Integrin-mediated transfection with peptides containing arginine-glycine-aspartic acid domains.
Two synthetic peptides comprising an RGD moiety for integrin binding and a polylysine moiety for DNA binding were tested for transfection efficiency under a variety of different conditions. Binding of target cells to the peptide was shown to be strongly dependent on cyclisation of the peptides via cysteine residues. Low (10 microM) concentrations of chloroquine, added to assist endocytic exit, unexpectedly reduced transfection efficiency in two of the cell lines tested. COS-7 and ECV304. However, transfection efficiency increased at higher chloroquine concentrations and exceeded that in the absence of chloroquine in the case of the COS-7 and A375M cell lines. With the ECV304 cell line, optimum transfection occurred in the absence of chloroquine. Transfection efficiency of the peptides was greatest at peptide:DNA ratios of 4:1 (w/w), which were calculated to generate complexes containing approximately 5000 peptide molecules per plasmid. This represented approximately a 6:1 ratio of positive to negative charges. Peptide 5 was shown to have a higher transfection efficiency under most conditions, possibly because of more efficient stabilisation of cyclisation by two cysteine-cysteine bonds.
Hart
S L
SL
Transplantation Biology Unit, Institute of Child Health, University College Medical School, London, UK.
Collins
L
L
Gustafsson
K
K
Fabre
J W
JW
D000818:Animals; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004247:DNA; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics*; D010957:Plasmids; D011971:Receptors, Immunologic; D014162:Transfection
D016023:Integrins; D009842:Oligopeptides; D011971:Receptors, Immunologic; C047981:arginyl-glycyl-aspartic acid; D002738:Chloroquine; D004247:DNA