The oxidative damage to the human telomere: effects of 5-hydroxymethyl-2'-deoxyuridine on telomeric G-quadruplex structures

As part of the genome, human telomeric regions can be damaged by the chemically reactive molecules responsible for oxidative DNA damage. Considering that G-quadruplex structures have been proven to occur in human telomere regions, several studies have been devoted to investigating the effect of oxidation products on the properties of these structures. However only investigations concerning the presence in G-quadruplexes of the main oxidation products of deoxyguanosine and deoxyadenosine have appeared in the literature. Here, we investigated the effects of 5-hydroxymethyl-2’-deoxyuridine (5-hmdU), one of the main oxidation products of T, on the physical–chemical properties of the G-quadruplex structures formed by two human telomeric sequences. Collected calorimetric, circular dichroism and electrophoretic data suggest that, in contrast to most of the results on other damage, the replacement of a T with a 5-hmdU results in only negligible effects on structural stability. Reported results and other data from literature suggest a possible protecting effect of the loop residues on the other parts of the G-quadruplexes

Bacterial model membranes under the harsh subsurface conditions of Mars

Biomembranes are a key component of all living systems. Most research on membranes is restricted to ambient physiological conditions. However, the influence of extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments, is less well understood. The deep subsurface of Mars is thought to harbour high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments. Here, we investigated the combined effects of high concentrations of Mars-relevant salts, including sodium and magnesium perchlorate and sulphate, and high hydrostatic pressure on the stability, structure, and function of a bacterial model membrane. To this end, several biophysical techniques have been employed, including calorimetry, fluorescence and CD spectroscopy, confocal microscopy, and small-angle X-ray scattering. We demonstrate that sulphate and perchlorate salts affect the properties of the membrane differently, depending on the counterion present (Na+vs. Mg2+). We found that the perchlorates, which are believed to be abundant salts in the Martian environment, induce a more hydrated and less ordered membrane, strongly favouring the physiologically relevant fluid-like phase of the membrane even under high-pressure stress. Moreover, we show that the activity of the phospholipase A2 is strongly modulated by both high pressure and salt. Compellingly, in the presence of the chaotropic perchlorate, the enzymatic reaction proceeded at a reasonable rate even in the presence of condensing Mg2+ and at high pressure, suggesting that bacterial membranes could still persist when challenged to function in such a highly stressed Martian environment

Thrombin–aptamer recognition: a revealed ambiguity

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 α-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

Selective Binding of Distamycin A Derivative to G-Quadruplex Structure [d(TGGGGT)]4

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 (compound 1) and the [d(TGGGGT)]4 quadruplex. Additionally, to compare the binding behaviour of netropsin and compound 1 to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)]4 has been performed. Experiments show that netropsin and compound 1 are able to bind to [d(TGGGGT)]4 with 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 compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity

Design, synthesis, biophysical and biological studies of trisubstitutednaphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)3 binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UVmelting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer

A new modified thrombin binding aptamer containing a 5′–5′ inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d((3′)GGT(5′)-(5′)TGGTGTGGTTGG(3)′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d((3′)GGT(5′)-(5′)TGGTGTGGTTGG(3′)) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d((3′)GGT(5′)-(5′)TGGTGTGGTTGG(3′)) continues to display an anticoagulant activity, even if decreased with respect to the TBA

Gut microbiota composition in COVID-19 hospitalized patients with mild or severe symptoms

Background and aimCOVID-19, the infectious disease caused by SARS-CoV-2 virus that has been causing a severe pandemic worldwide for more than 2 years, is characterized by a high heterogeneity of clinical presentations and evolution and, particularly, by a varying severity of respiratory involvement. This study aimed to analyze the diversity and taxonomic composition of the gut microbiota at hospital admission, in order to evaluate its association with COVID-19 outcome. In particular, the association between gut microbiota and a combination of several clinical covariates was analyzed in order to characterize the bacterial signature associate to mild or severe symptoms during the SARS-CoV-2 infection.Materials and methodsV3–V4 hypervariable region of 16S rRNA gene sequencing of 97 rectal swabs from a retrospective cohort of COVID-19 hospitalized patients was employed to study the gut microbiota composition. Patients were divided in two groups according to their outcome considering the respiratory supports they needed during hospital stay: (i) group “mild,” including 47 patients with a good prognosis and (ii) group “severe,” including 50 patients who experienced a more severe disease due to severe respiratory distress that required non-invasive or invasive ventilation. Identification of the clusters of bacterial population between patients with mild or severe outcome was assessed by PEnalized LOgistic Regression Analysis (PELORA).ResultsAlthough no changes for Chao1 and Shannon index were observed between the two groups a significant greater proportion of Campylobacterota and Actinobacteriota at phylum level was found in patients affected by SARS-CoV-2 infection who developed a more severe disease characterized by respiratory distress requiring invasive or non-invasive ventilation. Clusters have been identified with a useful early potential prognostic marker of the disease evolution.DiscussionMicroorganisms residing within the gut of the patients at hospital admission, were able to significantly discriminate the clinical evolution of COVID-19 patients, in particular who will develop mild or severe respiratory involvement. Our data show that patients affected by SARS-CoV-2 with mild or severe symptoms display different gut microbiota profiles which can be exploited as potential prognostic biomarkers paving also the way to new integrative therapeutic approaches