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12 p.-6 fig.-1 schem., CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells., H2020 Marie Skłodowska-Curie Actions. Grant Numbers: 642157, 814102 Mizutani Foundation for Glycoscience. Grant Number: 2014-2015

Nucleic acid aptamers are innovative and promising candidates to block the hallmark event in the prion diseases, that is the conversion of prion protein (PrP) into an abnormal form; however, they need chemical modifications for effective therapeutic activity. This communication reports on the development and biophysical characterization of a small library of chemically modified G-quadruplex-forming aptamers targeting the cellular PrP and the evaluation of their anti-prion activity. The results show the possibility of enhancing anti-prion aptamer properties through straightforward modifications.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

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.

Certain G-quadruplex forming guanine-rich oligonucleotides (GROs), including AS1411, are endowed with cancer-selective antiproliferative activity. They are known to bind to nucleolin protein, resulting in the inhibition of nucleolin-mediated phenomena. However, multiple nucleolin-independent biological effects of GROs have also been reported, allowing them to be considered promising candidates for multi-targeted cancer therapy. Herein, with the aim of optimizing AS1411 structural features to find GROs with improved anticancer properties, we have studied a small library of AS1411 derivatives differing in the sequence length and base composition. The AS1411 derivatives were characterized by using circular dichroism and nuclear magnetic resonance spectroscopies and then investigated for their enzymatic resistance in serum and nuclear extract, as well as for their ability to bind nucleolin, inhibit topoisomerase I, and affect the viability of MCF-7 human breast adenocarcinoma cells. All derivatives showed higher thermal stability and inhibitory effect against topoisomerase I than AS1411. In addition, most of them showed an improved antiproliferative activity on MCF-7 cells compared to AS1411 despite a weaker binding to nucleolin. Our results support the hypothesis that the antiproliferative properties of GROs are due to multi-targeted effects.

In the search for new drug-like selective G-quadruplex binders, a bioinspired design focused on the use of nucleobases as synthons in a multicomponent reaction was herein proved to be viable and successful. Hence, a new class of multifunctionalized imidazo[2,1-i]purine derivatives, easily synthesized with a convergent approach, allowed for the identification of the first dual BCL2/c-MYC gene promoter G-quadruplex ligand. Biophysical studies involving circular dichroism melting experiments, microscale thermophoresis measurements, NMR titrations, and computational docking calculations, as well as biological investigations including cytotoxicity and apoptotic assays, and quantitative polymerase chain reaction and Western blot analyses, were performed to assess the potency and to characterize the binding mode of the newly identified lead compound. The absence of toxicity toward normal cells, together with the small molecular weight (congruent to 500 Da), the water solubility, the ease of functionalization, and the selectivity profile, are promising and desirable features to develop G-quadruplex binders as safe and effective anticancer agents.

Microrganisms from sea ice, glacial and subglacial environments are currently under investigation due to their relevant ecological functions in these habitats, and to their potential biotechnological applications. The cold-adapted Colwellia psychrerythraea 34H produces extracellular polysaccharides with cryoprotection activity. We here describe the purification and detailed molecular primary and secondary structure of the exopolysaccharide (EPS) secreted by C. psychrerythraea 34H cells grown at 4 °C. The structure was determined by chemical analysis and NMR. The trisaccharide repeating unit of the EPS is constituted by a N-acetyl quinovosamine unit and two residues of galacturonic acid both decorated with alanine. In addition, the EPS was tested in vitro showing a significant inhibitory effect on ice recrystallization. In-depth NMR and computational analysis suggest a pseudohelicoidal structure which seems to prevent the local tetrahedral order of the water molecules in the first hydration shell, and could be responsible of the inhibition of ice recrystallization.\ud As cell cryopreservation is an essential tool in modern biotechnology and medicine, the observations reported in this paper could pave the way for a biotechnological application of Colwellia EPS.

