2-Aminoisobutylic Acid-Stabilized Helical Peptides for Selective Mitochondrial Gene Delivery

Abstract Efficient gene delivery to mitochondria remains a major challenge in the development of organelle-targeted therapeutics. Here, we report the rational design and evaluation of helix stabilized mitochondria-targeting peptides (MTPs) via the incorporation of 2-aminoisobutyric acid (Aib), a noncanonical residue known to promote helical structure and increase membrane permeability. Three essential peptides were newly synthesized by systematically substituting alanine or glycine residues with Aib in a peptide derived from the mitochondrial outer membrane protein mitoNEET. Structural analyses using molecular dynamics (MD) simulations and circular dichroism (CD) spectroscopy confirmed increased helical content in the Aib-substituted peptides. Compared with the unmodified peptide, the Aib-substituted peptides exhibited significantly increased HeLa cell uptake and mitochondrial localization. Furthermore, conjugation of the designed MTPs with a cationic arginyl−histidine repeat peptide enabled efficient delivery of plasmid DNA into mitochondria, leading to successful expression of mitochondrial-localized reporter genes. These findings demonstrate that Aib-induced helix stabilization is a powerful strategy for increasing mitochondrial gene delivery and provide a versatile platform for peptide-based mitochondrial therapeutics.