Experiments difficult however requiring relatively

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experiments difficult, however, requiring relatively high concentrations of ma- terial. Nonetheless, the sensitivity of luminescent lifetimes to coordination and indeed solvation is providing a novel spectroscopic handle to explore binding sites and structures of the macromolecules. Another quite novel luminescent
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2+ 481 Figure 8.12 Two spectroscopic probes of nucleic acids: A-Ru(DIP)/+ and Ru(bpYhdppz2+. handle has been phenanthroline and diphenylphenanthroline complexes of cop- per(I).61 These complexes are extremely valuable cleavage probes, as we will see later; to characterize better their interactions with the helix, luminescence experiments are being explored. A problem here has been the nonphysiological conditions necessary to achieve detectable luminescence. Nonetheless, studies with the copper complexes demonstrate how the whole range of transition-metal chemistry and spectroscopy is beginning to be applied in sorting through nu- cleic-acid interactions. B. Metallofootprinting Reagents Probably the most widespread application of metal nucleic-acid chemistry in the biology community has been the utilization of metal complexes for chemical footprinting. The footprinting technique (Figure 8.11) was developed by biologists 62 as a means of locating protein-binding sites on DNA.32P-end-la- beled double-stranded DNA fragments could be digested with a nuclease, such as DNAse, in the presence or absence of DNA-binding protein. After electro- phoresis of the denatured digests and autoradiography, one would find a "foot- print," that is, the inhibition of cleavage by DNAse, at the spot bound by protein, in comparison to a randomly cleaved pattern found on the DNA in the absence of binding protein. Although DNAse is still widely used, this footprint- ing reagent has some disadvantages: (i) the nuclease is not sequence-neutral in its cleavage, resulting in lots of noise in the footprinting background; and (ii) since the nuclease is itself a large protein, its ability to provide high-resolution footprinting patterns of smaller molecules is quite limited. Several metal complexes now serve as high-resolution, sequence-neutral chemical footprinting reagents. Some of these reagents are shown in Figure 8.13. The first, as mentioned previously, was MPE-Fe(II)Y The complex con-
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482 8+ MPE-Fe(II) Figure 8.13 Examples of metallofootprinting reagents. Rh(phihbpy3+, a photofootprinting intercalator, and MPE-Fe(II), a sequence-neutral intercalating agent. tains a sequence-neutral DNA binding moiety, the intercalator methidium, and a tethered DNA redox cleaving moiety, Fe(EDTA). The methidium, in binding nonspecifically to DNA, delivers the hydroxyl radicals, generated via Fenton chemistry at the Fe(II) center in the presence of peroxide and a reducing agent, to the DNA backbone in a random manner. Since the complex is small, high resolution can be achieved. Indeed, MPE-Fe(II) has been shown to footprint small natural products that bind to DNA, in addition to footprinting much larger DNA-binding peptides and proteins.
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