Non-small cell lung cancer (NSCLC) requires biomarkers that are both sensitive and actionable, as well as network-level therapies. In this context, microRNAs (miRs) have emerged as a unique bridge to meet these needs: their dysregulated signatures discriminate healthy individuals from NSCLC patients, while serving as regulators, each miR can coordinate multiple gene targets, making them compelling therapeutic levers. Leveraging this duality, the present thesis harnesses that property to test a “one-hit, multiple-target” strategy centered on miRs (miR-21, miR-155-3p, miR-3196), integrating sensitive detection with therapeutic modulation. To this end, multiple miRs were initially profiled in NSCLC liquid biopsies (LBs). Subsequently, molecular beacons (MBs) were engineered and qualified for the direct detection of two NSCLC-associated miRs (miR-21/miR-155) in biological samples, laying the groundwork for a diagnostic platform; additionally, one probe was integrated into a microchip, enabling a point-of-care diagnostic prototype. Therapeutically, miR-155-3p was silenced with an antisense strategy, and miR-3196 was reactivated by destabilizing a G-quadruplex (G4) within its precursor using a G4 ligand (phenylpyrrolocytosine; PhpC). To enable efficient intracellular delivery, gold nanoparticles (AuNPs) were developed to deliver both oligonucleotides and PhpC ligand, with complete physicochemical and cellular characterization. Overall, the expression profile analysis identified upregulation of miR-21/miR-155 and downregulation of miR-3196, with clinical correlations. The MB assays achieved high specificity and low-nanomolar sensitivity, enabling rapid readouts. Additionally, the AuNPs targeting miR-155-3p produced efficient cellular uptake and robust knockdown, attenuating oncogenic signaling and outperforming lipid transfection. Moreover, to the best of our knowledge, we have identified, for the first time, a G4-forming region within the human precursor of miR-3196 that restricts its maturation. Subsequently, using the G4 ligand, PhpC, unfolded this structure and restored mature miR-3196. In addition, PhpC-functionalized AuNPs preserved activity, enhanced intracellular persistence, and maintained low acute cytotoxicity across the tested ranges. These studies establish a theragnostic framework for NSCLC: MBs enable minimally invasive detection and real-time pharmacodynamic tracking, while miR modulation, via miR-155 inhibition and G4-guided rescue of miR-3196, may yield broad antitumor effects from a single molecular hit. Furthermore, the AuNP delivery overcomes key barriers to nucleic-acid and small-molecule therapeutics. Collectively, the work advances a translatable, miR-centered one-hit/multi-target paradigm and outlines clear next steps toward in vivo validation and clinical integration.

RNA G-quadruplexes (G4s) are promising drug targets due to their high cellular abundance. G-rich RNA regions inherently form G4 structures, while GC-rich sequences adopt stem-loop conformations, and their dynamic equilibrium critically influences RNA function. MicroRNAs (miRs), key regulators of protein expression, undergo processing by Dicer, which specifically recognizes stem-loop structures in precursor miRs (pre-miRs). Notably, some pre-miRs containing G4-forming sequences influence Dicer cleavage, suggesting that G4s can directly regulate miR production. Moreover, pre-miRs with G4 structures present promising targets for small molecules. This research focuses on identifying and modulating G4 structure in pre-miR-3196 to restore normal lung cancer (LC) levels, offering a potential therapeutic strategy. Firstly, bioinformatic analyses indicated the presence of G4 motifs in pre-miR-3196. We then demonstrated in vitro that this RNA sequence folds into stable G4s by a combination of biophysical and biochemical assays. Then, we demonstrated the formation of these G4s in human cancer cells by confocal imaging before showing that these G4s can be modulated using the RNA G4 destabilizer PhpC, which impacts the miR-3196 biogenesis. These findings highlighted the possibility of using G4s to control the expression of mature miR-3196 and revealed the potential of using the destabilizer PhpC to adjust its G4 structure.

Lung cancer (LC) is the leading cause of cancer-related death worldwide. Although the diagnosis and treatment of non-small cell lung cancer (NSCLC), which accounts for approximately 80% of LC cases, have greatly improved in the past decade, there is still an urgent need to find more sensitive and specific screening methods. Recently, new molecular biomarkers are emerging as potential non-invasive diagnostic agents to screen NSCLC, including multiple microRNAs (miRNAs) that show an unusual expression profile. Moreover, peripheral blood mononuclear cells’ (PBMCs) miRNA profile could be linked with NSCLC and used for diagnosis. We developed a molecular beacon (MB)-based miRNA detection strategy for NSCLC. Following PBMCs isolation and screening of the expression profile of a panel of miRNA by RT-qPCR, we designed a MB targeting of up-regulated miR-21-5p. This MB 21-5p was characterized by FRET-melting, CD, NMR and native PAGE, allowing the optimization of an in-situ approach involving miR-21-5p detection in PBMCs via MB. Data show the developed MB approach potential for miR-21-5p detection in PBMCs from clinical samples towards NSCLC.