligonucleotide aptamers • G4-forming aptamers in therapeutic applications as drugs and drug delivery systems • G4-forming aptamers in detection and diagnostic applications

An artificial alanine-based amino acid {(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoic acid, here named TioxAla}, bearing a substituted triazolyl-thione group on the side chain and able to bind RNA biomedical targets, was here chosen as a valuable scaffold for the synthesis of new platinum complexes with potential dual action owing to the concomitant presence of the metal centre and the amino acid moiety. Three new platinum complexes, obtained from the reaction of TioxAla with K2PtCl4, were characterized by mass spectrometry, nuclear magnetic resonance and UV–vis spectroscopy: one compound (Pt1, bis-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate-O,S} platinum(II)) consisted of two amino acid units coordinating the Pt(II) ion; the other two, Pt2 [potassium dichloro-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate (O,S)} platinum(II)] and Pt3 [potassium dichloro-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate (O,N)} platinum(II)], were isomers bearing one TioxAla unit, and two chlorides as Pt-ligands. Pt coordination involved preferentially the amino, carboxylic and thione functions of TioxAla. By preliminary antiproliferative assays, a moderate cytotoxic activity on cancer cells was observed only for Pt2 and Pt3, while no anticancer activity was found for both the chloride-free complex (Pt1) and TioxAla. This cytotoxicity, however lower than that of cisplatin, well correlated with the marked ability, here found only for Pt2 and Pt3 complexes, to bind DNA sequences either in random coil or in structured forms (duplex and G-quadruplex), as verified by spectroscopic and spectrometric analysis.

Among polyphenols, trans-resveratrol (tRES) and trans-polydatin (tPD) exert multiple biological effects, particularly antioxidant and antiproliferative. In this work, we have investigated the interaction of tPD with three cancer-related DNA sequences able to form G-quadruplex (G4) structures, as well as with a model duplex, and compared its behaviour with tRES. Interestingly, fluorescence analysis evidenced the ability of tPD to bind all the studied DNA systems, similarly to tRES, with tRES displaying a higher ability to discriminate G4 over duplex with respect to tPD. However, neither tRES nor tPD produced significant conformational changes of the analyzed DNA upon binding, as determined by CD-titration analysis. Computational analysis and biological data confirmed the biophysical results: indeed, molecular docking evidenced the stronger interaction of tRES with the promoter of c-myc oncogene, and immunoblotting assays revealed a reduction of c-myc expression, more effective for tRES than tPD. Furthermore, in vitro assays on melanoma cells proved that tPD was able to significantly reduce telomerase activity, and inhibit cell proliferation, with tRES producing higher effects than tPD.

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF165 binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.

Background trans-Resveratrol (tRES) is a polyphenolic stilbene found in plant products which has attracted great attention because of its antioxidant, anti-inflammatory and anticancer properties. Methods The possible correlation between tRES-induced suppression of melanoma cell growth and its influence on telomerase expression has been investigated by biological assays. Moreover, in order to gain new knowledge about possible mechanisms of action of tRES as antineoplastic agent, its interaction with biologically relevant secondary structure-forming DNA sequences, its aggregation properties and copper-binding activity have been studied by CD, UV and fluorescence spectroscopies. Results Biological assays have confirmed that growth inhibitory properties of tRES well correlate with the reduction of telomerase activity and hTERT gene transcript levels in human melanoma cells. Biophysical studies in solution have proved that tRES binds all the studied DNA model systems with low affinity, however showing high ability to discriminate G-quadruplex vs. duplex DNA. In addition, tRES has shown no propensity to form aggregates in the explored concentration range and has been found able to bind Cu2+ ions with a 2:1 stoichiometry. Conclusions From these biological and biophysical analyses it has emerged that tRES produces cytotoxic effects on human melanoma cells and, at a molecular level, is able to bind Cu2+ and cancer-involved G-quadruplexes, suggesting that multiple mechanisms of action could be involved in its antineoplastic activity. General significance Expanding the knowledge on the putative mechanisms of action of tRES as antitumour agent can help to develop novel, effective tRES-based anticancer drugs.

G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. Background: G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy.Methods: A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MIT assays.Results: A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4 forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells.Conclusions: The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands.General significance: Expanding the repertoire of the available G4-targeting chemotypes with improved physicochemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "Gquadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. (C) 2017 Elsevier B.V. All rights reserved.

Contrary to what was believed for many years, cis-PtI2(NH3)2, the diiodido analogue of cisplatin, displays high in vitro antiproliferative activity toward a set of tumour cell lines, overcoming resistance to cisplatin in a platinum-resistant cancer cell line. In the context of a general reappraisal of iodinated Pt(II) derivatives, aiming at a more systematic evaluation of their chemical and biological profiles, here we report on the reactivity of cis-PtI2(NH3)2 with selected DNA model systems, in single, double strand or G-quadruplex form, using cisplatin as a control. A combined approach has been exploited in this study, including circular dichroism (CD), UV-visible spectroscopy and electrospray mass spectrometry (ESI-MS) analyses. The data reveal that cis-PtI2(NH3)2 shows an overall reactivity towards the investigated oligonucleotides significantly higher than cisplatin.