G-quadruplex (G4) nucleic acid structures are involved in a number of different diseases and their drug-induced stabilization is deemed to be a promising therapeutic approach. Herein is reported a proof of principle study on the use of nano differential scanning fluorimetry for a rapid and accurate analysis of G4-stabilizing ligands, exploiting the fluorescence properties of a 2-aminopurine modified G4-forming oligonucleotide.

International audience; Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a positive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La-module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La-module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisation of its La-module and here we report the assignment of 1 H, 15 N and 13 C resonances resulting from our studies.

Background: DNA G-quadruplex (G4) structures represent potential anti-cancer targets. In this study, we compared the effect of two G4-targeting compounds, C066-3108 and the gold standard BRACO-19. Methods: In breast and prostate cancer cells, cytotoxicity induced by both molecules was measured by a sulforhodamine B assay. In breast cancer cells, cycle, apoptosis, the formation of G4 structures, calreticulin and high mobility group box 1 (HMGB1), as well as T cell activation, were analyzed by flow cytometry and adenosine triphosphate (ATP) by luminescence. Results: Both ligands inhibited cell survival and induced DNA damage. In MCF-7 cells, G4 ligands increased the subG0/G1 phase of the cell cycle inducing apoptosis and reduced intracellular ATP. In untreated MCF-7 cells, we observed a slight presence of G4 structures associated with the G2/M phase. In MDA-MB231 cells, G4 ligands decreased the G1 and enhanced the G2/M phase. We observed a decrease of intracellular ATP, calreticulin cell surface exposure and an increase of HMGB1, accompanied by T cell activation. Both compounds induced G4 structure formation in the subG0/G1 phase. Conclusions: Our data report similar effects for both compounds and the first evidence that G4 ligands induce the release of danger signals associated with immunogenic cell death and induction of T cell activation.

Phytosterols are known to reduce plasma cholesterol levels and thereby reduce cardiovascular risk. Studies conducted on human and animal models have demonstrated that these compounds have also anti-inflammatory effects. Recently, an experimental colitis model (dextran sulphate sodium-induced) has shown that pre-treatment with phytosterols decreases infiltration of inflammatory cells and accelerates mucosal healing. This study aims to understand the mechanism underlying the colitis by analysing the end-products of the metabolism in distal colon and liver excised from the same mice used in the previous work. In particular, an unsupervised gas chromatography-mass spectrometry (GC-MS) and NMR based metabolomics approach was employed to identify the metabolic pathways perturbed by the dextran sodium sulphate (DSS) insult (i.e. Krebs cycle, carbohydrate, amino acids, and nucleotide metabolism). Interestingly, phytosterols were able to restore the homeostatic equilibrium of the hepatic and colonic metabolome.

International audience; Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C-H +-C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue-type pathway or a chain extrusion-type pathway.; La spectrométrie de mobilité ionique (IMS) est devenue populaire pour caractériser le pliage des biomolécules. De nombreuses études ont montré que les protéines repliées en solution restent repliées en phase gazeuse, alors que les protéines dépliées en solution adoptent des conformations plus étendues en phase gazeuse. Ici, nous discutons du caractère général de ce principe. Nous avons étudié les ADN simple brins (séquences télomériques humaines riches en cytosine avec répétitions CCCTAA), qui se replient en une structure de motif intercalé (i-motif) d'une manière dépendant du pH, grâce à la formation de paires de bases C-H +-C. Comme la formation de motifs i-motifs est favorisée à faible force ionique, nous avons pu étudier le comportement des structures i-motifs ESI-IMS-MS à pH ~5,5 sur une large gamme de concentrations en acétate d'ammonium (15 mM à 100 mM). Les expériences de contrôle consistent soit en la même séquence à pH ~7,5 (à ce pH la séquence est dépliée), soit en des variantes de séquence qui ne peuvent pas former d'i-motifs (répétitions CTCTAA). Les résultats surprenants proviennent des expériences de contrôle. Nous avons constaté que la force ionique de la solution avait un effet plus important sur la compacité des structures en phase gazeuse que l'état de repliement de la solution. Cela signifie que les ions produits par électrospray gardent une mémoire du processus d'acquisition de la charge, et que celui-ci est influencé par la concentration de l'électrolyte. Nous discutons de ces résultats à la lumière de la répartition de l'analyte entre l'intérieur des gouttelettes et la surface des gouttelettes, réaprtition qui à son tour influence la probabilité pour la molécule d'être ionisée par une voie de type résidu chargé ou par une voie d'extrusion de la chaîne.

