Coupling of nuclear localization signals to plasmid DNA and specific interaction of the conjugates with importin alpha.
The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear targeting of proteins. We have developed a chemical strategy for covalent coupling of NLS peptides to plasmid DNA. A p-azido-tetrafluoro-benzyl-NLS peptide conjugate was synthesized. This conjugate was used to covalently associate NLS peptides to plasmid DNA by photoactivation. Reporter gene was expressed after transfection of the plasmid-NLS conjugates in NIH 3T3 cells. The conjugates interacted specifically with the NLS-receptor importin alpha, but plasmid-NLS conjugates were not detected in the nucleus, by fluorescence microscopy, after cytoplasmic microinjection.
Ciolina
C
C
UMR 133 CNRS/Rhône-Poulenc Rorer and Rhône-Poulenc Rorer Gencell, Centre de Recherche de Vitry Alfortville, B.P.14, 13 quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France.
Byk
G
G
Blanche
F
F
Thuillier
V
V
Scherman
D
D
Wils
P
P
D016475:3T3 Cells; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D008845:Microinjections; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals / Q000235:genetics / Q000528:radiation effects; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000528:radiation effects; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D014162:Transfection; D014466:Ultraviolet Rays; D028901:alpha Karyopherins
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D005982:Glutathione Transferase