Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography

Abstract

Plasmid DNA used for nonviral therapeutic gene transfer After extensive washing of the column, purified plasmid or nucleic acid vaccination has to be highly purified, devoid DNA is eluted using an alkaline buffer. The binding conof contaminating components such as bacterial proteins, ditions of the plasmid DNA on to the column have been endotoxins, or bacterial chromosomal DNA. We have optimized, as well as the hybridization sequence and the developed a new affinity chromatography technique for linker group between the matrix and the third strand oligoplasmid DNA purification: triple-helix affinity chromatonucleotide. The THAC technique makes it possible to purify graphy (THAC). This technique is based on the sequencein one step supercoiled plasmid DNA, and to significantly specific interaction of an oligonucleotide forming a triplereduce the level of contaminating RNA, endotoxins and helix with plasmid DNA. The oligonucleotide was covalently chromosomal DNA. In particular, a 100-fold reduction of linked to a chromatographic matrix, thus providing a rechromosomal DNA contamination over that obtained with usable affinity support. By inserting a suitable homopurine conventional techniques can be achieved through a single sequence in the plasmid DNA, it is possible to obtain a additional THAC step. Further improvements of THAC triple-helix interaction that will only be stable at mild acidic technology are possible, and we anticipate that this techpH and that will dissociate in alkaline conditions. A crude nique can be scaled up for integration into a full commerlysate from a recombinant E. coli, or a pre-purified plasmid cial-scale DNA production process. DNA, is thus applied at acidic pH on to a THAC column.

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer

“…DNA concentration was measured by reverse phase HPLC analysis, using a Poros R2/H column (100 × 4.6 mm; PerSeptive Biosystems, Cambridge, MA, USA) followed by UV absorbance at 260 nm, as previously described. 22 Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA), Gibco-BRL (Life Technologies, Cergy Pontoise, France) or Amersham (Amersham, Les Ulis, France).…”

“…Wils et al 22 developed an affinity chomatography method for the purification of plasmid DNA, based on sequence-specific formation of a triple helix between immobilized oligonucleotides and a specific sequence present on the plasmid. This method could be used for large-scale production of minicircles.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…However, the triple-helix interaction is only possible if a suitable target homopurine sequence has been previously inserted in the pDNA Wils et al 1997). Some reports provide evidence that it is possible to purify sc pDNA with THAC and to significantly reduce the level of contaminating RNA, endotoxins and gDNA in a single step (Wils et al 1997).…”

“…Triple helix affinity chromatography (THAC) is also an alternative affinity technique to purify pDNA, based on the sequence-specific interaction of a triple-helix forming oligonucleotide with pDNA Wils et al 1997).…”

Affinity purification of plasmid DNA by temperature-triggered precipitation

“…One representative technique is based on the formation of a triple helix between an oligonucleotide covalently linked to a chromatographic matrix and the complementary sequence present on the plasmid DNA to be purified 9 . The limitations of this method include the relatively slow helix formation and the need for high ionic strength and low pH 2 .…”