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 (SL) 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 (SGLY) with glycine spacers between modified ornithine residues for enhanced flexibility and iron-binding. We also synthesized marine siderophore analogs (MGLY and MALA) inspired by natural moanachelins. Using circular dichroism spectroscopy, spectrophotometric assays, and molecular dynamics simulations, we confirmed iron binding. Growth recovery experiments showed SGLY recognition and internalization via the TonB-dependent transport system, likely using hydroxamate siderophore pathways. The MGLY and MALA siderophores showed lower growth promotion than SGLY, indicating less efficient internalization. Molecular docking revealed high affinity of SGLY 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.