Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide.
We have recently described a new method for attaching padlock oligonucleotides to supercoiled plasmid DNA at specific sequences. A variant of this method has been developed in order to allow the coupling of targeting moieties to plasmids using a convenient strategy. After sequence-specific winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, the extremities of a triplex-forming oligonucleotide hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a hairpin oligonucleotide using T4 DNA ligase. Any targeting moiety may be attached to the hairpin oligonucleotide. This strategy was used to attach an NLS peptide to a luciferase-expressing plasmid. Despite the presence of the padlock oligonucleotide, the reporter gene was efficiently expressed after transfection of the plasmid in HeLa or T24 cells, using either cationic lipids or cationic polymers as transfecting agents. However, no increase in gene expression could be observed as a result of peptide attachment. Nevertheless, the coupling strategy described in this paper may find applications as a tool for plasmid functionalization in other targeting experiments, and may lead to 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, 75231 Paris Cedex 05, France.
Hélène
Claude
C
Escudé
Christophe
C
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D016503:Drug Delivery Systems / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000627:therapeutic use; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000627:therapeutic use; D010957:Plasmids / Q000008:administration & dosage* / Q000737:chemistry; D014162:Transfection / Q000379:methods* / Q000592:standards
D003432:Cross-Linking Reagents; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; D010455:Peptides; C072792:S-(N-methylcarbamoyl)cysteine; D003545:Cysteine