G-Quadruplex-binding compounds are currently perceived as possible anticancer therapeutics. Here, starting from a promising lead, a small series of novel hydrazone-based compounds were synthesized and evaluated as G-quadruplex binders. The in vitro G-quadruplex-binding properties of the synthesized compounds were investigated employing both human telomeric and oncogene promoter G-quadruplexes with different folding topologies as targets. The present investigation led to the identification of potent G-quadruplex stabilizers with high selectivity over duplex DNA and preference for one G-quadruplex topology over others. Among them, selected derivatives have been shown to trap G-quadruplex structures in the nucleus of cancer cells. Interestingly, this behavior correlates with efficient cytotoxic activity in human osteosarcoma and colon carcinoma cells.

G-quadruplex (G4) and i-motif (iM) are four-stranded non-canonical nucleic acid structural arrangements. Recent evidences suggest that these DNA structures exist in living cells and could be involved in several cancer-related processes, thus representing an attractive target for anticancer drug discovery. Efforts toward the development of G4 targeting compounds have led to a number of effective bioactive ligands. Herein, employing several biophysical methodologies, we studied the ability of some well-known G4 ligands to interact with iM-forming DNA. The data showed that the investigated compounds are actually able to interact with both DNA in vitro, thus acting de facto as multi-target-directed agents. Interestingly, while all the compounds stabilize the G4, some of them significantly reduce the stability of the iM. The present study highlights the importance, when studying G4-targeting compounds, of evaluating also their behavior toward the i-motif counterpart.

Target selectivity is one of the main challenges in the search for small molecules able to act as effective and non-toxic anticancer and/or antiviral drugs. To achieve this goal, handy, rapid and reliable High Throughput Screening methodologies are needed. We here describe a novel functionalization for the solid phase synthesis of oligonucleotides on Controlled Pore Glass, including a flexible hexaethylene glycol spacer linking the first nucleoside through the nucleobase via a covalent bond stable to the final deprotection step. This allowed us preparing fully deprotected oligonucleotides still covalently attached to their supports. In detail, on this support we performed both the on-line synthesis of different secondary structure-forming oligonucleotides and the affinity chromatography-based screenings of conformation-selective G-quadruplex ligands. By using a fluorescent core-extended naphthalene diimide with different emitting response upon binding to sequences folding into G-quadruplexes of different topologies, we have been able to discriminate not only G-quadruplex vs. duplex DNA structures, but also different G-quadruplex conformations on the glass beads by confocal microscopy.

Targeting of G-quadruplex-forming DNA in the BCL2 gene promoter to inhibit the expression of anti-apoptotic Bcl-2 protein is an attractive approach to cancer treatment. So far, efforts made in the discovery of molecules that are able to target the BCL2 G-quadruplex have succeeded mainly in the identification of ligands with poor drug-like properties. Here, a small series of furo[2,3-d]pyridazin-4(5H)-one derivatives were evaluated as a new class of drug-like G-quadruplex-targeting compounds. Biophysical studies showed that two derivatives could effectively bind to BCL2 G-quadruplex with good selectivity. Moreover, one such ligand was found to appreciably inhibit BCL2 gene transcription, with a substantial decrease in protein expression levels, and also showed significant cytotoxicity toward the Jurkat human T-lymphoblastoid cell line.

