SOLID-STATE 2D-NMR INVESTIGATION OF DNA &ITS COMPLEXES

We propose to apply a two-dimensional solid-state NMR
technique, recently invented by the PI, to the determination of
the structure of DNA and of bound molecules associated with it.
The technique uses magic-angle spinning to acquire high-
resolution NMR spectra of oriented DNA films produced by a
fiber-spinning technique, while encoding the orientations of each
group in the polymer as sideband intensities in the 2D spectra.
These intensities can be directly used to give the orientational
distribution function of individual groups: without prior
assumptions these functions can be combined to generate the
structure of the molecule. The proposed technique has some
advantages over existing physical methods of structural
determination, and because it probes molecular orientations
rather than dimensions, is likely to be complementary to them.
Preliminary calculations show that the spectra distinguish
between the major conformations of DNA, between different
models of the structure of these conformations, and between
different alignments of bound drug molecules relative to the DNA
fiber. We propose first of all to construct an apparatus for the
reproducible production of films of oriented native DNA in the
various forms which can be produced by altering electrolyte
concentration and ambient humidity, and to analyses via 2D-NMR
the structure of these conformers, refining and removing
ambiguities in existing models. We shall then examine the
conformation of synthetic DNAs of defined sequence, and
compare their structures with those of native materials. Finally,
we shall synthesize a series of isotopically-labelled DNA-binding
ligands, among them several clinically important cytotoxic and
antiviral drugs, and determine the structure of their complexes
with high-molecular-weight DNA. This will be possible at very
low ratios of drug molecules to DNA base pairs. The ultimate
goal is to establish a new physico-chemical tool to aid in the
characterization and development of novel pharmacological
materials, and to add to the understanding of the processes of
genetic replication, transcription and regulation.