Figure 1.

Schemes illustrating minicircle (MC) generation and triplex DNA (TriD)-mediated purification. (a) MC generation was conducted according to established protocol.¹⁰ Briefly, in the overnight culture of Escherichia coli strain ZYCY10P3S2T transfected with MC producing plasmid (PP), MC generation is induced by addition of L-arabinose which induces expression of both ΦC31 recombinase, which mediates DNA recombination between attB and attP, and endonuclease I-SceI, which mediates destruction of plasmid backbone circle (PB); consequently, MC becomes the only episomal circular DNA to be isolated using affinity column. (b) TriD-mediated MC Purification. (i) MC product is incubated with biotinylated DNA olgionucleotide (Olig), which forms triplex DNA complex with corresponding TriD forming sequences in the PP and PB. (ii) Subsequently, the TriD complex is removed by streptovidin-coated magnetic beads. (iii) The MC solution is collected and MC is precipitated with ethanol and reconstituted in TE. attB & attP, bacterial and bacteriophage attachment sites, respectively; attL & attR, the hybrid sequences; the tobacco pipe-like shape, biotinylated Olig; the solid circle, streptovidin bead; the diamond, TriD forming sequence.