Targeting mRNA G-quadruplexes with peptide nucleic acid conjugates to regulate gene expression in vivo
Zeljka Krpetic 1*, Andy Miah 1, Rosa Arrigo 1
Biosensors and Nanotheranostics 4(1) 1-8 https://doi.org/10.25163/biosensors.4110002
Submitted: 02 January 2025 Revised: 07 March 2025 Published: 08 March 2025
Abstract
Background: Guanine-rich nucleic acids can fold into stable secondary structures known as G-quadruplexes (G-4) in the presence of cations. These structures are commonly found in the 5′-untranslated region (5′-UTR) of many genes, where they can regulate translation. The N-RAS proto-oncogene, which plays a critical role in neuroblastoma RAS viral RNA translation, contains an RNA G-quadruplex in its 5′-UTR, making it a potential target for therapeutic intervention. Methods: This study investigated the stabilization of the RNA G-quadruplex in the 5′-UTR of N-RAS using PNA1-Heterocycle-PNA2 conjugates. Biophysical techniques, including Circular Dichroism (CD) spectroscopy and Isothermal Titration Calorimetry (ITC), were employed to assess the binding affinity and stabilization effect of the conjugates on the RNA G-quadruplex. Additionally, in vitro translation assays were conducted to evaluate the impact of G-quadruplex stabilization on oncogenic protein expression. Results: The results demonstrated that PNA1-Heterocycle-PNA2 conjugates effectively stabilized the G-quadruplex by targeting both the quadruplex structure and adjacent single-stranded RNA regions with sequence specificity. CD spectroscopy confirmed the structural stabilization of the RNA G-quadruplex, while ITC analysis revealed strong binding interactions. Furthermore, in vitro assays showed a significant reduction in oncogenic protein expression upon G-quadruplex stabilization. Conclusion: This study highlights the potential of PNA-conjugates as therapeutic agents for gene modulation. By stabilizing the RNA G-quadruplex in the 5′-UTR of the N-RAS transcript, these conjugates can suppress oncogenic protein translation, offering a novel approach for targeted cancer therapy.
Keywords: G-quadruplex, RNA stability, peptide nucleic acid, gene regulation, oncogene translation
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