1. Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9
- Author
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Mathilde Bourdeloux, Patrizia Alberti, Elisabeth Met, Fabienne Burlina, Gérard Chassaing, François Loll, Tula Saison-Behmoaras, Céline Cordier, Fatima Boutimah, Florian Dupuy, HAL UPMC, Gestionnaire, Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université Pierre et Marie Curie - Paris 6 (UPMC)
- Subjects
Peptide Nucleic Acids ,lcsh:Medicine ,HIV Infections ,Peptide ,Cell-Penetrating Peptides ,Biochemistry ,Cytosol ,Nucleic Acids ,Molecular Cell Biology ,Internalization ,lcsh:Science ,Peptide sequence ,Glycosaminoglycans ,media_common ,chemistry.chemical_classification ,Multidisciplinary ,Chemistry ,Transfection ,3. Good health ,Cell Processes ,Gene Targeting ,Cellular Structures and Organelles ,Cellular Types ,Research Article ,media_common.quotation_subject ,CHO Cells ,Endosomes ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Arginine ,Endocytosis ,Cell Line ,Cricetulus ,Animals ,Humans ,Amino Acid Sequence ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,Base Sequence ,Oligonucleotide ,lcsh:R ,Biology and Life Sciences ,Cell Biology ,Oligonucleotides, Antisense ,HIV-1 ,Cell-penetrating peptide ,Pinocytosis ,lcsh:Q ,Peptides ,Cytometry ,HeLa Cells ,Conjugate - Abstract
International audience; Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine-and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.
- Published
- 2014
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