Benigno, Daniela (2024) Design, synthesis and structural characterization of G-Quadruplex analogues with biological or catalytic properties. [Tesi di dottorato]

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Abstract

G-quadruplexes (G4s) are highly stable and polymorphic non-canonical nucleic acid secondary structures occurring naturally in specific human genomic regions, such as telomeres and promoters, participating in several cellular processes. The core unit of a G4 is a planar squared arrangement of four guanines (G-tetrad) interconnected by eight Hoogsteen hydrogen bonds. Two or more G-tetrads can stack on each other, generating the G4, which is further stabilized by cations accommodated in its central cavity. Certain exogenous G4-forming oligonucleotides serve as scaffolds of aptamers, synthetic nucleic acids able to adopt distinctive three-dimensional structures and bind, with high affinity and specificity, a wide variety of biological targets, making them a highly investigated class of compounds for therapeutic purposes. Moreover, G4s have recently been identified as catalytic species and applied in various reactions. This PhD thesis project fits perfectly into this context and is subdivided into two parts, namely Part I, focusing on G-quadruplex forming aptamers, and Part II on G-quadruplexes as DNAzymes. The studies disclosed in Part I aimed principally at designing, synthesizing and investigating the structural and biological properties of several G-rich oligonucleotide analogues in order to identify innovative aptamers with interesting pharmacological potential. Chapter I provides a general aptamer description, highlighting their various applications in different research areas. Instead, investigations focusing on different aptamers are reported in the next chapters. Chapter II concerns design, synthesis and investigation of Thrombin Binding Aptamer (TBA) oligonucleotide analogues. TBA represents one of the most investigated aptamers for its anticoagulant activity and its additional worthy antiproliferative properties. To improve thermal stability, enhance nuclease resistance and increase anticoagulant or antiproliferative activity, the effects of different chemical modifications in the G4 core or the addition of extra G-tetrads, have been evaluated. Chapter III is focused on the study of structural and biological properties of several analogues of the aptamers T30923 and T30175. They are mainly known because of their aptitude to target with high affinity the HIV-1 integrase. Furthermore, several studies have shown that the T30923 aptamer has also antiproliferative properties, while for T30175, no research concerning potential antiproliferative properties has ever been reported, although its sequence is strictly correlated to that of T30923. Therefore, in this chapter, the antiproliferative potential of analogues of the aptamers T30923 and T30175, and the involvement in their biological activity of the residues not belonging to the central core of stacked guanosines have been examined. Aptamers, due to their targeting properties and high specificity for different types of tumor cells, can also be conjugated with chemotherapeutics to form aptamer-drug conjugates (ApDCs) construct. A novel drug delivery vehicle (floxuridine, FdU), based on the conjugation of 5-fluoro-2′-deoxyuridine to the T30923 aptamer sequence, is analyzed in Chapter IV highlighting the potential for targeted and synergistic anticancer responses. Another interesting G4 aptamer is T40214, which targets Signal Transducer and Activator of Transcription 3 (STAT3) signaling. STAT3 is a key mediator of the oncogenic machinery, frequently activated in many types of human cancer. Chapter V is dedicated to designing, synthesizing and investigating oligonucleotide analogues of T40214 to improve the biological resistance and enhance the ability to affect the STAT3 biochemical pathway. Furthermore, a new G4-forming oligonucleotide with a sequence closely similar to T40214, apart from an extra cytidine in the second position, has been designed as the parent of a series of derivatives to identify novel antiproliferative aptamers. In Part II, the reported study focuses on the catalytic properties of G4‐DNAzymes. This topic is broadly introduced in Chapter I exploiting the peroxidase activity of G4-hemin complexes and G4 catalytic performances in asymmetric reactions. In this regard, the natural human telomeric G4 HT21 has been extensively utilized for enantioselective Diels-Alder, Friedel-Crafts, aldol reaction and sulfoxidation. Finally, starting from HT21, in order to improve the catalytic properties and study the role of the residues in the loops, the effect of the replacement of natural residues in the loops with chemically modified monomers has been evaluated in Chapter II.

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