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.

Figure 1

(A) Description of the plasmid and oligonucleotides used in this study. Sequences of the 59mer TFO and the 17mer template are shown. U is a biotinylated uridine. The 59mer oligonucleotide can form a triple helix by binding to an 18 bp oligopurine·oligopyrimidine target sequence (termed ORI in this paper), located in the f1 phage replication origin. The extremities of this TFO can hybridize to the 17mer template oligonucleotide. The sites for the restriction enzymes DraIII, KpnI and HindIII are also indicated. (B) Structure of the triplex-specific ligand BQQ.

Figure 2

Measurement of the yield of padlock oligonucleotide by gel shift assay. Plasmids were treated in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of an excess of 5′-phosphorylated TFO to allow for padlock formation, as described in Materials and Methods. After purification by spermidine precipitation, samples were heated in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 2 µM BQQ and digested with DraIII and KpnI to produce a short restriction fragment containing the ORI sequence. Samples were then loaded on a 6% native polyacrylamide gel containing 10 mM MgCl₂, stained with SybrGreen I and imaged with a camera. I and II refer to the restriction fragments of 2592 and 432 bp, respectively. III corresponds to fragment II shifted by the presence of a catenated padlock oligonucleotide.

Figure 3

Streptavidin binding to a biotinylated padlock oligonucleotide, as revealed by gel shift assay. A 5′-phosphorylated oligonucleotide with (lanes 3 and 4) or without (lanes 5–7) a biotinylated uridine was catenated to the pGA2 plasmid. Samples 2, 4, 6 and 7 were incubated in the presence of streptavidin before spermidine purification, as described in Materials and Methods. Samples were then incubated in the presence of 2 µM BQQ and digested with DraIII and KpnI, analyzed on a 6% native polyacrylamide gel containing 10 mM MgCl₂ and revealed by SybrGreen I staining. In lane 7, a plasmid catenated to a non-biotinylated padlock was incubated in the presence of streptavidin and purified by spermidine compaction, then an equal amount of plasmid catenated to a biotinylated oligonucleotide was added to the sample before digestion. Bands I, II and III are described in the legend to Figure 2. IV corresponds to fragment II shifted by the presence of a biotinylated padlock oligonucleotide linked to streptavidin.

Figure 4

Images of the complex between the padlock oligonucleotide and the supercoiled pGA2 plasmid. (a and b) Annular dark field EM visualization of the biotinylated padlock oligonucleotide, revealed with gp32 single strand-binding protein (a) or streptavidin–ferritin conjugate (b). (c and d) The biotinylated padlock oligonucleotide was revealed with unmodified streptavidin using AFM. At high ionic strength (10 mM Tris, 5 mM MgCl₂, 50 mM NaCl) (c) two supercoiled plectonemic forms complexed with streptavidin and a naked open circular form can be seen in the field. At low ionic strength (10 mM Tris, 5 mM MgCl₂) (d) the plasmid adopts a toroidal form and the streptavidin can be clearly seen. We cannot exclude the possibility that the plasmid shown here is not an open circular form, as it is not possible to distinguish the supercoiled and open circular forms under these conditions. The arrows indicate the position of the padlock oligonucleotide. The scale marker on EM views indicates 100 nm.