Grant: CIHR Fellowship

The proposed research aims at using oxidative desulfurization chemistry to activate functional RNA (siRNA, ribozymes, riboswitches and aptasensors). The 2-thiouridine (2sU) unit within an RNA strand is susceptible to reaction with hydrogen peroxide (H2O2). Analysis of the chemical reaction revealed an H2O2-mediated oxidative desulfurization pathway, heavily dependent on the solution pH. At pH 8, 2sU is converted to canonical uridine (U), whereas at low pH, the main product is 4-pyrimidone (2hU; uridine lacking a 2-keto functionality). Interestingly, the desulfurized products have a dramatic shift in the base-pairing preferences (for instance, 2sU pairs more strongly with A compared to U). This interdisciplinary research aims at using the desulfurization chemistry to fine tune structure and functionality of RNA-based technology. Certain RNA sequences are capable of carrying out enzymatic function (e.g. ribozymes) or bind to pre-selected targets (e.g. short interfering RNA (siRNA) or RNA aptamers), which are generally dependent on specific internucleotide interactions. Given that the presence of 2sU can disrupt critical base pairs necessary for RNA activity, the project aims at using a novel redox switching mechanism to either silence or activate functional RNA sequences as biomedical tools. The biocompatibility of such 2-thio modifications within RNA has precedence in the literature, making this avenue promising for in vivo applications. The approach outlined will pave the way to utilizing redox-sensitive RNA probes as tools in an unparalleled fashion. Preliminary work has shown that a specific aptamer (Spinach), transcribed using 2sU, instead of U, is effectively silenced. Activity is recovered upon desulfurizing the 2sU-containing aptamer, which further supports and bolsters the proposed research described herein.