The asymmetric binding of Actinomycin D to DNA hexamers Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/pc289n14k

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  • The solution structure determination of DNA molecules has been an important part of structural biology. NMR solution structures are a complement to structures solved via X-ray crystallography; the two methods are the only ways of obtaining three dimensional coordinates of macromolecules. Because of the nature of the molecule, the solution structure determination of DNA has been a challenging task. Assignments are the first and most important part of NMR structures, and can be simplified for DNA with the use of the rotating frame Overhauser spectroscopy (ROESY) experiment. The ROESY technique can be used for unambiguous assignments of H2' and H2" protons and for distinguishing the three main forms of DNA duplexes: A-form, B-form and Z-form. Many types of DNA have been examined using NMR spectroscopy, including drug-bound DNA complexes. Most previous studies of complexes of the anti-cancer drug Actinomycin D (ActD) and DNA used self- complementary sequences to identify stabilizing features. The studies presented in this thesis use non-self-complementary DNA hexamers to identify the two orientations in the binding of the asymmetric ActD drug. The largest preference of asymmetric binding was found for the d(CCGCCG)•d(CGGCGG) sequence; however, NMR spectral complications prevented the structure elucidation of this complex. Instead the solution structure was determined for the complex with the next largest orientational preference, ActD:d(CTGCGG)•d(CCGCAG), which has 67% of ActD molecules intercalated with the benzenoid side of ActD in the first strand. The solved structure identifies unusual DNA features, which could be due to the bound drug inducing structural changes to the B-DNA duplex or the presence of conformational motion. For seven of the eight sequences, the orientation of ActD intercalation within the DNA duplex was identified. The largest preference occurs when the benzenoid intercalation site is followed by a guanine. When this guanine is replaced by an inosine, a reduction in the asymmetric binding of ActD is observed, indicating that the guanine NH₂ group plays a role in the intermolecular contacts. Thus, the two orientations of ActD binding are not present in equal concentrations although their structures are similar, and the preference of orientation is influenced by the asymmetric DNA sequence flanking the intercalation site.
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