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The design of modified nucleic acid aptamers is improved by considering thermodynamics and kinetics of their association/dissociation processes. Locked Nucleic Acids (LNA) is a promising class of nucleic acid analogs. In this work the thermodynamic and kinetic properties of a LNA quadruplex formed by the TGGGT sequence, containing only conformationally restricted LNA residues, are reported and compared to those of 2'-OMe-RNA (O-RNA) and DNA quadruplexes. The thermodynamic analysis indicates that the sugar-modified quadruplexes (LNA and O-RNA) are stabilized by entropic effects. The kinetic analysis shows that LNA and O-RNA quadruplexes are characterized by a slower dissociation and a faster association with respect to DNA quadruplex. Interestingly, the LNA quadruplex formation process shows a second-order kinetics with respect to single strand concentration and has a negative activation energy. To explain these data, a mechanism for tetramer formation with two intermediate states was proposed.

The ends of chromosomes, termed telomeres, is composed of telomeric DNA and various telomere-associated proteins. Although most of telomeric DNA is double-stranded, the extreme 3'-end of the telomere consists of single-stranded G-rich DNA overhangs [1]. Human telomeric DNA contains tandem repeats of the sequence TTAGGG and the guanine-rich strand folds into an intramolecular quadruplex structure in Na+ solution. The structure of the telomeric DNA has been extensively studied in recent years due to the identification of a possible biological role and potential biotechnological and therapeutic applications. These structures seem to be involved in the recombination at G-rich sequences, telomere protection and elongation and transcriptional regulation [2]. The ability to chemically synthesize oligonucleotide analogues has opened up the opportunity to observe changes in their structures and activities that occur even upon single residue substitution [3]. The incorporation of modified bases into oligonucleotides may indeed produce useful changes in physical and biological properties of the resulting DNA fragments. In this work, the physico-chemical properties of quadruplex structures of the human telomeric repeat oligonucleotide d(TAGGGT), d(AGGGT) and modified sequences were compared. The adenine was substituted by 2'-deoxy-8-(propyn-1-yl)adenosine (AAPr) and by 8-bromodeoxyadenosine (A ABr).

Here we propose a new method to assign relative configurations of stereocenters in small organic molecules by using residual dipolar couplings; the main advantage of this method is that spatial proximity of the stereocenters is not required.

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effect of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility to work in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.

Ferroptosis is a recently recognized form of regulated cell death involving lipid peroxidation. Glutathione peroxidase 4 (GPX4) plays a central role in the regulation of ferroptosis through the suppression of lipid peroxidation generation. Connections have been reported between ferroptosis, lipid metabolism, cancer onset, and drug resistance. Recently, interest has grown in ferroptosis induction as a potential strategy to overcome drug resistance in hematological malignancies. GATA-1 is a key transcriptional factor controlling hematopoiesis-related gene expression. Two GATA-1 isoforms, the full-length protein (GATA-1FL) and a shorter isoform (GATA-1S), are described. A balanced GATA-1FL/GATA-1S ratio helps to control hematopoiesis, with GATA-1S overexpression being associated with hematological malignancies by promoting proliferation and survival pathways in hematopoietic precursors. Recently, optical techniques allowed us to highlight different lipid profiles associated with the expression of GATA-1 isoforms, thus raising the hypothesis that ferroptosis-regulated processes could be involved. Lipidomic and functional analysis were conducted to elucidate these mechanisms. Studies on lipid peroxidation production, cell viability, cell death, and gene expression were used to evaluate the impact of GPX4 inhibition. Here, we provide the first evidence that over-expressed GATA-1S prevents K562 myeloid leukemia cells from lipid peroxidation-induced ferroptosis. Targeting ferroptosis is a promising strategy to overcome chemoresistance. Therefore, our results could provide novel potential therapeutic approaches and targets to overcome drug resistance in hematological malignancies.

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.

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.

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.

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.

