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

Abstract

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.

Figure 1.

Schematic representation of the TBA G-quadruplex and chemical structure of 5-fluoro-2′-deoxyuridine (F). Guanosines in syn and anti glycosidic conformations are in dark and light grey, respectively.

Figure 2.

CD spectra at 20°C of modified TBAs and their natural counterpart (solid line) at 100 μM ODN strand concentration in a buffer solution 10 mM KH₂PO₄/K₂HPO₄, 70 mM KCl (pH 7.0). (A) TBA-F4 (dotted line) and TBA-F13 (dashed line); (B) TBA-F3 (dotted line) and TBA-F12 (dashed line); (C) TBA-F9 (dotted line) and TBA-F7 (dashed line).

Figure 3.

Aromatic and imino protons regions of the ¹H-NMR spectra (500 MHz, T = 25°C) of the modified TBAs and their natural counterpart in 10 mM KH₂PO₄/K₂HPO₄, 70 mM KCl and 0.2 mM EDTA (pH 7.0).

Figure 4.

Bottom view representations of the molecular models of the quadruplexes formed by TBA-F13 (left) and TBA-F4 (right). The structures are oriented with the 5′ and 3′-ends upward. The TGT loop and the G1-G6-G10-G15 tetrad have been omitted for clarity. Backbones and bases are depicted in coloured ‘stick’ (carbons, green; nitrogens, blue; oxygens, red; hydrogens, white; fluorine, yellow). The F13 (left) and F4 (right) residues and the respectively overlying G11 and G2 are reported in ball and sticks, in order to point out the effectiveness of the stacking interaction between them.

Figure 5.

PT values of the modified TBAs and their natural counterpart at different ODN concentrations and incubation times (A and C: 3 min.; B and D: 15 min.). See experimental section for details. ***P < 0.001 versus vehicle, °P < 0.05, °°P < 0.01, °°°P < 0.001 versus TBA (n = 3).