Grant: Discovery Grants Program - Individual

This research program investigates new approaches for lowering the levels of gene expression using short interfering RNAs (siRNAs) as substrates for the natural RNA interference (RNAi) pathway. Natural siRNAs fall short in that they are a) unstable to their environment and are degraded easily; b) exhibit relatively poor cell membrane permeability; c) exhibit off-target effects which causes non-specific toxicity; d) activate the immune system inadvertently; and e) have poor biodistribution. This research program continues to offer novel innovative methods to combat these challenges by making unique chemical modifications within the siRNA to alter its stability and specificity profile. The overall long-term goals of the research program are to understand, characterize, and predict the impact that chemical modifications have on the efficacy, potency, and specificity of siRNAs. The short-term objectives of this interdisciplinary research program utilize organic chemistry, biophysical analysis, and cell biology to generate new classes of chemically-modified siRNAs. This will be achieved by studying siRNAs bearing triazole backbones and functionalized spacer groups. In our previous work, we synthesized siRNAs bearing neutrally-charged triazole and amide backbones, and these chemically-modified siRNAs were highly active as gene-silencing substrates. These studies were the first to highlight RNAi compatibility with neutrally-charged backbone modifications within the Watson-Crick region of the siRNA. This proposal focuses on expanding the scope of backbone modifications by synthesizing second-generation triazole-backbone siRNA derivatives. These include the synthesis of a) chiral triazole-backbone modifications, b) polymeric triazole modifications, and c) triazole-backbone modifications in conjunction with base-modified variants. These molecules are designed to improve the structure-activity relationship of the siRNA and this work will provide new knowledge in designing novel backbone-modified siRNAs. In another proof-of-concept study that we published, we discovered a promiscuous area within the central region of siRNAs that are highly active when up to three nucleobases were replaced with a non-cleavable carbon-based spacer linker. Given this high-level of promiscuity, this approach involves expanding the scope of chemical functionality located at the centre of the siRNA duplex in order to fine-tune the chemical and functional characteristics of the duplex RNA. Molecular functional groups such as amines, amides, alcohols will be installed in order to improve the overall charge of the molecule. Functional groups such as allyl and alkyne groups will be installed due to their compatibility and reactivity with metal-catalyzed reactions used to covalently link molecules of interest such as cholesterol, guanidium groups and other related modifications to this area. In addition, other variants that will be studied that span the central region of the siRNA include moieties such as aromatic groups, amino acids, fluorescent probes and molecules that isomerize when photo-induced. These studies will provide new knowledge in an area of siRNA development that has not been explored and will generate new chemically-modified siRNAs with novel properties. The potential of harnessing this natural pathway would have enormous impact on research and medicine for not only Canada, but also worldwide. The research outlined in this proposal will provide new and novel approaches to chemically-modify siRNAs and will provide an outstanding interdisciplinary training environment in organic chemistry, bioorganic chemistry, biophysical chemistry, and biochemistry for both graduate and undergraduate students.