In vitro efficient transfection by CM₁₈-Tat₁₁ hybrid peptide: a new tool for gene-delivery applications.
Cell penetrating peptides (CPPs) are actively researched as non-viral molecular carriers for the controlled delivery of nucleic acids into cells, but widespread application is severely hampered by their trapping into endosomes. Here we show that the recently introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1-7 of Cecropin-A, 2-12 of Melittin, and 47-57 of HIV-1 Tat protein) can be exploited to obtain a self-assembled peptide-DNA vector which maintains the CM18-Tat11 ability to enter cells and destabilize vesicular membranes, concomitantly yielding high DNA transfection efficiency with no detectable cytotoxic effects. Different peptide-DNA stoichiometric ratios were tested to optimize vector size, charge, and stability characteristics. The transfection efficiency of selected candidates is quantitatively investigated by the luciferase-reporter assay. Vector intracellular trafficking is monitored in real time and in live cells by confocal microscopy. In particular, fluorescence resonant energy transfer (FRET) between suitably-labeled peptide and DNA modules was exploited to monitor complex disassembly during endocytosis, and this process is correlated to transfection timing and efficiency. We argue that these results can open the way to the rational design and application of CM18-Tat11-based systems for gene-delivery purposes.
NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Pisa, Italy.
Fabrizio
F
Salomone
Francesco
F
Cardarelli
Giovanni
G
Signore
Claudia
C
Boccardi
Fabio
F
Beltram
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D057054:Molecular Imaging; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism*
D057846:Cell-Penetrating Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C586882:cecropin A(1-7)-melittin(2-12)-Tat(47-57) peptide; D054322:tat Gene Products, Human Immunodeficiency Virus; C497137:tat peptide (47-57), Human immunodeficiency virus 1; D004247:DNA