Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer

► We characterise the pH responsive behaviour of nucleic acid delivery peptides. ► We determine their pH response in solution, bound to DNA/RNA cargo or membranes. ► All peptides bound to DNA/RNA or membranes respond at pH found in endocytosis. ► The most effective peptides avoid self‐association in solution during endocytosis. ► The peptide pH response in solution determines nucleic acid transfer efficacy.