Napolitano, Ettore (2023) G-quadruplex-forming aptamers for diagnostics and theranostics in anticancer strategies. [Tesi di dottorato]

Anteprima

Testo napolitano_ettore_35.pdf Download (4MB) | Anteprima

Abstract

In this PhD thesis, I focused on the development of innovative tools based on G-quadruplex forming oligonucleotide aptamers recognizing specific proteic markers for early detection and treatment of tumours and/or inflammations. In the first part of this work, I investigated V7t1 and 3R02, two G-quadruplex forming aptamers well known to recognize the Vascular Endothelial Growth Factor 165 (VEGF165) – an angiogenic protein, overexpressed in cancer cells, involved in growth and metastases of solid tumours – in their interaction with a novel cyanine dye, designed as an analogue of thiazole orange, here named CyOH. With an in-depth biophysical analysis comprising techniques like gel electrophoresis, circular dichroism (CD), UV-vis and fluorescence spectroscopy, I demonstrated that CyOH was able to interact with the two selected aptamers, giving in both cases a marked fluorescence light-up exclusively when bound to their dimeric forms. Interestingly, both V7t1 and 3R02 recognized VEGF165 with higher affinity in the conditions that allowed them structuring into dimeric G-quadruplexes, i.e. after simple dilution in a Na+-rich pseudo-physiological buffer, mimicking the extracellular environment, without any thermal treatment. Moreover, the fluorescent light-up of the probe was unaltered when the dimeric aptamer-CyOH complexes bound to the target protein. These complexes, tested on MCF-7 cancer cells using non-tumorigenic MCF-10A cells as control, showed an efficient internalization and colocalization with a fluorescently-labelled anti-VEGF-A antibody, which allowed both recognition and detection of the target. Our experiments clearly showed the potential of the studied systems as tools for anticancer theranostic strategies, combining the therapeutic potential of the G-quadruplex forming anti-VEGF aptamers with the diagnostic efficacy of the selective fluorescence light-up produced by CyOH. Novel V7t1 modified aptamers carrying pyridine-containing ligandosides were also synthesized with the aim of favouring the VEGF recognition after metal-induced dimerization. Biophysical characterization was carried out, obtaining preliminary results. However, a deeper analysis will be necessary to find the best conditions to reach the set goal. In the second part of the thesis, I focused on the development of aptameric systems selective for High-Mobility Group Box 1 (HMGB1), a cytokine involved in the pathogenesis of various inflammatory and immune diseases, as well as cancer. In the search for novel, effective anti-HMGB1 aptamers, we relied on the SELEX (Systematic Evaluation of Ligands by EXponential enrichment) technology, identifying 14 G-quadruplex forming aptamers from a properly designed library of guanine-rich oligonucleotides. By using UV-vis spectroscopy, CD, gel electrophoresis and size exclusion chromatography techniques, we demonstrated that the selected sequences, in pseudo-physiological conditions mimicking the extracellular environment, were able to fold into stable G-quadruplex structures, showing high polymorphism both in terms of G-quadruplex topology (parallel and hybrid G-quadruplex conformations typically coexist) and molecularity (both monomeric and dimeric species were found). In properly designed binding assays, some of these oligonucleotides showed very high affinity against the target HMGB1. Moreover, in vitro cellular assays on NIH3T3 fibroblasts showed, for these aptamers, an ability to inhibit the protein-induced cell migration directly related to their affinity for the target protein. Interestingly, in accordance with the findings obtained in the first part of this thesis, the best conditions for protein inhibition were the ones allowing the aptamers to adopt dimeric G-quadruplexes, i.e. dilution in a Na+-rich pseudo-physiological buffer without any thermal treatment. This is particularly interesting because it goes against the dogma of the “annealing procedure” typically used in literature to let the aptamers correctly fold into the specific three-dimensional shape for target interaction. Particularly valuable were the results obtained on the so-called L12 aptamer, whose dimeric form proved to be very active as HMGB1 inhibitor in chemotaxis assays (IC50 values in the low nM range), providing a useful lead candidate for future in vivo studies.

Downloads

Actions (login required)

Modifica documento