A phytochemical investigation of Tragopogon tommasinii Sch.Bip. (Asteraceae, Cichorieae) yielded a total of 21 natural products, two simple phenolic acids (4-hydroxybenzoic acid and p-coumaric acid), four caffeic acid derivatives (chlorogenic acid, 3-O-caffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid), six flavonoids (luteolin, luteolin 7-O-glucoside, vitexin, orientin, quercetin 3-O-glucoside, and isorhamnetin 3-O-glucoside), three simple bibenzyls [2-carboxyl-5-hydroxy-3-methoxy-4′-β-glucopyranosyl-oxybibenzyl, 3-caffeoyl-(9 → 5)-β-apiosyl-(1 → 6)-β-glucopyranosyloxy-5,4′-dihydroxy-3′-methoxybibenzyl, 3-caffeoyl-(9 → 5)-β-apiosyl-(1 → 6)-β-glucopyranosyloxy-4′-dihydroxy-5,3′-dimethoxybibenzyl], three phtalides [3-(4-β-glucopyranosyloxybenzyl)-7-hydroxy-5-methoxyphtalide, 7-β-glucopyranosyloxy-(S)-3-(4-hydroxybenzyl)-5-methoxyphtalide, and 7-(1 → 6)-α-rhamnosyl-β-glucopyranosyloxy-(S)-3-(4-hydroxybenzyl)-5-methoxyphtalide], two cannabispiradienone derivatives [3-O-β-glucopyranosyldemethoxycannabispiradienone and 3-caffeoyl-(9 → 5)-β-apiosyl-(1 → 6)-β-glucopyranosyloxydemethoxycannabispiradienone], and tetra-N-coumaroyl spermine. The three bibenzyls, the latter two benzylphthalides, and both cannabispiradienone derivatives represent new natural compounds and all compounds, except the caffeic acid derivatives and the flavonoids were new for T. tommasinii. The structures were established by HR mass spectrometry, extensive 1D and 2D NMR spectroscopy, and CD spectroscopy. Moreover, the potential anti-inflammatory activities of the new compounds were assayed using human neutrophils and their production of IL-1b, IL-8, TNF-α and MMP-9 as well as the expression of TLR-4, respectively.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea. Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Background Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. Methods Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. Results The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. Conclusions A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. General significance The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Background: The quality grade of an olive oil is defined according to the results of analytical and organoleptic examinations.The increasing attention towards both olive oil quality and quality verification methods prompted us to undertake a "critical" analysis of analytical and sensory data supplied by an International Certificated Body (ICB), relative to commercial olive oils produced in Mediterranean areas and purchased in Italy and in USA. Methods: ICB data included chemical analyses namely free acidity, peroxide index, spectrophotometric UV evaluation, fatty acid ethyl esters and stigmadiens content and organoleptic evaluations carried out by nine official International Olive Council labs according to EEC Regulation 2568/91. Results: The results of the chemical analyses, except the fatty acid ethyl ester content, obtained from the nine labs were consistent giving rise to the same quality grade. In nearly all samples, the fatty acid ethyl ester content was close to the threshold established for extra virgin olive oils indicating a non-excellent quality of the olive oils. Organoleptic evaluations, commonly called panel test, given by the nine labs were not consistent. Conclusions: The EEC Regulation 2568/91 does not give any indication on the way to report the uncertainty of the results, and in the case of extra virgin olive oils with a borderline value, the way to report the fatty acid ethyl ester content, with or without the uncertainty, can create confusion in defining the olive oil quality grade. Panel test seemed to work well only in the case of extremely good olive oils, whereas, in commercial extra virgin olive oils with borderline value of fatty acid ethyl ester content, a different sensory sensibility seems to be in the different IOC labs. [Figure not available: see fulltext. Caption: Panel test and chemical analyses of commercial olive oils: a comparative study.].

