If cleavage is carried out in the presence of the

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the autoradiograph. If cleavage is carried out in the presence of the protein, however, those sites that are bound by the protein are protected from cleavage by steric considerations, producing a shadow or footprint of the protein-binding site on the gel. Both the site-specific and footprinting experiments are illustrated schematically in Figure 8.11. This very powerful technology was first applied by Dervan and coworkers in demonstrating the application of methidium-propyl- FeEDTA (MPE-Fe) as a chemical footprinting reagent. 22 ,S? Tris(phenanthroline) metal complexes have been shown to cleave DNA nonspecifically, and their derivatives have been applied either as sensitive photofootprinting reagents, or as site-specific cleaving molecules, as we will see. IV. APPLICATIONS OF DIFFERENT METAL COMPLEXES THAT BIND NUCLEIC ACIDS Both the spectroscopy and the chemical reactivity of transition-metal complexes, coupled to biochemical assays, can therefore be exploited to obtain a wide range of useful reagents to probe nucleic acids. Here some specific applications are described.
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polyacrylamide gel A: cleavage by a sequence specific metal complex M _t _ --l'--- __ ~. denature and electrophorese A B c c: cleavage by a nonspecific metal complex in the presence of protein MMM(~ t + + + + 479 B: cleavage by a nonspecific metal complex M M M M M t + + + + t __ denature and electrophorese denature and electrophorese Figure 8.11 DNA cleavage by metal complexes. Shown schematically is the method used with single-base resolution to discover the sites where metal complexes are bound on double helical DNA. After the metal complex is bound to several sites on a radioactively end-labeled (*) DNA fragment and activated to permit strand cleavage at the binding sites, the nicked DNA is denatured and electrophoresed on a high-resolution polyacrylamide gel, and the gel submitted to autoradiogra- phy. From the molecular weights of the end-labeled denatured fragments, the positions of cleav- age and therefore binding by the metal complex may be deduced. The results in lane A show the cleavage pattern observed for a metal complex that binds to a specific site. The results in lane B show cleavage observed for a nonspecifically bound metal complex that binds and there- fore cleaves at every base site. Lane C illustrates a footprinting experiment. When protein is bound to DNA at a specific site, it protects the DNA from cleavage by the metal complex at its binding site, thus producing a "footprint" in the gel: the absence of end-labeled fragments of those lengths that are protected from cleavage as a result of protein binding. A. Spectroscopic Probes As discussed above, the tris(phenanthroline)ruthenium(II) complexes offer a novel spectroscopic probe of nucleic acids, since their luminescence is increased upon intercalation into the double helix. As a result the complexes provide a simple luminescent stain for DNA in fluorescent microscopy experiments. More inter- esting, perhaps, is the conformational selectivity of derivatives of tris(phenanthro-
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480 8 / METAUNUCLEIC-ACID INTERACTIONS line)ruthenium. Ru(DIPh
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