C-terminal modification of carrier peptides affects transient gene expression in mitochondria

Artificial peptide-DNA complexes enable the transient expression of transgenes in mitochondria; however, the mechanism by which DNA is released from such complexes after delivery remains poorly understood. We previously established particle bombardment-assisted peptide-mediated gene transfer as a method for DNA delivery in plant mitochondria. In this study, we examined the effects of C-terminal modification of a mitochondria-targeting carrier peptide on transient gene expression in mitochondria. Plasmid DNA encoding NanoLuc (Nluc) was complexed with C-terminally amidated cytcox-KH (cytcox-KH-NH₂) or non-amidated cytcox-KH (cytcox-KH-COOH) and delivered to Brassica campestris leaves. Introduction of the cytcox-KH-COOH-based complex considerably elevated transient Nluc activity compared to that of the cytcox-KH-NH₂-based complex. Both peptides demonstrated comparable DNA-binding capacities, DNase I protection, and transcriptional activity, indicating similar interactions with plasmid DNA. However, the cytcox-KH-COOH-based complex exhibited increased susceptibility to trypsin digestion. This result demonstrates that C-terminal modification modulates transient gene expression in the mitochondria by altering the protease sensitivity of the peptide-DNA complexes. Consequently, controlled peptide degradability is a crucial parameter contributing to transient gene expression in mitochondria.