| For over a decade, the use of double-stranded short interfering RNAs (siRNAs) to silence the expression of genes associated with disease at the translational level has gained much attention. Using a highly conserved endogenous pathway within cells, siRNA technology displays a high degree of target specificity and potency. However, traits required for the successful design of siRNA-based therapeutics such as resistance to nuclease-mediated degradation, improved cell membrane permeability and reduced off-target toxicity, are compromised by the native structure of duplex RNA's charged backbone. This study therefore focused on synthesizing novel and neutrally-charged triazole-linked nucleoside dimer analogs which were incorporated throughout siRNA duplexes using DMT-phosphoramidite chemistry, in order to attenuate the negative contributions of RNA's native backbone. In order to further elucidate the mechanism of the RNA interference (RNAi) pathway, additional siRNAs were synthesized containing commercially available abasic alkyl spacers. Through the robust copper(I)-catalyzed Huisgen [3+2] cycloaddition, azide and alkyne nucleic acid monomers were joined through the heterocyclic linkage in nearly quantitative yields, producing the novel triazole-linked uracil-uracil (UtU) and cytosine-uracil (CtU) dimers. Results from cell-based assays indicate that triazole-modified siRNAs are capable of silencing the transiently-expressed reporter gene firefly luciferase and the endogenous gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a dose-dependent manner. In addition, modifying the 3&... |
