title: Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides.
abstract: Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure-activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.
authors.affiliation: Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University , Svante Arrhenius väg 16B, 10691 Stockholm, Sweden.
authors.forename: Tõnis
authors.identifier: 0000-0002-6440-7577
authors.initials: T
authors.lastname: Lehto
authors.affiliation: Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University , Svante Arrhenius väg 16B, 10691 Stockholm, Sweden.
authors.forename: Luis
authors.initials: L
authors.lastname: Vasconcelos
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu , Riia 23a, 51010 Tartu, Estonia.
authors.forename: Helerin
authors.initials: H
authors.lastname: Margus
authors.affiliation: Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University , Svante Arrhenius väg 16B, 10691 Stockholm, Sweden.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Figueroa
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu , Riia 23a, 51010 Tartu, Estonia.
authors.forename: Margus
authors.initials: M
authors.lastname: Pooga
authors.affiliation: Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University , Svante Arrhenius väg 16B, 10691 Stockholm, Sweden.
authors.forename: Mattias
authors.initials: M
authors.lastname: Hällbrink
authors.affiliation: Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University , Svante Arrhenius väg 16B, 10691 Stockholm, Sweden.|Institute of Technology, University of Tartu , Nooruse 1, 50411 Tartu, Estonia.
authors.forename: Ülo
authors.initials: Ü
authors.lastname: Langel
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D005227:Fatty Acids / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D055666:Lipopeptides / Q000737:chemistry*; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D057846:Cell-Penetrating Peptides; D005227:Fatty Acids; D055666:Lipopeptides; D009841:Oligonucleotides; C560878:PepFect14 peptide