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

Backbone modified TBA analogues endowed with antiproliferative activity

Background: Thrombin binding aptamer (TBA) is endowed with antiproliferative properties but its potential development is counteracted by concomitant anticoagulant activity. Methods: Five oligonucleotides (ODNs) based on TBA sequence (GGTTGGTGTGGTTGG) and containing L-residues or both L-residues and inversion of polarity sites have been investigated by NMR and CD techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay), and their resistance in fetal bovine serum have been tested. Results: CD and NMR data suggest that investigated ODNs are able to form right- and left-handed G-quadruplex structures. All ODNs do not retain anticoagulant activity characteristic of TBA but are endowed with a significant antiproliferative activity against two cancerous cell lines. Their resistance in biological environment after six days is variable, depending on ODN. Conclusions: A comparison between results and literature data suggests that antiproliferative activity of TBA analogues investigated could depends on two factors: a) biological pathways and targets different from those already identified or proposed for other antiproliferative G-quadruplex aptamers, and b) contribution of the guanine-based degradation products. General significance: Modified TBA analogues containing L-residues and inversion of polarity sites lose the anticoagulant activity but gain antiproliferative properties against two cancer cell lines

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

Synthesis, structural studies and biological properties of new TBA analogues containing an acyclic nucleotide

A new modified acyclic nucleoside, namely N(1)-(3-hydroxy-2-hydroxymethyl-2-methylpropyl)-thymidine, was synthesized and transformed into a building block useful for oligonucleotide (ON) automated synthesis. A series of modified thrombin binding aptamers (TBAs) in which the new acyclic nucleoside replaces, one at the time, the thymidine residues were then synthesized and characterized by UV, CD, MS, and (1)H NMR. The biological activity of the resulting TBAs was tested by Prothrombin Time assay (PT assay) and by purified fibrinogen clotting assay. From a structural point of view, nearly all the new TBA analogues show a similar behavior as the unmodified counterpart, being able to fold into a bimolecular or monomolecular quadruplex structure depending on the nature of monovalent cations (sodium or potassium) coordinated in the quadruplex core. From the comparison of structural and biological data, some important structure-activity relationships emerged, particularly when the modification involved the TT loops. In agreement with previous studies we found that the folding ability of TBA analogues is more affected by modifications involving positions 4 and 13, rather than positions 3 and 12. On the other hand, the highest anti-thrombin activities were detected for aptamers containing the modification at T13 or T12 positions, thus indicating that the effects produced by the introduction of the acyclic nucleoside on the biological activity are not tightly connected with structure stabilities. It is noteworthy that the modification at T7 produces an ON being more stable and active than the natural TBA

Spectroscopic properties of two 5′-(4-dimethylamino)azobenzene conjugated G-quadruplex forming oligonucleotides

The synthesis of two 5'-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5'-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5'-end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer

Outstanding effects on antithrombin activity of modified TBA diastereomers containing an optically pure acyclic nucleotide analogue

Herein, we report optically pure modified acyclic nucleosides as ideal probes for aptamer modification. These new monomers offer unique advantages in exploring the role played in thrombin inhibition by a single residue modification at key positions of the TBA structure

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13

Aptamer-Based Biosensors for the Analytical Determination of Bisphenol A in Foodstuffs

Bisphenol A (BPA) is a synthetic compound utilized to manufacture plastics for Food Contact Materials (FCMs) or resins for the inside of food containers. Since it was recognized as an Endocrine-Disrupting Chemical (EDC), its implications in pathologies, such as cancer, obesity, diabetes, immune system alterations, and developmental and mental disorders, have been widely documented. Diet is considered the main source of exposure for humans to BPA. Consequently, continuous monitoring of the levels of BPA in foods is necessary to assess the risk associated with its consumption in one’s diet. So far, many reviews have been published on biosensors and aptamer-based biosensors, but none of them focus on their applications in their analyses of bisphenols in food matrices. With this review, the authors aim to fill this gap and to take a snapshot of the current state-of-the-art research on aptasensors designed to detect BPA in food matrices. Given that a new TDI value has recently been proposed by the EFSA (0.04 ng/kg), the search for new sensitive tools for the quantitative analysis of BPA is more topical and urgent than ever. From this perspective, aptasensors prove to be a good alternative to traditional analytical techniques for determining BPA levels in food