Chemical Synthesis and Applications of Nucleic Acid Analogues

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Nucleic acids are the fundamental molecules of life, carrying the genetic blueprints for all living things on Earth. Refined through billions of years of evolution, they achieve an ideal balance between complexity and elegance, a perfect marriage of form and function. As such, synthetic DNA and RNA oligonucleotides and their analogues are incredibly useful tools for molecular biology, functional genomics, and biochemistry research. We will pursue three specific aims within this proposed NSERC Discovery Grant program. First, we will synthesize and characterize branched RNA analogues in order to study the structure and properties of the human lariat RNA debranching enzyme (hDbr1), the only enzyme known to hydrolyze 2’,5’-phosphodiester bonds of lariat RNA introns; carry out structure-guided synthesis of non-hydrolyzable RNA branchpoint analogues through the introduction of sugar and base modifications to create conformations that mimic that of the lariat RNA branchpoint observed in nature; determine the structures of Dbr1•inhibitor complexes, and test the toxicity of Dbr1 inhibitor compounds in neuronal cell lines. Second, we will design synthetically modified DNA and RNA structures that are able to "switch off" genes (e.g., disease-causing genes) by intercepting mRNA, by virtue of their expected increased binding affinity for RNA and stability in biological media. Third, we plan to study folded genomic DNA structures, i.e., the G-quadruplexes and i-motifs. While the former structures are already known to control gene expression, the biological role of i-motifs remains elusive. We propose to correlate, for the first time, telomerase activity with transient formation of G-quadruplexes and putative i-motifs within duplex DNA. The stabilization of i-motif structures at physiological conditions by our proposed approach will open new research directions on i-motifs and G-quadruplexes. Our proposed research program will capitalize on the expertise and research advances obtained during the previous granting period to further advance the state of the art and shed light on several clinically relevant questions in bioorganic chemistry. This proposed program minimizes risk through building on previous achievements, while still continuing our lab's theme of boldly pushing the leading edge of nucleic acids research. Our scientific approach combines solution and solid-phase synthetic methods, drug discovery, spectroscopic analysis, and molecular biology techniques. These studies expose my students to a wide range of topics in nucleic acid chemistry and biology research, and to strategies used for identifying candidate molecules and developing them into useful probes and/or therapeutic drugs.