A novel method for monitoring the cytosolic delivery of peptide cargo.
The intracellular delivery of a diverse array of cargos can be mediated by conjugation to cell-penetrating peptides (CPPs). To date, delivery of cargos into the cytosol via CPPs has been measured indirectly and normally, has been inferred from changes in biological activity. We describe a novel method to directly assay CPP-mediated delivery of peptide cargo into the cytosol, and use this method to define the kinetics of this process. The CPP and the cargo are differentially labeled with the fluorophores FAM (carboxyfluorescein), and TAMRA (carboxytetramethylrhodamine) respectively, and coupled via a disulfide bond to promote quenching of FAM fluorescence by the proximal TAMRA. Delivery of the peptide pair to cells produces an increase in FAM fluorescence within 10 min, consistent with its rapid transfer into the reducing environment of the cytosol, separation of the two components, and concomitant dequenching. The fluorescence-based assay described here can thus be used to select a CPP module that is optimized for efficient delivery of particular cargos designed to modify molecular targets in the cytosol.
Cheung
Joanne C
JC
Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Canada M5G 1X8.
Kim Chiaw
Patrick
P
Deber
Charles M
CM
Bear
Christine E
CE
D000595:Amino Acid Sequence; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010873:Pinocytosis; D011485:Protein Binding; D021381:Protein Transport; D012235:Rhodamines / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D004220:Disulfides; D005456:Fluorescent Dyes; D019812:Heparan Sulfate Proteoglycans; D016877:Oxidants; D010455:Peptides; D012235:Rhodamines; D006861:Hydrogen Peroxide; D019793:Fluorescein