Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells.

Abstract

For application of DNA in gene medicine plasmid or viral DNA is usually used as a vector for the gene of interest. To generate DNA with a minimum of foreign DNA sequences, we used the prokaryotic telomerase, protelomerase TelN, of bacteriophage N15. This is a novel enzyme with cleaving-joining activity, which is required for the formation of linear prophage DNA with closed ends in lysogenic bacteria. Acting on a telomere resolution site telRL, the protelomerase converts circular plasmid DNA into linear covalently closed dumbbell-shaped molecules ("doggybones") in a single-step enzyme reaction. Two such sites were inserted into an expression plasmid flanking a gene of interest. This is cleaved and joined by means of the protelomerase, yielding linear closed mini DNA coding for green fluorescent protein (EGFP) or interleukin-12 (IL-12). Upon transient transfection of human embryonal kidney cells, EGFP was expressed at higher levels from linear closed molecules than from linear open molecules generated by restriction endonucleases for comparison. The level of transcription was comparable to that observed for the parental plasmid DNA. To test whether the linear closed mini DNA molecules are functional in vivo the B16F10/C57BL/6 melanoma metastasis model was applied, where injection of IL-12-expressing DNA inhibits metastasis formation in the lung. The anti-metastatic effect of the IL-12-expressing linear closed DNA was equal or higher than that of the parental plasmid DNA. Therefore, the TelN/ telRL system is well suited to generate linear closed mini DNA with high stability and a minimum of foreign nucleotide sequences.