Structural Optimization of Guanidinium-Linked Morpholino Phosphorodiamidate Morpholino Oligonucleotide Chimeras to Improve In Vitro Antisense Efficacy

Although phosphorodiamidate morpholino oligomers (PMOs) are potent and stable antisense agents, their neutral backbone limits cellular uptake and biodistribution, requiring specialized delivery strategies to improve therapeutic efficacy. Chimeric guanidinium-linked morpholino oligomers (GMOs) with PMOs are designed to enhance cellular delivery yet compromise hybridization with the target sequence due to backbone rigidity. To address this, we designed flexible GMO-PMO chimeras by incorporating sarcosine, β-alanine, and ethanamine as internucleotide spacers in the oligonucleotide. Thermal melting studies on a 19-mer polythymidine (polyT) sequence demonstrate that these flexible GMO-PMOs partially restore duplex stability in a modification-dependent manner. This structural restoration is also observed in biologically relevant mixed GMO-PMO sequences targeting Nanog, a gene linked to stemness and cancer biology, while retaining their B-type global geometry as confirmed by circular dichroism analysis. The optimized ethanamine-modified Nanog chimera elicited strong, dose-dependent silencing, with up to 95% repression in MCF7 and ∼73% in HCT116 cells at 1 μM after 48 h. Importantly, both rigid and flexible GMO-PMOs exhibit similar lysosomal sequestration, varying in a cell-line-dependent manner. Collectively, flexible GMO-PMO chimeras mitigate the long-standing trade-off between carrier-free PMO delivery and duplex stability, establishing a robust platform for antisense gene silencing and potential therapeutic applications.