Platform-dependent performance of P2A and IRES linkers in multicistronic gene expression

Efficient co-expression of multiple genes within individual cells is essential for advances in biotechnology and clinical applications. While 2 A peptides and internal ribosome entry sites (IRESs) are widely used linkers, their performance in synthetic mRNA systems—particularly with modified nucleotides—is insufficiently characterized compared to DNA-based platforms. We systematically compared bicistronic gene expression mediated by ribosome skipping peptide P2A and four viral IRES types across plasmid DNA and synthetic mRNA platforms. Plasmids were engineered with two open reading frames linked by P2A or four distinct IRES types. mRNAs were generated via in vitro transcription with or without N1-methylpseudouridine (m1Ψ) substitution. Expression efficiency was quantified through dual-luciferase assays, using firefly luciferase as a control. We observed distinct linker-dependent expression patterns between DNA and RNA platforms. P2A generally provided superior overall protein expression in both plasmid DNA and m1Ψ –modified mRNA systems, with the exception of unmodified mRNA, in which P2A and the poliovirus (PV; type I) IRES produced comparable downstream gene expression. Full substitution of uridine with m1Ψ markedly impaired protein expression driven by all IRES. Notably, even partial m1Ψ (25%) was sufficient to disrupt type I IRES activity, whereas P2A constructs displayed a gradual increase in protein output with increasing levels of modification. These results indicate that nucleotide modifications fundamentally alter linker performance. Our study highlights the importance of linker-dependent effects in the design of RNA-based expression systems, providing insights for optimizing multi-gene expression strategies. These findings may contribute to the development of next-generation mRNA-based therapeutics, including vaccines.