Chemical Methods for Illuminating Cellular DNA

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Fluorescent probes have revolutionized the study of biological macromolecules in their native environments. However, as compared to the extensive methodologies developed for proteins, relatively few imaging strategies are currently available for cellular nucleic acids. These methods are limited by their negative impact on native systems and/or inability to be applied in live cells and animals. My program aims to develop non-toxic fluorescent probes for characterizing nucleic acid structures and dynamics in living cells. We design and synthesize new fluorophores and suitable metabolic precursors for their introduction into cells. In addition to the study of DNA folding and ligand binding interactions, a number of probes developed in my lab have been commercialized and used by many other research groups working in the fields of regenerative medicine, genome architecture, stem cell biology, and virology. My group is currently moving to McGill University. An award from NSERC will be critical for re-establishing my rigorous research and training program in Canada, where we aim to: 1) synthesize fluorescent nucleobase analogues with sufficient brightness to probe DNA dynamics in single molecules and living cells; 2) develop fluorogenic nucleoside triphosphate analogues that report DNA or RNA synthesis in real time; 3) conduct bioorthogonal synthesis of fluorescent nucleobase analogues in genomic DNA; 4) develop new fluorogenic probes that selectively react with bioorthogonal functional groups present in cellular G-quadruplex structures. Our new probes and techniques for studying biopolymer synthesis, structure, and dynamics will provide unprecedented insights into the behavior of cellular nucleic acids. The visualization of DNA/RNA dynamics in living cells will require the development of much brighter fluorescent nucleobase analogues than are currently available. The C-nucleosides proposed here constitute a novel family of intensely bright nucleobase analogues containing a single C-H----O or C-H----N interaction in the Watson-Crick interface that supports their faithful enzymatic incorporation into cellular nucleic acids. We will also develop copper-free bioorthogonal chemical reactions between two small precursors that generate a fluorescent nucleobase as the product in the chromatin of live cells. The fluorescence properties of these probes will provide specific information about structural context (ex. duplex, single-stranded, i-motif, or G-quadruplex), and can also be used to track real-time DNA-drug and DNA-metal binding reactions in live cells. Future applications of our research are very broad and include the development of theranostic agents for cancer analysis and treatment. In addition to excellent student training opportunities that build a highly qualified workforce, this program has the potential to benefit Canadian society by generating new business and health care opportunities in pharmaceuticals, diagnostics, and biotechnology.