Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment

Abstract

Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1-5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro) 3-(Gly) 3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-Pro NH2) 3-(Gly) 3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-Pro Gu) 3-(Gly) 3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-Pro Gu)2-(l-Arg)4-l-Pro Gu-(Gly) 3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-Pro Gu) 3-(Gly) 3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-Pro NH2 and l-Pro Gu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-Pro Gu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-Pro Gu-containing peptide 3 may be a useful candidate as a gene delivery carrier.