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Fast screening assays for cell-penetration properties of peptides

Cell-penetrating peptides (CPP) have served as efficient vectors for translocation of hydrophilic molecules into living cells. The assays for selection of peptides with cell-penetrating ability have been laborious and we optimised these for using in HTS. Translocation of fluorescein-labelled peptides into cells was screened by the emission signal of cells in 96-well plates after quenching the extra-cellular fluorescence of not-internalised peptides. For the fast screening of unlabelled peptides we designed and optimised an assay based on the changes in the cellular uptake of protein-binding fluorescent dyes upon the internalisation of CPPs.

The peptides with CPP properties were further characterised for the ability to carry into cell interior cargo molecules using similar assays. The cargoes were coupled to transport peptide non-covalently, by forming a complex between biotin-tagged CPP and labelled avidin/streptavidin. The protein was quantified in cells by the label of protein, a fluorescent dye or enzyme activity, and all studied CPP showed protein delivery activity. Few CPPs were even capable of forming a stable complex with cargo (protein, oligonucleotide, plasmid DNA) without using biotin-avidin interaction, and subsequent cellular delivery.

The specificity of CPP-mediated delivery in vivo could be further characterised by using Cre-recombinase (coupled to peptides covalently or non-covalently) in respective reporter mice.

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University of Tartu
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