Fluorescent and photochemical properties of a single zinc finger conjugated to a fluorescent DNA-binding probe

A Single zinc finger derived from the DNA-binding domain of the glucocortic oid receptor (GR) has been tethered to the intercalating fluorophore thiazo le orange, and the DNA recognition characteristics of the conjugate have be en examined. DNA sequence specificity for the peptide-dye conjugate, determ ined by steady-state fluorescence measurements and photoactivated DNA cleav age experiments, reproduce the binding features of response element recogni tion found in the native GR, The thiazole orange is able to intercalate and fluoresce when the conjugate binds, at concentrations where little fluores cence is observed from either the conjugate alone or the conjugate mixed wi th DNA lacking the zinc finger target sequence. The conjugate preferentiall y targets a 5'-TGTTCT-3' sequence (the native glucocorticoid receptor eleme nt) with a dissociation constant of about 25 nM, Lower binding affinities ( up to 10-fold) are observed for single site variants of this sequence, and much lower affinity (40-50-fold) is observed for binding to the estrogen re sponse element (which differs from the glucocorticoid receptor element at t wo positions) as well as to nonspecific DNA. Footprinting reactions show a 4-6 base pair region that is protected by the zinc finger moiety. Photoclea vage assays reveal a several base pair region flanking the recognition sequ ence where the tethered thiazole orange moiety is able to intercalate and s ubsequently cleave DNA upon visible light exposure. Thiazole orange is also shown to oxidize the 5'-G of remote GG sequences, depending on the details of the intervening DNA sequence, Small synthetic protein-dye conjugates su ch as this one are potentially useful for a variety of purposes including s equence-specific probes that work under physiological conditions (without m elting and hybridization of DNA), sequence-specific photocleavage agents, a nd self-assembling components in electron and energy transfer systems that utilize DNA as a scaffold and/or photochemical medium.