Criscuolo, Andrea (2025) Exploring multivalency effects in G-quadruplex-forming aptamers for diagnostics and theranostics. [Tesi di dottorato]

Documento PDF PhD_thesis_Andrea_Criscuolo.pdf Visibile a [TBR] Amministratori dell'archivio Download (6MB) | Richiedi una copia

Abstract

This doctoral research explored the design, synthesis, characterization and functional optimization of G-quadruplex-forming DNA aptamers targeting High Mobility Group Box 1 (HMGB1), a multifunctional chromatin-associated protein implicated in inflammation, cancer, and immune regulation. Considering the intrinsic stability and tunability of G-quadruplex (G4) architectures, this project aimed to develop nucleic acid-based tools with high affinity, selectivity, and biological efficacy against extracellular HMGB1. Through a SELEX-inspired selection strategy directed toward guanine-rich sequences derived from the telomeric truncation tel26, a family of novel G4-forming aptamers was identified and systematically characterized by spectroscopic, chromatographic, and biological assays. Among them, the aptamer named L12 emerged as the lead candidate, exhibiting exceptional thermodynamic stability and enzymatic resistance, along with affinity for HMGB1 in the low nanomolar range. Further investigations revealed that L12 spontaneously forms parallel dimeric G4 assemblies, whose properties are significantly enhanced compared to its monomeric form in terms of both protein recognition and inhibitory potency. Cellular assays confirmed its ability to suppress HMGB1-induced fibroblast and cancer cell migration without producing cytotoxic effects. Subsequent studies demonstrated that thermal annealing, commonly used to promote structural homogeneity, irreversibly disrupts the dimeric G4 organization, leading to monomeric species with reduced stability and activity, highlighting the functional importance of non-covalent parallel dimeric G4 structures. To stabilize this architecture, a series of covalently linked L12 dimers were rationally designed using synthetic linkers of variable length and nature. These constructs preserved the favourable spatial arrangement of the constituting G4 domains, yielding improved conformational homogeneity, higher affinity for the target protein, and enhanced enzymatic resistance. Furthermore, photoactive G4 ligands were explored to achieve light-controlled covalent stabilization of the preformed non-covalent dimeric species, establishing a proof of concept for ligand-photoresponsive aptamer systems. To expand applicability toward therapeutic delivery and biosensing, the L12 aptamer was also anchored onto biocompatible silica nanoparticles, generating hybrid nanoconjugates that integrate molecular recognition with nanoscale engineering. These aptamer–functionalised nanoparticle systems exploit multivalency and cooperative binding, offering improved stability and targeting efficiency in complex biological environments. Overall, this PhD thesis establishes a structure-driven framework for the development of functional G-quadruplex-based aptamers and demonstrates how multivalency, obtained either by covalent stabilization, or nano conjugation can synergistically enhance the biochemical and biological performance. The here described findings lay the groundwork for next generation theranostic platforms targeting biologically relevant targets and related biomolecular pathways, where tailor-made nucleic acid design, photochemistry, and nanotechnology converge to achieve selective and controllable molecular recognition.

Downloads

Actions (login required)

Modifica documento