The occurrence of a G-triplex folding intermediate of thrombin binding aptamer (TBA) has been recently predicted by metadynamics calculations, and experimentally supported by Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) data collected on a 3′ end TBA-truncated 11-mer oligonucleotide (11-mer-3′-t-TBA). Here we present the solution structure of 11-mer-3′-t-TBA in the presence of potassium ions. This structure is the first experimental example of a G-triplex folding, where a network of Hoogsteen-like hydrogen bonds stabilizes six guanines to form two G:G:G triad planes. The G-triplex folding of 11-mer-3′-t-TBA is stabilized by the potassium ion and destabilized by increasing the temperature. The superimposition of the experimental structure with that predicted by metadynamics shows a great similarity, with only significant differences involving two loops. These new structural data show that 11-mer-3′-t-TBA assumes a G-triplex DNA conformation as its stable form, reinforcing the idea that G-triplex folding intermediates may occur in vivo in human guanine-rich sequences. NMR and CD screening of eight different constructs obtained by removing from one to four bases at either the 3′ and the 5′ ends show that only the 11-mer-3′-t-TBA yields a relatively stable G-triplex., Nucleic Acids Research, 42 (21), ISSN:1362-4962, ISSN:0301-5610

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.

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.

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.

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.

Under certain conditions, specific DNA sequences have the potential to adopt noncanonical secondary structures, such as i-motifs. Interestingly, these DNA stretches are not randomly located throughout the genome but rather frequently clustered in regulatory regions of oncogenes and in telomeres, the terminal regions of chromosomes. Recent evidences suggest that i-motif DNA structures exist in living cells and could be involved in a variety of biological processes, such as replication, regulation of oncogene expression, and telomere functions. Therefore, the targeting of i-motif DNA is an emerging research area in medicinal chemistry. Bringing these noncanonical structures into focus as targets for anticancer drug design and gene regulation processes could be crucial for a better understanding of their biological functions and to open the way to new, effective strategies for cancer treatment.

A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known G-quadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility.

The S100 subfamily of EF-hand proteins is distinguished by the binding of Zn(2+) in addition to Ca(2+). In an effort to understand the role of Zn(2+) in modulating the activity of S100 proteins, we have carried out heteronuclear NMR studies of Zn(2+)-bound S100A2 and obtained near complete resonance assignments. This analysis revealed an equilibrium between multiple isoforms due to cis-trans isomerism of proline residues in flexible regions of the protein. The secondary structure of S100A2 has been determined based on the NMR chemical shift index (CSI) technique. The protein is found to possess essentially the same secondary structure found in other S100 proteins such as S100A6 and S100B. Homology models have been built based on the high resolution three-dimensional structures of other S100 proteins. The models predict two Zn(2+) binding clusters, one involving residues His17-Cys21-Cys93 and the other Cys2-His39, and with Cys86 participating in either the N-terminal or the C-terminal binding site.

More than 40 years after its isolation, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. The Na(+),K(+)-ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K(+) and remarkable rise of cytosolic Na(+) levels. In addition, a slight permeability to Ca(2+) is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca(2+) concentration gives rise to downstream events ultimately leading to cell death. The widely accepted recognition of the dependence of important cellular events on calcium ion concentration propelled us to investigate the occurrence of palytoxin-Ca(2+) complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca(2+) is preferentially coordinated. This study constitutes the first characterization of a calcium complex with palytoxin and, as such, is expected to support the investigation of the toxin molecular bioactivity.

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by 1H NMR, and with a yield of 30-300 mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K+ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A1 > B1 > B > A > B4 > J 1. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.

Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven. © 2012 American Chemical Society.

