Naoya Ohmori

Binding of Cationic α-Helical Peptides to Plasmid DNA and Their Gene Transfer Abilities into Cells

Polycationic reagents such as cationic lipids and poly-l-lysine are widely used for gene transfer into cells in vitro and show promise as vectors for in vivo gene therapy applications as nonviral gene transfer techniques. We have developed a novel transfection method using cationic amphiphilic α-helical oligopeptides with repeated sequences. Oligopeptide has the advantages of being easily designed and modified because of its simple structure. In this study, we synthesized five kinds of peptides of which the total chain length and the width of the hydrophobic region were changed. The binding of the peptides to plasmid DNA was evaluated by agarose gel electrophoresis. It was found that the long and/or hydrophobic peptides can strongly bind to the DNA. The formation of large aggregates with a 0.5–5-μm diameter, which consisted of the long peptides and the DNA, was observed by electron microscopy. The transfection abilities of the peptides were determined by the expression of luciferase from its cDNA in COS-7 cells. The long peptides showed high transfection abilities. As a result, it could be said that the transfection ability of these peptides was parallel to their ability to form aggregates with DNA. Furthermore, the transfection ability was increased by the addition of chloroquine in the transfection procedure. This result indicated that the internalization of the peptide-DNA aggregates would be mediated by the endocytosis pathway.

Interaction of bundled Ser-rich peptides with phospholipid bilayer

Design of bundled peptides has become of interest in recent year, because such peptides are useful not only as models of natural proteins but also as functional materials. We previously reported that an amphiphilic α-helical 24 mer peptide 46 showed strong activity toward phospholipid membrane [1]. Bundled peptides, 4α-46 composed of 4 fragments of the 46 chains and 4α-46S in which 24 Ser residues are present instead of the Leu and Ala residues in 4α-4 6, had much stronger membrane perturbation activity than 46 [2]. In the present study, we synthesized more hydrophilic bundled peptides containing 36 Ser residues, [Trp2]and [Trp12]-4α-46 S9, and examined their properties.

Interaction of bundled Ser-rich amphiphilic peptides with phospholipid membranes

To investigate properties of hydrophilic bundled peptides and their interactions with phospholipid membranes, bundled peptides named [Trp2]‐ and [Trp12]‐4α‐46S9, which are composed of four fragments of amphiphilic 24‐mer peptide, were designed and synthesized. Tryptophan (Trp) was introduced at the 2nd position from the N‐terminal or at the centre (12th) of the helix to monitor the peptide–lipid interaction. Circular dichroism measurements indicated that the peptides had low α‐helicities in a buffer solution (pH 7.4) and also in the presence of dipalmitoyl‐dl‐3‐phosphatidylcholine (DPPC) vesicles. In the presence of DPPC/dipalmitoyl‐dl‐3‐phosphatidylglycerol (DPPG) (3:1) vesicles, the measurement could not be taken because of turbidity induced by vesicle aggregation. Both peptides had moderate perturbation activity for both the neutral and acidic vesicles at 25°C. The perturbation patterns at 50°C were much different from those at 25°C and the maximum activity reached 100% at a low peptide concentration. The results of the measurement of membrane fusion activity of peptides showed a similar tendency to that found in the perturbation experiment. A quenching experiment indicated that the Trp2 and Trp12 residues in [Trp2]‐ and [Trp12]‐4α‐46S9 were scarcely embedded in neutral lipid membranes. Copyright