G-quadruplex stabilizers are an established opportunity in anticancer chemotherapy. To circumvent the antiproliferative effects of G4 ligands, cancer cells recruit PARP enzymes at telomeres. Herein, starting from the structural similarity of a potent G4 ligand previously discovered by our group and a congeneric PARP inhibitor, a library of derivatives was synthesized to discover the first dual G4/PARP ligand. We demonstrate that a properly decorated thieno[3,2-c]quinolin-4(5H)-one stabilizes the G4 fold in vitro and in cells, induces a DNA damage response localized to telomeres, inhibits PARylation in cells, and has an antiproliferative effect in BRCA2 deficient tumor cells.

Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2′ and C4′ atoms are linked through a methylene bridge and the sugar is blocked in a 3′-endoconformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5′-TGGGT-3′ is presented refined at 1.7 Å resolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3′-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3′-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO center dot) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 50,8-cyclo-20-deoxyguanosine (50,8-cdG) and 50,8-cyclo-20-deoxyadenosine (50,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT](4) (TG4T), d[AGGG(TTAGGG)(3)] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)(3)A] (mutTel24) were exposed to HO center dot radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L> OC >> SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (similar to 50% solution folded) and mutTel24 (similar to 90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33 angstrom with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.

Recent findings have unambiguously demonstrated that DNA G-rich sequences can adopt a G-quadruplex folding in living cells, thus further validating them as crucial targets for anticancer therapy. Herein, to identify new potent G4 binders as antitumor drug candidates, we have targeted a 24-nt G4-forming telomeric sequence employing a receptor-based virtual screening approach. Among the best candidates, in vitro binding experiments allowed identification of three novel G4 ligands. Among them, the best compound features an unprecedented binding selectivity for the human telomeric DNA G-quadruplex with no detectable binding for other G4-forming sequences present at different genomic sites. This behavior correlates with the detected ability to generate DNA damage response in tumor cells at the telomeric level and efficient antiproliferative effect on different tumor cell lines at low micromolar concentrations. © 2013 American Chemical Society.

Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the energetics of thermally-induced transitions of DNA and other biological macromolecules. Therefore, DSC has been used to study the thermodynamic stability of several nucleic acids structures. G-quadruplexes are among the most important non-canonical nucleic acid architectures that are receiving great consideration. This article reports examples on the contribution of DSC to the knowledge of G-quadruplex structures. The selected case studies show the potential of this method in investigating the structure stability of G-quadruplex forming nucleic acids, and in providing information on their structural complexity. Indeed, DSC can determine thermodynamic parameters of G-quadruplex folding/unfolding processes, but it can also be useful to reveal the formation of multiple conformations or the presence of intermediate states along the unfolding pathway, and to evaluate the impact of chemical modifications on their structural stability. This article aims to show that DSC is an important complementary methodology to structural techniques, such as NMR and X-ray crystallography, in the study of G-quadruplex forming nucleic acids.

The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested.

Olive fruits from 19 varieties and different areas of Bejaia province (Algeria) were used to produce monovarietal olive oils in laboratory. The olive oils were analyzed using both traditional chemical analyses and nuclear magnetic resonance (NMR) methodology. The investigation involved pigment content determination, tocopherol analysis, fatty acid composition, and chromatographic determination of phenolic compounds. Chlorophyll, carotenoids, tocopherols and the content of oleic acid turned out to be variety dependent. The extra-virgin olive oils (EVOOs) were analyzed as a whole and as phenolic extract by NMR. The study gave general indication on olive oil quality and information about geographical origin of the samples. Overall, the results obtained in the present work reveal that Algerian monovarietal olive oils produced in Bejaia province have the potential to produce blends that may compete with other Mediterranean products.

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

The chemical synthesis and 1H NMR analysis of a novel bicyclic uridine derivative, with a 18-crown-6 ether moiety fused at the ribose 2- and 3-positions, as first example of a hitherto unknown class of ribose-modified nucleosides, are here described. NMR-based conformational analysis studies showed for the modified nucleoside a marked preference for an N-type sugar puckering and the nucleobase in the anti conformation, with the uracil favouring the coordination of a sodium ion hosted in the crown ether.

