Activation of gene expression by triplex-directed psoralen crosslinks.

Abstract

Psoralen-conjugated triplex-forming oligonucleotides (pso-TFOs) can target photochemical adducts to specific DNA sequences. Here, we have used pso-TFOs to activate gene expression on a plasmid. We designed a pso-TFO adapter, consisting of a single-stranded TFO for targeting DNA, linked to a double-stranded hairpin segment that contains a hybrid ecdysone response element (E/GRE) enhancer for binding activated ecdysone receptors. When targeted to the 5' flanking region of a minimal promoter, this pso-TFO adapter increased the expression of a downstream reporter gene three- to four-fold. Gene activation, however, was independent of both the E/GRE hairpin of the adapter and ecdysone receptors, suggesting it was due to an intrinsic effect of triplex. Gene activation was dependent on psoralen photo-crosslinking. Gene activation by pso-TFOs in which the psoralen was linked to the TFO via a disulfide bond was similar before and after detachment of the TFO and its release from the triplex. These results indicate that psoralen photo-crosslinks play a prominent role in activation. Gene activation was undiminished in XPA, XPD and XPG human cell lines, indicating that activation was not dependent on the complete nucleotide excision repair (NER) pathway. Collectively, these results demonstrate that TFOs can be used to direct psoralen crosslinks adjacent to a gene as a way of activating gene expression.