Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR , hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary –yet not sufficient– step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.
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