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.

The effect of l-thymidine, acyclic thymine and 8-bromoguanine on the stability of model G-quadruplex structures

“…On the contrary 8-Br-dG double substitutions in the TBA produced a decrease in the affinity of the TBA variants for thrombin. From these experiments we can conclude that one single modification of guanine by 8-BrdG is allowed for thrombin recognition but double modification is detrimental for thrombin binding and it confirms that thrombin binding is not dependent of the thermal stability of TBA variants as shown by other authors [17,19]. …”

“…Although there is no clear relationship between the structure and the biophysical properties in the antiproliferative behavior [16] recent research has taken this direction [17][18][19].…”

Spectroscopic Properties of Two 5′-(4-Dimethylamino)Azobenzene Conjugated G-Quadruplex Forming Oligonucleotides

“…In particular, G 8 and T 9 in the T 7 G 8 T 9 loop stack on the G-quartet at the 5′-end whereas T 4 and T 13 , in the T 12 T 13 and T 3 T 4 loops, stack on the G-quartet at the 3′-end. Structural studies, performed by means of both X-ray and NMR techniques, showed that T 4 , G 8 , T 9 , and T 13 have an important role in stabilizing the TBA-Q [ 25 , 26 , 27 , 28 ].…”

“…Ultimately, the whole set of our results constitutes a solid basis of chemical and physical knowledge about (i) the Q-folding capacity of the two sequences T30695 and TBA in comparison with those of the corresponding (4-dimethylamino)azobenzene conjugated ones, about (ii) the structural behavior of the unmodified Qs and their variants in solution with different solvents and, finally, on (iii) their photoresponsive properties. This information may be useful in any future biological and biotechnological application envisaged for these two aptamers, taking into account that both T30695 and TBA have an important biological activity, since T30695 binds to and inhibits the HIV-1 integrase at nanomolar concentrations [ 24 , 29 , 30 ], whereas TBA [ 27 , 28 , 31 , 32 , 33 , 34 ] binds to and inhibits the thrombin, causing an anticoagulant effect.…”

Development of an Efficient G‐Quadruplex‐Stabilised Thrombin‐Binding Aptamer Containing a Three‐Carbon Spacer Molecule

“…was able to improve the functionality of TBA. In the first study, they introduced 5′‐fluoro‐2′‐deoxyuridine residues to positions T4 and T13, achieving remarkable improvements in melting temperatures and anticoagulant activity . In the second study, they added an extra residue at the 3′‐end or at both ends of the original TBA sequence, linked through 3′–3′ or 5′–5′ phosphodiester bonds, resulting in strong improvements in the thermal stability of TBA …”