Triple helix-forming oligonucleotides target psoralen adducts to specific chromosomal sequences in human cells

The ability to target photochemical adducts to specific genomic DNA sequenc es in cells is useful for studying DNA repair and mutagenesis in intact cel ls, and also as a potential mode of gene-specific therapy, Triple helix-for ming DNA oligonucleotides linked to psoralen (psoTFOs) were designed to del iver UVA-induced psoralen photoadducts to two distinct sequences within the human interstitial collagenase gene. A primer extension assay demonstrated that the appropriate psoTFO selectively damages a collagenase cDNA target. Site-specific genomic psoTFO DNA adducts were detected by a single-strand ligation PCR assay. The adduct, formed at a single site by a psoTFO in puri fied genomic DNA, contrasted with the multiple sites that were damaged with in the observed segment of the collagenase gene upon treatment with free ps oralen and subsequent photoactivation, When treated with psoTFOs, both repa ir-deficient fibroblasts from xeroderma pigmentosum complementation group A and HT1080 fibrosarcoma cells exhibited site-specific DNA adducts followin g UVA irradiation. Addition of phorbol ester, a transcriptional activator o f the collagenase gene, to xeroderma pigmentosum cells did not detectably a lter the initial levels of damage produced by psoTFOs, suggesting-that furt her stimulation of transcription neither improves accessibility of psoTFOs to their targets nor enhances removal of non-covalently bound psoTFOs.