Modulation of the Properties of Nucleic Acids through Structural Modifications

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Nucleic acids are crucial for many fundamental biological processes. They carry genetic information, and transmit it through replication, transcription and translation. These molecules are also recognized for their regulatory roles in many cellular processes. This proposal aims at approaches to introduce unique structural moieties into nucleic acids and consequently, confer properties to these molecules that will further our knowledge on the fundamental biological processes. In this regard, three chemical methods will be investigated. First, a methodology to metabolically label nucleic acids will be explored. This chemistry is based on the inverse Electron Demand Diels-Alder reaction between tetrazine and strained alkenes. It is proposed that a strained alkene moiety will be incorporated into nucleosides and subsequently, incorporated into nucleic acids enzymatically. Upon this incorporation, tetrazine that is covalently linked to a BODIPY fluorophore (non-fluorescent in the form of tetrazine-BODIPY conjugate) will be added, where an inverse Electron Demand Diels-Alder reaction takes place to restore the fluorescence. As such reactions are typically very fast, this chemistry will virtually allow for real-time tracking of cellular processes involving nucleic acids, and importantly, with minimal fluorescent background. Second, a chemical mimetic of restriction enzymes that will cleave nucleic acids in a sequence-specific manner is proposed. This approach is based on the Bergman cyclization of enediyne moieties that generate reactive biradicals during the cyclization. By covalently linking enediyne moieties to synthetic oligonucleotides, Bergman cyclization of enediyne can be directed towards the desired cleavage site due to the complementarity of the oligonucleotides with the target nucleic acids. This approach will be particularly useful in fragmenting large nucleic acids, such as genomic DNAs, in cell and molecular biology studies. Last, a methodology to regulate nucleic acid properties by light will be explored through the incorporation of cyclic azobenzene into nucleic acids. Due to the structural changes in cyclic azobenzene as a result of external light stimuli, nucleic acids bearing this modification will respond to light of specific wavelengths. This chemistry will be explored for spatiotemporal regulation of processes such as hybridization and transcription, and in the long term gene functions in living systems. The proposed research will provide a training ground for over a dozen much-needed highly qualified personnel. Work from the proposed research will further our knowledge on some fundamental biological processes that will benefit the wellbeing of Canadians, and contribute to Canada's knowledge-driven economy.