Troisi, Romualdo (2021) Structural and functional properties of complexes involving G-quadruplex-based DNA aptamers. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: Structural and functional properties of complexes involving G-quadruplex-based DNA aptamers
Creators:
CreatorsEmail
Troisi, Romualdoromualdo.troisi@unina.it
Date: 8 July 2021
Number of Pages: 160
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 33
Coordinatore del Corso di dottorato:
nomeemail
Lombardi, Angelinaalombard@unina.it
Tutor:
nomeemail
Sica, FilomenaUNSPECIFIED
Date: 8 July 2021
Number of Pages: 160
Keywords: Structural Biology; Oligonucleotide aptamers; Coagulation factors.
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/02 - Chimica fisica
Date Deposited: 22 Jul 2021 16:04
Last Modified: 07 Jun 2023 10:40
URI: http://www.fedoa.unina.it/id/eprint/13730

Collection description

Despite significant advances in the prevention and treatment of thrombosis, this disease is still one of the leading causes of death worldwide. The anticoagulant drugs often used to control blood coagulation in many cases show adverse effects. Oligonucleotide aptamers have shown to be alternative specific anticoagulants, which are characterized by nonimmunogenicity and nontoxicity. Interestingly, a subclass of these aptamers that adopts a G-quadruplex-based structure is able to effectively modulate the activity and the generation of the human α-thrombin (thrombin). In particular, anti-thrombin aptamers that recognize either thrombin exosite I (TBA, NU172) or exosite II (HD22) were selected. Despite their excellent anticoagulant properties, new studies expand day-to-day in order to in-depth elucidate their mechanism of action and overcome some long-standing limitations, as the short circulating half-life in vivo. The research activity carried out in the frame of this PhD project has been focused on three intriguing aspects of the interaction between thrombin or its zymogen prothrombin and aptamers adopting a G-quadruplex or a mixed duplex/quadruplex structure. First, using a repertoire of different experimental and computational techniques, the impact that the binding of HD22_27mer aptamer at the exosite II has on the binding of TBA or NU172 at exosite I, and vice versa, was investigated. In particular, the crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer, extensive molecular dynamics simulations of different thrombin/aptamer complexes, and anticoagulant activity experiments were performed. Collectively, the findings provide a clear and detailed picture of the cooperative action that TBA or NU172 and HD22_27mer exert on thrombin inhibition. Secondly, structural studies on new analogues of thrombin binding aptamers were carried out. In particular, the crystal structures of the complexes between thrombin and three TBA variants, in which Thy3 contains functional substituents at N3 of the pyrimidine heterocycle, were solved. The results suggest an explanation for their higher binding affinity toward thrombin with respect to that of TBA. Moreover, a preliminary analysis of the crystal structures of the complexes between thrombin and two TBA variants, carrying modifications at 5ʹ and 3ʹ ends, was presented. Conversely, the structural and biochemical properties of NU172 variants incorporating hexitol nucleotides were investigated in solution, indicating the modification at the Thy9 as the most promising. Finally, a preliminary investigation of the interaction between prothrombin and some anti-thrombin aptamers recognizing exosite I was carried out. In particular, a thermodynamic analysis of the binding of TBA, RE31 and NU172 aptamers to thrombin and prothrombin was performed by means of ITC experiments. The results indicate the ability of the examined aptamers to recognize prothrombin pro-exosite I with an affinity similar to that shown for thrombin exosite I. Furthermore, the ability of prothrombin to act as molecular chaperone of aptamers was revealed by CD experiments. To increase the possibility to obtain crystallographic information on aptamer-prothrombin complexes, considering the high flexibility of the wild-type protein, the prethrombin-2 intermediate and two prothrombin mutants were recombinantly produced.

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