Investigating Noncanonical DNA Structures as Anti_Cancer Drug Targets

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.