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

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG3T) and its modified analogs containing a 50-50 or 30-30 inversion of polarity sites, namely d(30TG50-50G2T30), d(30T50- 50G3T30) and d(50TG30-30G2T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 50-30 backbone. Exchange of 15NH+ 4 ions between G-quadruplex and bulk solution is faster at the 30-end in comparison to the 50-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG3T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures

Improved performances of catalytic G-quadruplexes (G4-DNAzymes) via the chemical modifications of the DNA backbone to provide Gquadruplexes with double 3′-external G-quartets

Here we report on the design of a new catalytic G-quadruplex-DNA system (G4-DNAzyme) based on the modification of the DNA scaffold to provide the DNA pre-catalyst with two identical 3′-ends, known to bemore catalytically proficient than the 5′-ends. To this end, we introduced a 5′-5′ inversion of polarity site in the middle of the G4-forming sequences AG4A andAG6A to obtain d(3′AGG5′-5′GGA3′) (orAG2-G2A) and d(3′AGGG5′-5′GGGA3′) (or AG3-G3A) that fold into stable G4 whose tetramolecular nature was confirmed via nuclear magnetic resonance (NMR) and circular dichroism(CD) investigations. Both AG2-G2AandAG3-G3A display two identical external G-quartets (3′-ends) known to interact with the cofactor hemin with a high efficiency, making the resulting complex competent to performhemoprotein-like catalysis (G4-DNAzyme). A systematic comparison of the performances of modified and unmodified G4s lends credence to the relevance of the modification exploited here (5′-5′ inversion of polarity site), which represents a new chemical opportunity to improve the overall activity of catalytic G4s

Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/ target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protei

Subtyping patients with somatic tinnitus: modulation of tinnitus and history for somatic dysfunction help identify tinnitus patients with temporomandibular joint disorders

Objective: Determine in a cohort of patients with normal hearing and chronic tinnitus if self-reported history for temporomandibular joint (TMJ) dysfunction and a positive modulation of tinnitus in the TMJ region could be suggestive of an underlying TMJ disorder. Patients and Methods: The study included 226 patients presenting to the Head and Neck Service of our University Hospital. Following audiological and somatic tinnitus evaluation, patients were divided into two groups. The study group (n= 134) included subjects that met both the following criteria: A) a self-reported history for TMJ dysfunction and B) a positive modulation of tinnitus following somatic maneuvers in the TMJ region. The control group (n=92) included patients with similar demographic and tinnitus characteristics that did not meet the proposed criteria for somatic tinnitus. Afterwards, patients underwent clinical TMJ evaluation in the Service of Clinical Gnathology of our University. Results: One hundred thirty-one patients (57.9%) received a clinical diagnosis of TMJ disorder according to DC/TMD Axis I; 79.1% in the study group and 27.2% in the control group. Ninety-five (42.1%) patients were negative for TMJ disorders; 20.9% in the study group and 72.8% in the control group. A significantly higher number of TMJ disorders was found in patients in the study group compared to the control group (p<0.0001). Most patients had joint disorders (67.2%), followed by other (29.8%) and pain disorders (29%). Logistic regression analysis in the study group showed that female gender was more prevalent in patients with TMJ disorders. Conclusion: Our findings in patients with chronic tinnitus and normal hearing suggest that self-reported history for somatic dysfunction and modulation of tinnitus, when occurring simultaneously in the TMJ region, can be useful to preliminarily identify patients with TMJ disorders

Monomolecular G-quadruplex structures with inversion of polarity sites: new topologies and potentiality

In this paper, we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy and electrophoresis methods, on three oligonucleotide sequences, each containing one 3- 3 and two 5-5 inversion of polarity sites, and four G-runs with a variable number of residues, namely two, three and four (mTG2T, mTG3T andmTG4T with sequence 3-TGnT-5-5-TGnT-3-3-TGnT-5-5-TGnT-3 in which n = 2, 3 and 4, respectively), in comparison with their canonical counterparts (TGnT)4 (n = 2, 3 and 4). Oligonucleotides mTG3T and mTG4 T have been proven to form very stable unprecedented monomolecular parallel G-quadruplex structures, characterized by three side loops containing the inversion of polarity sites. Both G-quadruplexes have shown an all-syn G-tetrad, while the other guanosines adopt anti glycosidic conformations. All oligonucleotides investigated have shown a noteworthy antiproliferative activity against lung cancer cell line Calu 6 and colorectal cancer cell line HCT-116 p53−/−. Interestingly, mTG3T andmTG4T have proven to be mostly resistant to nucleases in a fetal bovine serum assay. The whole of the data suggest the involvement of specific pathways and targets for the biological activity

