Padlock oligonucleotides as a tool for labeling superhelical DNA.
Labeling of a covalently closed circular double-stranded DNA was achieved using a so-called 'padlock oligonucleotide'. The oligonucleotide was targeted to a sequence which is present in the replication origin of phage f1 and thus in numerous commonly used plasmids. After winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, a biotinylated oligonucleotide was circularized using T4 DNA ligase and in this way became catenated to the plasmid. A gel shift assay was developed to measure the extent of plasmid modification by the padlock oligonucleotide. A similar assay showed that a modified supercoiled plasmid was capable of binding one streptavidin molecule thanks to the biotinylated oligonucleotide and that this binding was quantitative. The catenated complex was visualized by electron and atomic force microscopies using streptavidin conjugates or single strand-binding proteins as protein tags for the padlock oligonucleotide. This method provides a versatile tool for plasmid functionalization which offers new perspectives in the physical study of supercoiled DNA and in the development of improved vectors for gene therapy.
Roulon
Thibaut
T
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U201, CNRS UMR8646, 43 Rue Cuvier, F-75231 Paris Cedex 05, France.
Coulaud
Dominique
D
Delain
Etienne
E
Le Cam
Eric
E
Hélène
Claude
C
Escudé
Christophe
C
D001483:Base Sequence; D019921:Biotinylation; D003090:Coliphages / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011088:DNA Ligases / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015316:Genetic Therapy / Q000379:methods; D007202:Indicators and Reagents / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D018741:Replication Origin / Q000235:genetics; D019809:Streptavidin / Q000378:metabolism; D013379:Substrate Specificity
D004278:DNA, Superhelical; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D009841:Oligonucleotides; C051802:triplex DNA; D004247:DNA; D019809:Streptavidin; D011088:DNA Ligases