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. / Graduation date: 2002
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29609 |
| Date | 15 June 2001 |
| Creators | Ivancic, Monika |
| Contributors | Hsu, Victor L. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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