Abstract:
This work focuses on the structure and interactions with ligands of noncanonical
DNA structures. These noncanonical structures include guanine quadruplexes (GQs), as
well as a novel type of quadruple helix. Sequences capable of forming these structures
are overrepresented at telomeres and replication perturbed loci, respectively. Better
understanding of these structures and ligand interactions will provide an improved
platform upon which mechanistic understanding and therapeutic treatments can be
designed.
Our discussion begins with the development and biophysical characterization of a
model for the smallest stable structure formed by the repeat sequence d(CAGAGG)n.
This sequence was one of many short purine rich tandem repeats recently identified by
the Brown lab to be associated with intrinsic replication stress and increased likelihood of
replication fork stalling. The resulting model is a monomolecular tetraplex containing
two stacked GCGC tetrads and three 4-nucleotide loops that connect the tetrads in an
antiparallel manner. This work describes the extension of the repeat to 3-nucleotide
overhangs on the 5’ and 3’ ends to improve the structure’s stability, as well as the results
of systematic mutations in the core tetrad and loop regions which further support the
model. Additional work to extend the model to longer, more biologically relevant repeat
lengths is discussed.
We sought further validation of the biophysical model, as well as elucidation of
its atomic details, through x-ray crystallography. A variety of constructs were screened
for crystal results, with varying degrees of success. The best crystals contain short
mutations to the 5’ and 3’ ends, and diffracted to 1.92 Å. However, due to a lack of a
suitable model for molecular replacement, poor anomalous redundancy, and striking
nonisomorphism, solution of the phase problem has been a major impediment. Results for
all phasing approaches are discussed below.
The final chapter addresses the potential for small molecule ligands, in particular
the highly cationic porphyrin TMPyP4, to stabilize the human telomere GQ structure
(Tel22) selectively as an anti-cancer therapeutic precursor. Five TMPyP4 derivatives
with varying cationic charge were assayed for binding strength and stoichiometry,
stabilization of Tel22, and selectivity for Tel22 over duplex DNA. A small decrease in
cationic charge, exemplified in porphyrin 4P3, resulted in both improved stabilization
and selectivity for Tel22 over TMPyP4.