Intracellular fate and nuclear targeting of plasmid DNA.
One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5-115 p-azidotetrafluorobenzyllissamine-NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide-NLS and plasmid lissamine-NLS conjugates interacted specifically with the NLS-receptor importin alpha. Plasmid-lissamine-NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid-lissamine-NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus.
Neves
C
C
UMR 133 CNRS/Rhône-Poulenc Rorer, Centre de Recherche de Vitry Alfortville, Vitry-sur-Seine, France.
Escriou
V
V
Byk
G
G
Scherman
D
D
Wils
P
P
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D018014:Gene Transfer Techniques; D008086:Lissamine Green Dyes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008845:Microinjections; D019913:Nuclear Localization Signals / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D010957:Plasmids / Q000235:genetics*; D014162:Transfection; D028901:alpha Karyopherins
D005456:Fluorescent Dyes; D008086:Lissamine Green Dyes; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010455:Peptides; D028901:alpha Karyopherins; D004247:DNA