Nucleic acids represent the alphabet of the cellular language and through their sequence and topology regulate vital cellular functions. In recent years, it has been found that many variations from the Watson–Crick duplex structure play key roles in many cellular processes. Examples are hairpins, cruciforms, parallel-stranded duplexes, triplexes, G-quadruplexes, and the i-motif. These structures can be formed by nucleotide sequences distributed throughout the whole human genome, their location is not random and often associated with human diseases. These complexes are formed from one to four strands, stabilized by base stacking and hydrogen bond interactions, with a variety of non-standard pairings. For instance, DNA triplexes can present G:G-C, A:A-T, C:G-C, and T:A-T pairings, with two strands in the standard Watson–Crick duplex structure (i.e. G-C and A-T) and the third one lying in the major groove of the duplex. In contrast, G-quadruplexes are four-stranded structures stabilized by stacking of two or more guanine tetrads (Figure 1). These examples highlight the structural polymorphism of DNA and suggest that other structures might exist, perhaps with specific cellular functions that are, to date, unknown. Herein, using metadynamics simulations, we have identified a stable folding intermediate of the thrombin binding aptamer (TBA) quadruplex. This intermediate is characterized by a “G-triplex” structure, having G:G:G triad planes stabilized by an array of Hoogsteen-like hydrogen-bonds (Figure 1). This kind of structure has been already hypothesized in other investigations on different DNA sequences, but never experimentally proven. Herein, for the first time, we have structurally and thermodynamically characterized this DNA structural motif, through a combination of biophysical experiments. Well-tempered metadynamics simulations have been used to study the folding of TBA, which is a 15-mer oligonucleotide (5’-dGGTTGGTGTGGTTGG-3’) organized in an anti-parallel monomolecular G-quadruplex with a chairlike structure (Figure 2a). This structure consists of two Gtetrads, able to coordinate a metal ion at the center, connected by two TT loops and a single TGT loop. Metadynamics accelerates the sampling, adding a bias on a few degrees of freedom of the system, called collective variables (CVs). In such a way, long time scale events, such as ligand/protein docking or protein/DNA folding, can be sampled in an affordable computational time and the free energy surface (FES) of the process can be computed. In the present case, the FES was calculated as a function of two CVs, the radius of gyration CV defined by the oxygen atoms of the guanines forming the G-tetrads and a second CV that counts the number of hydrogen bonds between these guanines (see Supporting Information). Looking at the FES obtained after approximately 80 ns of metadynamics simulation, three main energy minima can be identified (Figure 2b). The deepest one, basin A, corresponds to the experimental G-quadruplex structure of TBA. In the second minimum, basin B, TBA shows a partial opening of the 3’ end with residue G15

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human ??-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K(+) ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of ??-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na(+) or K(+) and a circular dichroism study of the structural stability of the aptamer both free and complexed with ??-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na(+) and K(+) on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein-aptamer interface. The present data, in combination with those previously obtained on the complex between ??-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies. © 2012 Bentham Science Publishers.

The emergence of nucleic acid four-stranded architectures, denominated G-quadruplexes as a prolific area of research, has led to an interest in the development of inexpensive methods for the rapid assessment of their structural characterization in solution. Research in this area is motivated by their potential impact in regulation of biological mechanisms and technological applications. For many applications, light absorption techniques, such as circular dichroism (CD) and UV, have been sufficient to discriminate the quadruplex fold from other architectures. CD is also useful to discriminate a single quadruplex topology from all other 25 generic folding topologies. Here we demonstrate the use of these techniques for characterizing three different types of G-quadruplex topologies classified through the sequence of glycosidic bond angles (GBA) adopted by guanosines of the G-quadruplex stem.

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)] 4, we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG BrGGT)] 4 and [d(TGGGG BrT)] 4. The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)] 4 with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far. © 2011 Elsevier Masson SAS. All rights reserved.

The present study has employed a combination of spectroscopic, calorimetric and computational methods to explore the binding of the three side-chained triazatruxene derivative, termed azatrux, to a human telomeric G-quadruplex sequence, under conditions of molecular crowding. The binding of azatrux to the tetramolecular parallel [d(TGGGGT)](4) quadruplex in the presence and absence of crowding conditions, was also characterized. The data indicate that azatrux binds in an end-stacking mode to the parallel G-quadruplex scaffold and highlights the key structural elements involved in the binding. The selectivity of azatrux for the human telomeric G-quadruplex relative to another biologically relevant G-quadruplex (c-Kit87up) and to duplex DNA was also investigated under molecular crowding conditions, showing that azatrux has good selectivity for the human telomeric G-quadruplex over the other investigated DNA structures.

Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are found in many regions of human genome and in the telomeres of all eukaryotic organisms. Since small molecules that target G-quadruplexes have been found to be effective telomerase inhibitors, the identification of new specific ligands for G-quadruplexes is emerging as a promising approach to develop new anticancer drugs. Distamycin A is known to bind to AT-rich sequences of duplex DNA, but it has recently been shown to interact also with G-quadruplexes. Here, isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound1) and the [d(TGGGGT)]4quadruplex. Additionally, to compare the binding behaviour of netropsin and compound1to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)]4has been performed. Experiments show that netropsin and compound1are able to bind to [d(TGGGGT)]4with good affinity and comparable thermodynamic profiles. In both cases the interactions are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound1decrease the affinity of the ligand toward the duplex, enhancing the selectivity.