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

2-aminopurine as a probe for quadruplex loop structures

Fluorescent reporter groups have served for many years as sensitive probes of macromolecular structure. Such probes can be especially useful in comparative studies such as detection of conformational changes and discrimination among structural models. Spectroscopic methods such as fluorescence are attractive because they are rapid, require small amounts of material, are nondestructive, can be carried out with commonly available equipment, and are relatively inexpensive. In addition, there is a rich library of theoretical and practical materials available to aid in data interpretation.The intrinsic fluorescence of most nucleic acids is too low to be useful in structural studies. Thus, it is necessary to incorporate a suitable reporter group to utilize fluorescence methods involving polynucleotide structure. A highly fluorescent adenine analog, 2-aminopurine, has long served in this capacity. The present article describes our use of 2-aminopurine as a probe of loop structures in quadruplex DNA. In particular, we show how knowledge of the relative intensity of 2-aminopurine emission as well as its sensitivity to exogenous quenching molecules such as acrylamide can aid in comparing crystal and solution structures of an oligonucleotide model of the human telomere and in discrimination among models containing tandem repeats of the telomeric quadruplex

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

A Super Stable Mutant of the Plant Protein Monellin Endowed with Enhanced Sweetness

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI

Binding of a type 1 RIP and of its chimeric variant to phospholipid bilayers: evidence for a link between cytotoxicity and protein/membrane interactions

Ribosome-inactivating proteins (RIPs) are enzymes, almost all identified in plants, able to kill cells by depurination of rRNAs. Recently, in order to improve resistance to proteolysis of a type 1 RIP (PD-L4), we produced a recombinant chimera combining it with a wheat protease inhibitor (WSCI). Resulting chimeric construct, named PD-L4UWSCI, in addition to present the functions of the two domains, shows also an enhanced cytotoxic action on murine cancer cells when compared to PD-L4. Since different ways of interaction of proteins with membranes imply different resulting effects on cells, in this study we investigate conformational stability of PD-L4 and PD-L4UWSCI and their interaction with membrane models (liposomes). Circular dichroism analysis and differential scanning calorimetry measurements indicate that PD-L4 and PD-L4UWSCI present high and similar conformational stability, whereas analysis of their binding to liposomes, obtained by isothermal titration calorimetry and differential scanning calorimetry, clearly indicate that chimera is able to interact with biomembranes more effectively. Overall, our data point out that WSCI domain, probably because of its flexibility in solution, enhances the chimeric protein interaction with membrane lipid surfaces without however destabilizing the overall protein structure. Analysis of interactions between RIPs or RIP based conjugates and lipid surfaces could provide novel insights in the search of more effective selective membrane therapeutics

Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2'-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the 'chair-like' conformation typical of the TBA. However, only ODNs TBA-F4: and TBA-F13: have shown a remarkable improvement both in the melting temperature (ΔTm ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications

Effects of abasic sites on structural, thermodynamic and kinetic properties of quadruplex structures

Abasic sites represent the most frequent lesion in DNA. Since several events generating abasic sites concern guanines, this damage is particularly important in quadruplex forming G-rich sequences, many of which are believed to be involved in several biological roles. However, the effects of abasic sites in sequences forming quadruplexes have been poorly studied. Here, we investigated the effects of abasic site mimics on structural, thermodynamic and kinetic properties of parallel quadruplexes. Investigation concerned five oligodeoxynucleotides based on the sequence d(TGGGGGT), in which all guanines have been replaced, one at a time, by an abasic site mimic (dS). All sequences preserve their ability to form quadruplexes; however, both spectroscopic and kinetic experiments point to sequence-dependent different effects on the structural flexibility and stability. Sequences d(TSGGGGT) and d(TGGGGST) form quite stable quadruplexes; however, for the other sequences, the introduction of the dS in proximity of the 3′-end decreases the stability more considerably than the 5′-end. Noteworthy, sequence d(TGSGGGT) forms a quadruplex where dS does not hamper the stacking between the G-tetrads adjacent to it. These results strongly argue for the central role of apurinic/apyrimidinic site damages and they encourage the production of further studies to better delineate the consequences of their presence in the biological relevant regions of the genome

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

Higher-order Quadruplex Structures

Structural studies have shown that four G-tracts along a DNA strand are the minimal requirement for intramolecular G-quadruplex formation. Longer DNA sequences containing multiple of four G-tracts could, in principle, form higher-order structures based on multiple G-quadruplex blocks. This later condition is abundantly verified for the telomeric single-stranded overhang (~ 200 nt) constituted of tens of TTAGGG repeats, thus opening new interesting questions about the structure of the “real” telomeric DNA. How many quadruplex units form in the human telomeric overhang ? Which type of quadruplex topologies ? Do they interact or not ? What about their binding properties ? Although, many of these questions are still unanswered, they recently began to be object of experimental and computational studies. The question about the existence and relevance of these higher-order quadruplex structures in the human genome is now an interesting and stimulating research topic in the quadruplex field. The recent results, the unsolved problems and the future prospective on the intramolecular telomeric higher-order quadruplex structures are the main topic of this review. Other studies on long telomeric RNA sequences and on other intramolecular (non telomeric) DNA higher order quadruplex structures are also presented