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 glucocorticoid receptor (GR) has been tethered to the intercalating fluorophore thiazole orange, and the DNA recognition characteristics of the conjugate have been examined. DNA sequence specificity for the peptide-dye conjugate, determined by steady-state fluorescence measurements and photoactivated DNA cleavage experiments, reproduce the binding features of response element recognition found in the native GR. The thiazole orange is able to intercalate and fluoresce when the conjugate binds, at concentrations where little fluorescence is observed from either the conjugate alone or the conjugate mixed with DNA lacking the zinc finger target sequence. The conjugate preferentially targets a 5'-TGTTCT-3' sequence (the native glucocorticoid receptor element) 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 response element (which differs from the glucocorticoid receptor element at two positions) as well as to nonspecific DNA. Footprinting reactions show a 4-6 base pair region that is protected by the zinc finger moiety. Photocleavage assays reveal a several base pair region flanking the recognition sequence where the tethered thiazole orange moiety is able to intercalate and subsequently 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 such as this one are potentially useful for a variety of purposes including sequence-specific probes that work under physiological conditions (without melting and hybridization of DNA), sequence-specific photocleavage agents, and self-assembling components in electron and energy transfer systems that utilize DNA as a scaffold and/or photochemical medium.
Thompson
M
M
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Woodbury
N W
NW
D001483:Base Sequence; D052160:Benzothiazoles; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism; D008027:Light; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D011804:Quinolines; D011960:Receptors, Estrogen / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D026082:Singlet Oxygen; D013379:Substrate Specificity; D013816:Thermodynamics; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D017186:Titrimetry; D016335:Zinc Fingers
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D015335:Molecular Probes; D010446:Peptide Fragments; D011804:Quinolines; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D013844:Thiazoles; C051000:thiazole orange; D026082:Singlet Oxygen; D006147:Guanine; D004247:DNA; D010100:Oxygen