Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display

• Published in: Bioconjugate chemistry Vol. 31; no. 10; pp. 2325 - 2338
• Main Authors: Bowen, John, Schloop, Allison E, Reeves, Gregory T, Menegatti, Stefano, Rao, Balaji M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.10.2020
• Subjects: Animals; Biological membranes; Cell Line; Cell-Penetrating Peptides - chemistry; Cell-Penetrating Peptides - genetics; Cell-Penetrating Peptides - pharmacokinetics; Confocal microscopy; Developmental stages; Drosophila melanogaster - embryology; Drug delivery; Drug delivery systems; Embryo cells; Embryos; Flow cytometry; Fruit flies; Gene Library; Humans; Mammalian cells; Mammals; Membranes; Mice; Models, Molecular; mRNA; NIH 3T3 Cells; Penetration; Peptides; Peptides, Cyclic - chemistry; Peptides, Cyclic - genetics; Peptides, Cyclic - pharmacokinetics; Permeability; Permeation; Pretreatment; RNA, Messenger - genetics; Stem cell transplantation; Stem cells; Synthetic peptides
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.0c00413

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo­[Glut-MRK­RHA­SRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.