A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

We have studied the molecular properties of in-vitro-transcribed sliced small interfering RNAs (tsli-siRNAs) as an alternative RNAi agent for chemically synthesized siRNA. We describe here a simple and cost-effective procedure for high-purity production of tsli-siRNA using bacteriophage T7 RNA polymerases. tsli-siRNAs exhibit potent gene knockdown effects, with efficacy comparable with that of chemically synthesized sli-siRNAs and classical siRNAs. Furthermore, we found that it is very easy to prepare potent tsli-siRNAs with modified bases, such as 2′-fluorine- or biotin-16-modified tsli-siRNAs. tsli-siRNAs can cause a mild innate immune response, which can be easily eliminated by alkaline phosphatase treatment. On the other hand, this feature, which can be useful as a trigger of the innate immune response, can be enhanced by polynucleotide kinase treatment. Because of the simplicity of preparation and purification, the procedure presented here could be useful for the production of RNAi or immunostimulatory reagents.