Modulating backbone flexibility in hydroxamate siderophores for improved iron chelation and peptide nucleic acid (PNA) delivery into bacteria

Peptide nucleic acid (PNA) is a synthetic oligonucleotide analog with a peptide-based backbone that selectively binds with high affinity to natural nucleic acids. PNA is a valuable tool in antisense technology with potential antibacterial applications. However, PNA cannot penetrate bacterial cells alone. To address this, we explored iron chelators - siderophores - as PNA carriers. Bacteria acquire iron through siderophores, which are transported via specific TonB-dependent receptors in the bacterial envelope. Previously, we demonstrated that a synthetic hydroxamate-type siderophore (S _L ) exploited this transport system to deliver PNA into bacterial cells, achieving a gene-silencing effect. However, this transport was limited to an Escherichia coli mutant with continuous iron uptake, and was not observed in wild-type E. coli . In this study, we developed a new synthetic siderophore (S _GLY ) with glycine spacers between modified ornithine residues for enhanced flexibility and iron-binding. We also synthesized marine siderophore analogs (M _GLY and M _ALA ) inspired by natural moanachelins. Using circular dichroism spectroscopy, spectrophotometric assays, and molecular dynamics simulations, we confirmed iron binding. Growth recovery experiments showed S _GLY recognition and internalization via the TonB-dependent transport system, likely using hydroxamate siderophore pathways. The M _GLY and M _ALA siderophores showed lower growth promotion than S _GLY , indicating less efficient internalization. Molecular docking revealed high affinity of S _GLY for E. coli receptors involved in the uptake of hydroxamate siderophores. However, upon conjugation to PNA, all three siderophores effectively delivered PNA into E. coli cells. We confirmed PNA-mediated gene silencing using fluorescence measurements and confocal microscopy.