Exploring New Potential Anticancer Activities of the G-Quadruplexes Formed by [(GTG2T(G3T)3] and Its Derivatives with an Abasic Site Replacing Single Thymidine

In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties

Structural and Biological Features of G-Quadruplex Aptamers as Promising Inhibitors of the STAT3 Signaling Pathway

: In this paper, we investigate the structural and biological features of G-quadruplex (G4) aptamers as promising antiproliferative compounds affecting the STAT3 signalling pathway. Targeting the STAT3 protein through high-affinity ligands to reduce its levels or activity in cancer has noteworthy therapeutic potential. T40214 (STAT) [(G3C)4] is a G4 aptamer that can influence STAT3 biological outcomes in an efficient manner in several cancer cells. To explore the effects of an extra cytidine in second position and/or of single site-specific replacements of loop residues in generating aptamers that can affect the STAT3 biochemical pathway, a series of STAT and STATB [GCG2(CG3)3C] analogues containing a thymidine residue instead of cytidines was prepared. NMR, CD, UV, and PAGE data suggested that all derivatives adopt dimeric G4 structures like that of unmodified T40214 endowed with higher thermal stability, keeping the resistance in biological environments substantially unchanged, as shown by the nuclease stability assay. The antiproliferative activity of these ODNs was tested on both human prostate (DU145) and breast (MDA-MB-231) cancer cells. All derivatives showed similar antiproliferative activities on both cell lines, revealing a marked inhibition of proliferation, particularly at 72 h at 30 µM. Transcriptomic analysis aimed to evaluate STAT's and STATB's influence on the expression of many genes in MDA-MB-231 cells, suggested their potential involvement in STAT3 pathway modulation, and thus their interference in different biological processes. These data provide new tools to affect an interesting biochemical pathway and to develop novel anticancer and anti-inflammatory drugs

Dermanyssus gallinae: the long journey of the poultry red mite to become a vector

: The possibility that Dermanyssus gallinae, the poultry red mite, could act as a vector of infectious disease-causing pathogens has always intrigued researchers and worried commercial chicken farmers, as has its ubiquitous distribution. For decades, studies have been carried out which suggest that there is an association between a wide range of pathogens and D. gallinae, with the transmission of some of these pathogens mediated by D. gallinae as vector. The latter include the avian pathogenic Escherichia coli (APEC), Salmonella enterica serovars Enteritidis and Gallinarum and influenza virus. Several approaches have been adopted to investigate the relationship between D. gallinae and pathogens. In this comprehensive review, we critically describe available strategies and methods currently available for conducting trials, as well as outcomes, analyzing their possible strengths and weaknesses, with the aim to provide researchers with useful tools for correctly approach the study of the vectorial role of D. gallinae

Improved thrombin binding aptamer analogues containing inversion of polarity sites: structural effects of extra-residues at the ends

In this paper, we report the investigations, based on NMR, molecular modelling, CD measurements and electrophoresis, of thrombin binding aptamer (TBA) analogues containing an extra-residue at the 3’-end or at both the ends of the original TBA sequence, linked through 3’–3’ or 5’–5’ phosphodiester bonds. The data indicate that most of the modified aptamers investigated adopt chair-like G-quadruplex structures very similar to that of the TBA and that stacking interactions occur between the 3’–3’ or 5’–5’ extra residues and the deoxyguanosines of the upper G-tetrad. A comparison of the thermodynamic data of TBA-A and TBA-T containing a 3’–3’ extra residue and their canonical versions clearly indicates that the 3’–3’ phosphodiester bond is fundamental in endowing the modified aptamers with remarkably higher thermal stabilities than the original TBA