Silence of the fish: injection of photoswitchable short interfering RNA oligonucleotides into Japanese medaka embryos (oryzias latipes) to photochemically control gene silencing

Abstract

The siRNA duplex functions by binding to and cleaving mRNA, a process known as the RNAi pathway. Existing siRNAs face challenges such as off-target effects and unpredictable prolonged gene-silencing. To address these issues, enhancing their therapeutic potential, siRNAs can be modified controlling their role in the RNAi pathway. An Ortho-functionalized, tetrafluorinatedazobenzene was integrated into the siRNA backbone via phophoramidite chemistry to generate a class of photoswitchable F-siRNAs. These F-siRNAs retained the ability to photoisomerize from an active trans to an inactive cis state through blue and green light respectively. This thesis reported on a novel technique to inject single cell medaka embryos with both Wt and F-siRNAs. Medaka are a small freshwater teleost fish that have a number of desirable features for use as a vertebrate model in gene silencing projects. These attributes include daily spawning, a number of useful genetic strains, a completely sequenced genome, and a transparent egg.