Padlock oligonucleotides as a tool for labeling superhelical DNA.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Roulon T,Coulaud D,Delain E,Le Cam E,Hélène C,Escudé C

doi

10.1093/nar/30.3.e12

keywords:

subject

Has Abstract

pub_date

2002-02-01 00:00:00

pages

E12

issue

3

eissn

0305-1048

issn

1362-4962

journal_volume

30

pub_type

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