Synthesis and characterization of the photophysical and photochemical properties of sequence specific DNA -binding probes.

摘要

The complexes designed in this work combine the sequence specific binding properties of native DNA-binding domains with intercalating cyanine dyes. DNA sequence specificity for the peptide-dye conjugate determined by steady state fluorescence measurements reproduce the binding features of response element recognition found in the native proteins. The cyanine dyes are able to intercalate and fluoresce when the conjugate binds, at concentrations where little fluorescence is observed from either the dye alone or the conjugate mixed with DNA lacking the appropriate cognate sequence. The conjugates exhibit higher binding affinity than either the dye or the nonlabeled peptide alone, but with lower affinity than would be expected from the product of the two individual equilibrium constants, suggesting noncooperative behavior. Photocleavage assays performed using the thiazole orange-zinc finger conjugate 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 cause both direct and long-range sequence specific cleavage. The thermodynamic the contributions of the polyelectrolyte and hydrophobic effects for specific and nonspecific DNA binding of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques. Nonspecific binding shows greater sensitivity to changes in salt concentration than specific binding. Conversely, specific binding shows a greater sensitivity to changes in temperature than nonspecific binding. Fluorescence resonance energy transfer studies were used to understand the self-assembly properties of the YOHin and TOTc3 conjugates on several DNA templates. 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 sequence-specific photocleavage agents, and self assembling components in electron and energy transfer systems that utilize DNA as a scaffold and/or photochemical medium.