Central position of histidine in the sequence of designed alternating polarity peptides enhances pH-responsive assembly with DNA.

Background: Self-assembling peptides hold great promise for medical applications, particularly as carriers for gene delivery, but their potential remains unrealized due to a limited understanding of how amino acid sequence positioning affects their properties. In this study, we designed and evaluated two alternating polarity peptides, RFH (RFRHRHRFR) and RHF (RHRFRFRHR), differing only in the position of their histidine and phenylalanine residues, to investigate the impact of sequence variation on pH-responsive DNA co-assembly and transfection efficiency.

Results: Both peptides formed stable co-assemblies with DNA at neutral pH. However, RHF retained its co-assembling ability at acidic pH, as confirmed by gel retardation studies. Coarse-grained molecular dynamics simulations further supported these findings, showing a reduced affinity of RFH for DNA and a sharper decrease in DNA binding when its histidine residues were protonated. Morphological analysis revealed that both co-assemblies underwent structural transitions with increasing N/P ratios, though their sizes and morphologies differed significantly. Biological studies demonstrated that both peptides achieved a higher transfection efficiency in 293T and HeLa cells compared to R₉, with a lower cytotoxicity than polyethyleneimine. Notably, RFH exhibited superior transfection performance at lower N/P ratios compared to RHF, likely due to its distinct histidine and phenylalanine arrangement and its pH-responsive co-assembly behavior with DNA.

Conclusions: These findings highlight the importance of histidine positioning within peptide sequences for tuning pH-responsiveness and optimizing DNA co-assembly and transfection efficiency. The results provide valuable insights for the rational design of efficient, safe, and pH-responsive peptide-based gene delivery systems.