Novel method for covalent fluorescent labeling of plasmid DNA that maintains structural integrity of the plasmid.
We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation. In contrast to nick-translated plasmid DNA, plasmid-lissamine conjugates appeared on gel as supercoiled DNA. Reporter gene was expressed after transfection of the plasmid-lissamine conjugates in NIH 3T3 cells, although gene transfer efficiency was decreased by 60% as compared with unlabeled DNA. Intracellular traffic of plasmid-lissamine conjugates was studied in transfected cells. After cytoplasmic microinjection, fluorescent plasmid did not diffuse from the site of injection and appeared to be progressively degraded in the cytoplasm.
Neves
C
C
UMR 133 CNRS/Rhône-Poulenc Rorer, Centre de Recherche de Vitry Alfortville, B.P.14, 13 quai Jules Guesde 94403 Vitry-sur-Seine Cedex France.
Byk
G
G
Escriou
V
V
Bussone
F
F
Scherman
D
D
Wils
P
P
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D015870:Gene Expression; D008086:Lissamine Green Dyes / Q000138:chemical synthesis / Q000737:chemistry; D051379:Mice; D008845:Microinjections; D009282:Naphthalenesulfonates / Q000138:chemical synthesis / Q000737:chemistry*; D009285:Naphthoquinones / Q000138:chemical synthesis / Q000737:chemistry*; D010777:Photochemistry; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019076:Transgenes
D005456:Fluorescent Dyes; D008086:Lissamine Green Dyes; D009282:Naphthalenesulfonates; D009285:Naphthoquinones; C404822:p-azido-tetrafluoro-benzyl-lissamine; D004247:DNA