Abstract
Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure–activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.
Highlights
Transfection of siRNAs is nowadays widely employed to knock-down the cellular expression of any targeted protein
We recently showed that peptides composed only of arginine, leucine and tryptophan residues presented a random/helical structure in water with the ability to attain a helical conformation in the presence of anionic components [20]
These four peptides have nearly the same amount of tryptophan residues but differ more importantly in the amount of arginine or leucine residues. All together these resulted in longer peptides in combination with a higher number of charges for C6M1/ C6M1-L compared to the WRAP series (Fig. 1A)
Summary
Transfection of siRNAs is nowadays widely employed to knock-down the cellular expression of any targeted protein. The previously shown structural polymorphism of amphipathic peptides (helicoidal structure formation in a specific environment) [17] was demonstrated using a fully retro-inverso peptide [18] Another group suggested that amphipathic helical peptides composed of hydrophobic and cationic amino acids exposed on different sides of the helix could be typical CPPs for driving the uptake of siRNAs [19]. We recently showed that peptides composed only of arginine, leucine and tryptophan residues presented a random/helical structure in water with the ability to attain a helical conformation in the presence of anionic components (nucleic acids or membranemimicking compounds) [20]. All these observations could focus towards a crucial amphipathic structuration of CPPs for inducing the uptake of at least this class of short nucleic acid molecules, namely siRNAs
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