Design, Synthesis and Biophysical Characterisation of CPP-PNA Conjugates for Targeting Oncogenic microRNA-221.

Peptide nucleic acids (PNAs) are a promising platform targeting disease-associated microRNAs due to their high binding affinity, excellent sequence specificity and resistance to enzymatic degradation. However, their therapeutic application is often limited by inefficient cellular uptake. In this study, we present the design, synthesis and physicochemical characterisation of cell-penetrating peptide (CPP)-PNA conjugates that target the oncogenic microRNA miRNA-221-5p, as well as their preliminary biological evaluation. Anti-miRNA-221-5p PNAs functionalised for conjugation with CPPs via Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) or disulfide-mediated coupling were synthesised using solid-phase peptide synthesis. Circular dichroism and UV-melting analyses confirmed the formation of a stable, sequence-specific PNA-RNA heteroduplex with miRNA-221-5p. This demonstrated strong hybridisation and high thermodynamic stability compared to scrambled control sequences. Cell proliferation assays indicated modest but reproducible antiproliferative effects for the CPP-PNA conjugates co-incubated with chloroquine, with no evidence of acute cytotoxicity. These findings emphasise the potential of CPP-PNA systems for the intracellular delivery of antisense agents and highlight the importance of the linker nature in determining their biological performance.