A comparative study of the competitive cation exchange between the alkali metal ions K+, Rb+, and Cs+ and the Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2 was performed in aqueous solution by a combined use of the 23Na and 1H NMR spectroscopy. The titration data confirm the different binding affinities of these ions for the G-quadruplex and, in particular, major differences in the behavior of Cs+ as compared to the other ions were found. Accordingly, Cs+ competes with Na+ only for the binding sites at the quadruplex surface (primarily phosphate groups), while K+ and Rb+ are also able to replace sodium ions located inside the quadruplex. Furthermore, the 1H NMR results relative to the CsCl titration evidence a close approach of Cs+ ions to the phosphate groups in the narrow groove of [d(G4T4G4)]2. Based on a three-site exchange model, the 23Na NMR relaxation data lead to an estimate of the relative binding affinity of Cs+ versus Na+ for the quadruplex surface of 0.5 at 298 K. Comparing this value to those reported in the literature for the surface of the G-quadruplex formed by 5'-guanosinemonophosphate and for the surface of double-helical DNA suggests that topology factors may have an important influence on the cation affinity for the phosphate groups on DNA.

Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), 3′GGT5′-5′TGGTGTGGTTGG3′, which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.

Several researches have been devoted to structure-activity relationship and to post-SELEX modifications of the thrombin binding aptamer (TBA), one of the first aptamers discovered by the SELEX methodology. However, no studies on TBA dealing with the effects of introduction of inversion of polarity sites have been reported yet. In this frame, we have undertaken the synthesis and the study of a mini-library composed of several TBA analogues containing a 3'-3' or a 5'-5' inversion of polarity site at different positions into the sequence. Particularly, in this article, we present preliminary results about their structural and biological properties.

Differential scanning calorimetry (DSC) and circular dichroism (CD) techniques were used to investigate the physico-chemical properties of the quadruplexes formed by the two different truncations of human telomeric sequence d(TAGGGT) and d(AGGGT), where the adenines were substituted by 2′-deoxy-8-(hydroxyl)adenosine (A → A OH ). CD spectra show that the modified sequences are able to form parallel-stranded quadruplex structure. Analysis of the thermodynamic parameters reveals that the introduction of the modified adenine affects in different way the thermal stability of the [d(TAGGGT)] 4 and [d(AGGGT)] 4 quadruplexes.

The interest in DNA quadruplex structures has been fueled by the recognition that telomeres, the 3' single stranded guanine-rich overhangs found at the termini of chromosomes, are likely to form G-tetrads type structures important in cell senescence and cancer. In addition to their presence in telomeres, where they may play a role in maintaining the stability and integrity of chromosomes, guanine-rich regions are found in other region of the genome, amongst these is intron 6 of hTERT a gene codifying for the enzyme telomerase. Interestingly, the formation of G-quadruplexes in this region is involved in the down-regulation of telomerase activity caused by an alteration of the hTERT splicing pattern. Therefore, we have analyzed several sequences of that intron by (1)H-NMR and CD spectroscopy, and we have found that the sequence d(GGGGTGAAAGGGG) is able to fold in a single well-defined antiparallel quadruplex structure consisting of four G-tetrads, possessing a twofold symmetry, and containing four Gs in a syn glycosidic conformation.

A chemoproteomic-driven approach was used to investigate the interaction network between human telomeric G-quadruplex DNA and nuclear proteins. We identified novel G-quadruplex binding partners, able to recognize these DNA structures at chromosome ends, suggesting a possible, and so far unknown, role of these proteins in telomere functions.

The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

There is currently significant interest in the development of G-quadruplex-interactive compounds, given the relationship between the ability to stabilize these non-canonical DNA structures and anticancer activity. In this study, a set of biophysical assays was applied to evaluate the binding of six drug-like ligands to DNA G-quadruplexes with different folding topologies. Interestingly, two of the investigated ligands showed selective G-quadruplex-stabilizing properties and biological activity. These compounds may represent useful leads for the development of more potent and selective ligands.

