Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides

Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) hav e been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indic ate that these psoTFO bind to and photoreact with model target DNA sequence s following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 mu M. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks a re efficiently Formed on targets containing either 5'-ApT-3' or 5'-TpA-3' s equences adjacent to the TFO binding sequence. The dependence of adduct for mation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence o f a TFO, When psoralen is tethered to a TFO. the rate of monoadduct formati on exceeds that of crosslinking for all sequences studied, This contrasts w ith the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial p hotochemistry that generates monoadducts, but does not significantly. affec t interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cle avage by a restriction endonuclease when the psoralen covalently reacts dir ectly at the endonuclease site. The particular TFO studied do not completel y inhibit endonuclease activity when they are noncovalently bound or when t he covalent psoralen adduct does not coincide with the endonuclease site. O ur findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate ps oralen photoreactivity and DNA-protein interactions.