Rotondo, Martina Design and Development of Molecular Platforms for Targeting Nucleic Acids in Cancer. [Tesi di dottorato]

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Abstract

This thesis investigates the development and characterisation of peptide- and small molecule-based systems designed to recognise and modulate nucleic acids. The unifying focus is on molecular mechanisms relevant to cancer biology and therapeutic innovation. An integrated strategy combining synthetic organic chemistry, peptide chemistry, circular dichroism (CD), fluorescence spectroscopy, surface plasmon resonance, nuclear magnetic resonance (NMR) and cellular assays is employed to explore how molecular architecture and environmental conditions dictate the recognition of oncogenic nucleic acid targets. The first section of the thesis addresses the synthesis of Peptide Nucleic Acid (PNA) conjugates functionalised with Cell-Penetrating Peptides (CPPs) for modulating the activity of the oncogenic microRNA miR-221-5p, which is a key regulator involved in tumour progression and resistance pathways. CPP-PNA conjugates were obtained via CuACC and disulfide coupling strategies and evaluated in terms of internalisation and biological activity. Although hybridisation capability was preserved, internalisation assays revealed limitations related to aggregation that highlight the need for improved formulation. Nevertheless, these results establish a solid methodological basis for the future refinement of PNA-based gene-silencing tools. The second part of the study examined peptide fragments from Vimentin, an intermediate filament protein that plays a central role in epithelial-mesenchymal transition (EMT), cell motility, and metastatic dissemination. CD and NMR analyses revealed that Vimentin-derived peptides adopt different conformational states under membrane-mimetic conditions. Alanine-scanning variants revealed the critical importance of aromatic and basic residues in mediating interactions with G-quadruplex (G4) DNA – noncanonical structures associated with the transcriptional regulation of oncogenes – thus offering insight into how the intrinsically disordered regions of Vimentin may recognise nucleic acids in contexts relevant to cancer. The third part extends the investigation of G4 recognition to tetrazole-based small molecules and the peptide Rhau25, employing high-throughput synchrotron radiation circular dichroism (HT-SRCD) – a label-free technique ideally suited to rapid ligand screening. These studies revealed that ligand accessibility and induced structural changes are strongly influenced by ionic conditions, with Na+ promoting more flexible G4 conformations conductive to binding. Several tetrazole derivatives were found to selectively perturbed telomeric and regulatory G4s, while Rhau25 robustly remodelled G4 topologies. These findings identify promising hit compounds for the future development of G4-targeting anti-cancer ligands and validate HT-SRCD as an efficient discovery tool. Overall, this thesis integrates synthetic chemistry with structural and biophysical analysis to provide new mechanistic insights and methodological foundations for developing next-generation nucleic acid-targeting agents for use in cancer therapeutics.

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