The synthesis and preliminary structural studies of ODNs A8MeGGGT and TA8MeGGGT, where A8Me represents 2'-deoxy-8-methyladenosine, are reported.

Truncated sequences of human telomeric DNA can readily assemble to form parallel stranded quadruplexes containing A- and G-tetrads. The formation of an A-tetrad is highly context-dependent and the relationship between the formation of an A-tetrad and the glycosidic torsion angle of the adenosine residues implicated has not been completely clarified so far. In order to give a further insight in this issue we synthesized the modified oligomers d(ABrGGGT) and d(TABrGGGT), two different truncations of the human telomeric sequence containing a 8-bromoadenosine residue, named ABr. NMR data show that both the modified oligomers are able to perfectly fold into highly symmetric quadruplexes with all strands parallel to each other. Molecular modeling studies were performed on both [d(ABrGGGT)]4 and [d(TABrGGGT)]4, indicating that a bulky substituent, such as a bromine atom at the C8 position of adenines, can force the glycosidic bond to adopt a syn conformation, stabilizing the resulting quadruplexes.

The aim of this work is to compare the physicochemical properties of three oligonucleotidic sequences, d(TGGGT), d(TGGGGT) and d(TGGGGGT), which assemble to form quadruplex structures with the same molecularity, but containing three, four, and five G-quartets, respectively. The addition of one or two G-tetrads greatly increases both the enthalpy and Tm values of the quadruplex dissociation.

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. The guanine rich strand can fold into four-stranded G-quadruplex structures, which have recently become attractive for biomedical research. Indeed, the aptamers based on the quadruplex motif may prove useful as tools aimed at binding and inhibiting particular proteins, catalyzing various biochemical reactions, or even serving as pharmaceutically active agents. The incorporation of modified bases into oligonucleotides can have profound effects on their folding and may produce useful changes in physical and biological properties of the resulting DNA fragments. In this work, the adenines of the human telomeric repeat oligonucleotide d(TAGGGT) and d(AGGGT) were substituted by 2′-deoxy–8-(propyn-1-yl)adenosine (A→APr) or by 8-bromodeoxyadenosine (A→ABr). The biophysical properties of the resulting quadruplex structures were compared with the unmodified quadruplexes. NMR and CD spectra of the studied sequences were characteristic of parallel-stranded, tetramolecular quadruplexes. The analysis of the equilibrium melting curves reveals that the modifications stabilize the quadruplex structure. The results are useful when considering the design of novel aptameric nucleic acids with diverse molecular recognition capabilities that would not be present using native RNA/DNA sequences. © 2004 Wiley Periodicals, Inc. Biopolymers, 2005

In this paper we report the synthesis and the structural characterization of two modified oligodeoxyribonucleotides (ODNs), namely d(A8MeGGGT) and d(TA8MeGGGT), where A8Me represents a 8-methyl-2′-deoxyadenosine. Both ODNs have been studied by 1H NMR, CD spectroscopy and molecular modelling and shown to form fourfolds symmetric G-quadruplex structures, with all strands parallel and equivalent to each other. The complexes are characterized by thermal stabilities comparable to that of their natural counterparts. NOE patterns involving 8-methyl group in A8Me residues allowed us to define the main structural features at the 5′-end of the complexes. Particularly, inter- and intrastrand NOEs show a syn-orientation and a symmetrical arrangement of A8Me bases stacking on the adjacent G-tetrad.

Two new callipeltin-related acyclic peptides ( 2 and 3 ) have been isolated, together with callipeltins A–C from the marine sponge Latrunculia sp. collected at the Vanuatu Islands. The gross structures of new compounds were elucidated by spectroscopic data. The application of the Marfey's analysis on the new derivatives and on callipeltin A ( 1 ), allowed us to revise the configuration of two amino acid units in callipeltin A.