The synthesis of tetra-modified RNA for the multidimensional control of gene expression via light-activated RNA interference.

ABSTRACT

Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process are siRNA or dsRNA that have been modified with four photocleavable groups of dimethoxy nitro phenyl ethyl (DMNPE), located on the four terminal phosphate groups of the duplex RNA. These mediators can be easily synthesized and purified using two readily available products synthetic RNA oligonucleotides and DMNPE-hydrazone. The synthesis of the tetra-DMNPE-modified duplex RNA is made possible by a remarkable regiospecificity of DMNPE for terminal phosphates (over internal phosphates or nucleobases) that we have previously identified. The four installed DMNPE groups effectively limit RNAi until irradiation cleaves them, releasing native, active siRNA. By using the described protocol, any process that is mediated by RNAi can be controlled with light. Although other methods exist to control gene expression with light by using specialized reagents, this method requires only two commercially available products. The protocol takes ∼3 d in total for the preparation of modified RNA.