An acyclic pyrimidine analogue, containing a five member cycle fused on the pyrimidine ring, was synthesized and introduced at position 7 or 12 of the 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG, known as Thrombin Binding Aptamer (TBA). Characterization by 1H-NMR and CD spectroscopies of the resulting aptamers, TBA-T7b and TBA-T12b, showed their ability to fold into the typical antiparallel chair-like G-quadruplex structure formed by TBA. The apparent CD melting temperatures indicated that the introduction of the acyclic residue, mainly at position 7, improves the thermal stability of resulting G-quadruplexes with respect to TBA. The anticoagulant activity of the new molecules was then valued in PT assay and it resulted that TBA-T7b is more potent than TBA in prolonging the clotting time. On the other hand, in purified fibrinogen assay the thrombin inhibitory activity of both modified sequences resulted lower than that of TBA using human enzyme, while the potency trend was again reversed using bovine enzyme. Obtained structure-activity relationships were investigated by structural and computational studies. Taken together, our results reveal the active role of TBA residues T7 and T12 and the relevance of some amminoacids located in the anion binding exosite I of the protein in aptamer-thrombin interaction.

Investigating the role of T7 and T12 residues on biological properties of Thrombin Binding Aptamer: enhancement of anticoagulant activity by a single nucleobase modification / Borbone, Nicola; Bucci, Mariarosaria; Oliviero, Giorgia; Morelli, Elena; Amato, Jussara; D'Atri, Valentina; D'Errico, Stefano; Vellecco, Valentina; Cirino, Giuseppe; Piccialli, Gennaro; Fattorusso, Caterina; Varra, Michela; Mayol, Luciano; Persico, Marco; Scuotto, Maria. - In: JOURNAL OF MEDICINAL CHEMISTRY. - ISSN 0022-2623. - 55:23(2012), pp. 10716-10728. [10.1021/jm301414f]

Investigating the role of T7 and T12 residues on biological properties of Thrombin Binding Aptamer: enhancement of anticoagulant activity by a single nucleobase modification

BORBONE, NICOLA;BUCCI, MARIAROSARIA;OLIVIERO, GIORGIA;MORELLI, ELENA;AMATO, JUSSARA;D'ATRI, VALENTINA;D'ERRICO, STEFANO;VELLECCO, VALENTINA;CIRINO, GIUSEPPE;PICCIALLI, GENNARO;FATTORUSSO, CATERINA
;
VARRA, MICHELA
;
MAYOL, LUCIANO;PERSICO, MARCO;SCUOTTO, MARIA
2012

Abstract

An acyclic pyrimidine analogue, containing a five member cycle fused on the pyrimidine ring, was synthesized and introduced at position 7 or 12 of the 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG, known as Thrombin Binding Aptamer (TBA). Characterization by 1H-NMR and CD spectroscopies of the resulting aptamers, TBA-T7b and TBA-T12b, showed their ability to fold into the typical antiparallel chair-like G-quadruplex structure formed by TBA. The apparent CD melting temperatures indicated that the introduction of the acyclic residue, mainly at position 7, improves the thermal stability of resulting G-quadruplexes with respect to TBA. The anticoagulant activity of the new molecules was then valued in PT assay and it resulted that TBA-T7b is more potent than TBA in prolonging the clotting time. On the other hand, in purified fibrinogen assay the thrombin inhibitory activity of both modified sequences resulted lower than that of TBA using human enzyme, while the potency trend was again reversed using bovine enzyme. Obtained structure-activity relationships were investigated by structural and computational studies. Taken together, our results reveal the active role of TBA residues T7 and T12 and the relevance of some amminoacids located in the anion binding exosite I of the protein in aptamer-thrombin interaction.
2012
Investigating the role of T7 and T12 residues on biological properties of Thrombin Binding Aptamer: enhancement of anticoagulant activity by a single nucleobase modification / Borbone, Nicola; Bucci, Mariarosaria; Oliviero, Giorgia; Morelli, Elena; Amato, Jussara; D'Atri, Valentina; D'Errico, Stefano; Vellecco, Valentina; Cirino, Giuseppe; Piccialli, Gennaro; Fattorusso, Caterina; Varra, Michela; Mayol, Luciano; Persico, Marco; Scuotto, Maria. - In: JOURNAL OF MEDICINAL CHEMISTRY. - ISSN 0022-2623. - 55:23(2012), pp. 10716-10728. [10.1021/jm301414f]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/515145
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