EFFICIENT PURIFICATION OF PLASMID DNA FOR GENE-TRANSFER USING TRIPLE-HELIX AFFINITY-CHROMATOGRAPHY

Plasmid DNA used for nonviral therapeutic gene transfer or nucleic aci d vaccination has to be highly purified, devoid of contaminating compo nents such as bacterial proteins, endotoxins, or bacterial chromosomal DNA. We have developed a new affinity chromatography technique for pl asmid DNA purification: triple-helix affinity chromatography (THAC). T his technique is based on the sequence-specific interaction of an olig onucleotide forming a triple-helix with plasmid DNA. The oligonucleoti de was covalently linked to a chromatographic matrix, thus providing a reusable affinity support. By inserting a suitable homopurine sequenc e in the plasmid DNA, it is possible to obtain a interaction that will only be stable at mild acidic and that will dissociate in alkaline co nditions. A crude from a recombinant E. coli, or a pre-purified plasmi d DNA, is thus applied at acidic pH on to a THAC column. After extensi ve washing of the column, purified plasmid DNA is eluted using an alka line buffer. The binding conditions of the plasmid DNA on to the colum n have been optimized, as well as the hybridization sequence and the l inker group between the matrix and the third strand oligonucleotide. T he THAC technique makes it possible to purify in one step supercoiled plasmid DNA, and to significantly reduce the level of contaminating RN A, endotoxins and chromosomal DNA. In particular, a 100-fold reduction of chromosomal DNA contamination over that obtained with conventional techniques can be achieved through a single additional THAC step. Fur ther improvements of THAC technology are possible, and we anticipate t hat this technique can be scaled up for integration into a full commer cial-scale DNA production process.