Investigating the Role of T7 and T12 Residues on the Biological Properties of Thrombin-Binding Aptamer: Enhancement of Anticoagulant Activity by a Single Nucleobase Modification

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• 作者：Nicola Borbone ; Mariarosaria Bucci ; Giorgia Oliviero ; Elena Morelli ; Jussara Amato ; Valentina D鈥橝tri ; Stefano D鈥橢rrico ; Valentina Vellecco ; Giuseppe Cirino ; Gennaro Piccialli ; Caterina Fattorusso ; Michela Varra ; Luciano Mayol ; Marco Persico ; Maria Scuotto
• 刊名：Journal of Medicinal Chemistry
• 出版年：2012
• 出版时间：December 13, 2012
• 年：2012
• 卷：55
• 期：23
• 页码：10716-10728
• 全文大小：633K
• 年卷期：v.55,no.23(December 13, 2012)
• ISSN：1520-4804

文摘

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 chairlike 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 clotting time. On the other hand, in purified fibrinogen assay the thrombin inhibitory activity of both modified sequences was lower than that of TBA using human enzyme, whereas the potency trend was again reversed using bovine enzyme. Obtained structure鈥揳ctivity relationships were investigated by structural and computational studies. Taken together, these results reveal the active role of TBA residues T₇ and T₁₂ and the relevance of some amino acids located in the anion binding exosite I of the protein in aptamer鈥搕hrombin interaction.