Tolerance of RNA Interference Toward Modifications of the 5′ Antisense Phosphate of Small Interfering RNA

Bringing RNA interference (RNAi) under the control of light will allow the spacing, timing, and degree of gene expression to be controlled. We have previously shown that RNAi by small interfering (si) RNA can be modulated through randomly incorporated photolabile groups. Our and others interest is to find key locations on siRNA that can completely block RNAi until irradiation releases completely active siRNA. Some literature suggests that the 5′ phosphate of the antisense strand of siRNA cannot be modified without completely blocking RNAi. We have examined this site as a potential switch for light control of RNAi and present evidence that siRNA modified at the 5′ antisense phosphate can still cause RNAi, although not at the level effected by fully native siRNA. This contrasts with results from the literature, which suggest that modification of the 5′ antisense phosphate will completely abrogate RNAi in siRNA. We have used mass spectrometry to identify and quantitate possible impurities that may be responsible for residual RNAi and show that they are present at 1% or less. Our results suggest that there is an inherent tolerance of the RNAi machinery toward modification of the 5′ antisense phosphate.