D'Atri, Valentina (2013) Synthesis and Structural Studies on DNA G-Quadruplexes: from Nucleic Acid Aptamers to Higher Order Assemblies. [Tesi di dottorato]

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Abstract

Quadruple helices, or G-quadruplexes, are DNA secondary structures found in guanine rich oligonucleotide sequences, having a natural propensity to self-associate in coplanar arrays of four guanines, stabilized by Hoogsteen hydrogen bonding. The scientific interest towards these particular DNA structures is mainly due to the presence of guanine rich domains, potentially able to form G-quadruplexes, in important regions of the human genome, as gene promoters and telomeres, and to the fact that the G-quadruplexes can constitute the scaffold of aptamers. Aptamers are short DNA or RNA fragments capable to bind with high affinity specific proteins, as for example thrombin or HIV-proteins. On these grounds, aptamers-based synthetic oligonucleotides can represent a new class of pharmacologically interesting molecules, characterized by a high selectivity of action. Furthermore, the Gquadruplexes can have a potential use in nanotechnology. As a matter of fact, the overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligonucleotide strands, which can form higher-order assemblies by multimerization between G-quadruplex units. In these contests, my research studies have been mainly focused on: i) Investigation of the most prominent biological properties of selected Gquadruplexes and identification of bioactive sequences able to play a biological role as aptamers; and ii) Elucidation of the sequence-specific thermodynamic stability and physicalchemical mechanisms that underlie the G-quadruplexes multimerization. Specifically, during my PhD research studies, I focused on anti-HIV aptamers, and Thrombin Binding Aptamer (TBA) with the aim to identify the structural features required for their biological activity and reaching the way to improve them. Furthermore, I identify the CGGXGGT oligonucleotide sequence (where X = A, C, G, or T) able to lead to the formation of higher order G-quadruplex assemblies, that can potentially be used as DNA-based nanostructures. In order to achieve these objectives, it has been necessary to exploit the synthesis and the structural characterization of quadruplex-forming oligonucleotides, the analysis of the sequence-specific thermodynamic stability, the physical-chemical properties and the structural features of resulting G-quadruplexes. Furthermore, as regard the studies about aptamers, the evaluation of the binding properties to the selected proteins, and bioassays to ascertain the biological properties of the synthesized complexes have been undertaken.

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