Author(s): Moulton HM, Moulton JD, Moulton HM, Moulton JD, Moulton HM, Moulton JD, Moulton HM, Moulton JD
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Abstract Cationic transport peptides conjugated to steric blocking antisense oligomers (oligos) increase oligo uptake in eukaryotic cell lines, bacteria and mice. Recent reports of arginine-rich transport peptide conjugates strongly suggest that the mechanism of uptake is primarily endocytotic and that previous assay techniques produced confounding artifacts that led to the old non-endocytotic, membrane-penetrating peptide model. The artifacts result from fixing cells for fluorescent microscopy and from using non-trypsinized cells for flow cytometry. Fixing cells redistributes the peptide or peptide-oligo conjugates associated with the outside of cell membranes and trapped in endosomes, giving apparent diffuse cytosolic and nuclear fluorescence. Cationic peptides bound to the outer surface of cells, if not removed, skew fluorescence data obtained by flow cytometry, leading to the earlier conclusions. Upregulation assays now provide a tool for comparing the efficacy of conjugates, measuring oligo uptake by quantitating antisense activity of conjugates. These assays, developed in cell culture and mouse models, are faster and have higher signal-to-noise ratios than downregulation assays. Thus, a convenient and effective method now exists to screen transport peptides.
This article was published in Curr Opin Mol Ther
and referenced in Research & Reviews: Journal of Botanical Sciences