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

Hiroko Yamashita,Masaaki Kurihara,Yosuke Demizu,Makoto Oba,Nobumichi Ohoka,Takuma Kato,Masakazu Tanaka,Mikihiko Naito,Takayuki Hattori,Takashi Misawa

doi:10.1038/srep33003

Hiroko Yamashita, Masaaki Kurihara + Show 8 more

Open Access

https://doi.org/10.1038/srep33003

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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-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu 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-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier.

Highlights

L-Proline (l-Pro) is a genetically encoded amino acid
We speculated that the insertion of an l-Pro analogue with a guanidinylated side chain into Arg-based peptides could result in a peptide with a secondary structure that was able to adapt to the surrounding environment
Fmoc-l-Pro-OH, Fmoc-l-ProNH2-OH, and Fmoc-l-ProGu-OH were incorporated into R9 analogues to afford the desired peptides (1–5), all of which contained N-terminal fluorescein (FAM) labels and C-terminal Gly[3] linkers

Summary

Peptide that Exhibits Responsive

Changes in its Secondary Structure received: 16 May 2016 accepted: 18 August 2016 Published: 09 September 2016 in the Cellular Environment. During cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Oligoprolines form a characteristic helical structure called a polyproline helix[5,6], and l-Pro residues in peptide sequences sometimes induce specific secondary structural changes in an environment-dependent manner[7,8]. The cationic peptide B formed a stable helical structure and exhibited greater cell permeability than nona-arginine (R9)[20]. These studies suggested that the secondary structures and cell permeability of CPP are related (Fig. 1). We conducted an experiment in which the transportation of pDNA into HeLa cells was examined in the presence of the peptides

Results and Discussion

Methods

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