Deciphering the structure and dynamics of non-canonical DNA implicated in cancer - PROJECT SUMMARY The proposed research will improve the selectivity and efficacy of anticancer therapies by contributing new knowledge about non-canonical nucleic acid structures, G-quadruplexes (GQ) and i-motifs, and details of their interactions with small-molecule ligands. Bioinformatics studies have identified 700,000 sequences with GQ- forming potential in the human genome. The C-rich opposite strands are proposed to form i-motifs. There is now convincing biological evidence that GQs and i-motifs form in vivo and that these structures complement each other in regulating a variety of cancer-related biological processes. GQ nucleic acids have been firmly established as an important therapeutic target for cancer. The same evidence for i-motifs is steadily accumulating. Small molecules that bind selectively to GQ DNA and RNA and to i-motifs have been identified, and some have been shown to inhibit tumor cells growth; however, exact mechanisms underlying this inhibition are not known. Additionally, the number of selective i-motif ligands is low. Such ligands may ultimately become lead compounds for cancer intervention superior to conventional mutagenetic therapies. Nucleic acid-centered drug discovery programs suffer from limited structural information for GQs and i-motifs, especially in the presence of ligands. As of now, no structure of an i-motif-ligand complex has been reported. The situation is further complicated by high structural diversity of both GQs and i-motifs, their contradictory biological functions, and our limited ability to target their specific folding topology (e.g., parallel vs antiparallel GQs). To address these challenges, we propose to perform comprehensive crystallographic investigation of telomeric and oncogene promoter GQs and i-motifs, both alone and in complex with novel and commercially available selective small-molecule ligands. The diversity of interactions which provide stability to GQs and i-motifs will be determined. The details of ligand binding sites, as well as chemical and structural features of ligands essential for their affinity and selectivity will be identified. This work will be complemented by spectroscopic and calorimetric studies of the thermodynamic parameters of ligand binding. For GQ DNA, that is much more explored, structural studies will be complemented by rigorous kinetic exploration of ligand-assisted GQ folding. Kinetic information can help us identify the timescale of GQ formation and, thus, biological processes that can be affected by the presence of these structures. Collectively, the proposed work will enhance our understanding of GQ and i-motif structural plasticity, supply coordinates for drug discovery platforms, shed light on the origin of ligand selectivity for a specific DNA or RNA target, and guide the design of novel anticancer therapies all while providing transformative training to Swarthmore undergraduates.
