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

The development of effective delivery systems for peptide nucleic acids (PNAs) into bacterial cells remains a critical challenge in antisense therapeutics. We report the design and evaluation of hydroxamate siderophore-PNA conjugates that exploit bacterial iron uptake pathways for targeted delivery. We demonstrate that modulating the backbone flexibility of hydroxamate siderophores, through glycine or alanine spacers, enhances iron(III) binding affinity and PNA delivery efficiency into Escherichia coli cells. Molecular dynamics simulations revealed that glycine insertion increases backbone flexibility, enabling optimal coordination of all three hydroxamate groups to iron(III). Circular dichroism spectroscopy and iron(III) competition assays confirmed that the siderophores form stable Λ-configured ferric complexes, with the flexible siderophore showing superior iron(III)-binding affinity compared to the less flexible analog. Growth recovery experiments using E. coli mutants deficient in several transporters indicated recognition and internalization of the siderophores via the TonB-dependent hydroxamate pathway. Molecular docking demonstrated the affinity of these siderophores for E. coli hydroxamate receptors, with binding scores comparable to those of natural siderophores. All three siderophore carriers successfully delivered functional PNA targeting the mrfp reporter gene into bacterial cells, achieving sequence-specific gene silencing as confirmed by fluorescence measurements and confocal microscopy. Additionally, bacteriostatic activity was observed for the best-performing siderophore mimic conjugated with a PNA targeting the essential acpP gene.