Biosensors and Nanotheranostics

Bionanotechnology, Drug Delivery, Therapeutics | online ISSN 3064-7789
83
Citations
54.1k
Views
60
Articles
Your new experience awaits. Try the new design now and help us make it even better
Switch to the new experience
Figures and Tables
RESEARCH ARTICLE   (Open Access)

Targeting mRNA G-quadruplexes with peptide nucleic acid conjugates to regulate gene expression in vivo

Zeljka Krpetic 1*, Andy Miah 1, Rosa Arrigo 1

+ Author Affiliations

Biosensors and Nanotheranostics 4 (1) 1-8 https://doi.org/10.25163/biosensors.4110002

Submitted: 02 January 2025 Revised: 04 March 2025  Published: 08 March 2025 


5895.jpg?638753826687787889

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

References


Abes, R., Arzumanov, A. A., Moulton, H. M., Abes, S., Ivanova, G. D., Iversen, P. L., Gait, M. J., & Lebleu, B. (2007). Cell-penetrating peptide-based delivery of oligonucleotides: An overview. Biochemical Society Transactions, 35, 775-779.

Alter, J., Sennoga, C. A., Lopes, D. M., Eckersley, R. J., & Wells, D. J. (2009). Microbubble stability is a major determinant of the efficiency of ultrasound and microbubble-mediated in vivo gene transfer. Ultrasound in Medicine & Biology, 35, 976-984.

Balasubramanian, S., & Neidle, S. (2009). G-quadruplex nucleic acids as therapeutic targets. Nature Reviews Drug Discovery, 8(6), 478-489.

Biffi, G., Tannahill, D., McCafferty, J., & Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 5(3), 182-186.

Borden, M., & Sirsi, S. (2009). Microbubble compositions, properties and biomedical applications. Bubble Science, Engineering & Technology, 1(1-2), 3-17.

Bugaut, A., & Balasubramanian, S. (2012). 5’-UTR RNA G-quadruplexes: Translation regulation and targeting. Nucleic Acids Research, 40, 1-15. https://doi.org/10.1093/nar/gks1234

Bugaut, A., & Balasubramanian, S. (2012). 5′-UTR RNA G-quadruplexes: Translation regulation and targeting. Nucleic Acids Research, 40(11), 4727-4741.

Burge, S., Parkinson, G. N., Hazel, P., Todd, A. K., & Neidle, S. (2006). Quadruplex DNA: Sequence, topology, and structure. Nucleic Acids Research, 34(19), 5402-5415.

Chaudhuri, R., Lemos, B., & Tyagi, S. (2020). RNA G-quadruplexes and their potential as therapeutic targets. Nucleic Acids Research, 48(6), 2175–2186.

Chen, Y., Yang, D., & Chen, Y. (2018). Targeting oncogenic RNA G-quadruplex structures. Theranostics, 8(16), 4095-4106.

Di Antonio, M., McLuckie, K. I., Biffi, G., & Balasubramanian, S. (2020). Targeting RNA G-quadruplex structures with peptide nucleic acids. Angewandte Chemie International Edition, 59(5), 1680-1685.

Garg, T., Goyal, A. K., & Rath, G. (2018). Microbubbles: An evolving paradigm in therapeutics. Journal of Controlled Release, 279, 674-691.

Hänsel-Hertsch, R., Di Antonio, M., & Balasubramanian, S. (2017). DNA G-quadruplexes in the human genome: Detection, functions, and therapeutic potential. Nature Reviews Molecular Cell Biology, 18(5), 279-284.

Kumari, S., Bugaut, A., & Balasubramanian, S. (2008). Position and stability are determining factors for translation repression by an RNA G-quadruplex-forming sequence within the 5’ UTR of the NRAS proto-oncogene. Biochemistry, 47, 12664–12669. https://doi.org/10.1021/bi801365t

López-Méndez, B., García-López, A., & Cernaianu, A. (2016). Structural analysis of RNA G-quadruplexes in translation regulation. Journal of Biomolecular Structure and Dynamics, 34(2), 291–300.

Masiero, S., Trotta, R., Pieraccine, S., De Tito, S., Perone, R., Randazzo, A., & Spada, G. P. (2010). A non-empirical chromophoric interpretation of CD spectra of DNA G-quadruplex structures. Organic & Biomolecular Chemistry, 8(12), 2683-2692. https://doi.org/10.1039/c001545a

Meguellati, K., Koripelly, G., & Ladame, S. (2012). DNA-templated synthesis of trimethine cyanine dyes: A versatile fluorogenic reaction for sensing G-quadruplex formation. Angewandte Chemie International Edition, 49, 2738-2742.

Nielsen, P. E. (2007). Peptide nucleic acids and the origin and homochirality of life. Origins of Life and Evolution of Biospheres, 37, 323-328.

Nielsen, P. E. (2010). Peptide nucleic acids (PNA) in chemical biology and drug discovery. Chemistry & Biodiversity, 7, 786-804. https://doi.org/10.1002/cbdv.201000008

Pagano, B., Mattia, C. A., & Giancola, C. (2009). Applications of isothermal titration calorimetry in biophysical studies of G-quadruplexes. International Journal of Molecular Sciences, 10, 2935-2957.

Paul, A., Poulami, S., Yamuna, K., & Ladame, S. (2008). Combining G-quadruplex targeting motifs on a single peptide nucleic acid scaffold: A hybrid (3+1) PNA-DNA bimolecular quadruplex. Chemistry, 14, 8682-8689.

Vorlícková, M., Kejnovská, I., Sagi, J., Renciuk, D., Bednárová, K., Motlová, J., & Kypr, J. (2012). Circular dichroism and guanine quadruplexes. Methods, 57(1), 64-75. https://doi.org/10.1016/j.ymeth.2012.01.007

Yu, H., Guo, X., & Su, J. (2021). Peptide nucleic acid targeting of G-quadruplexes to regulate oncogenic translation. Biochemical Pharmacology, 188, 114570.


Article metrics
View details
4
Downloads
0
Citations
1332
Views
📖 Cite article

View Dimensions


View Plumx


View Altmetric



4
Save
0
Citation
1332
View
0
Share