Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells

Triple helix-forming oligonucleotides may be useful as gene-targeting reage nts in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection, Although encouraging, these measurements do not necessarily report tripler stability in cellular compar tments that support DNA functions such as replication and mutagenesis. We h ave devised a shuttle vector plasmid assay that reports the stability of tr iplexes on DNA that undergoes replication and mutagenesis. The assay is bas ed on plasmids with novel variant supF tRNA genes containing embedded seque nces for tripler formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids, At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (WA). After time for replication and mutagenesis, progeny plasmids were recovered and the freque ncy of plasmids with mutations in the supF gene determined. Site-specific m utagenesis by psoralen cross-links was dependent on precise placement of th e psoralen by the triple helix-forming oligonucleotide at the time of WA tr eatment. The results indicated that both pyrimidine and purine motif triple xes were much less stable on replicated DNA than on DNA in vitro or in tota l transfected DNA. Incubation of cells with amidoanthraquinone-based triple r stabilizing compounds enhanced the stability of the pyrimidine triplex.