Escherichia coli RNase III, which is involved in the maturation and degradation of diverse cellular, phage, and plasmid RNAs (6-9), digests long dsRNA to short duplex products ranging from ~12 to 15 bp in length with termini identical to those produced by Dicer (9). The average product length generated by RNase III digestion can be increased by altering digestion conditions, and it has recently been shown that these longer products (21 bp range) can effectively mediate RNAi in both mammalian cells (10) and mouse embryos (11). We demonstrate here that the siRNA cocktails produced by complete digestion by RNase III (~12 to 15 bp) are capable of silencing specific genes at levels comparable to chemically or enzymatically synthesized siRNAs.
Efficient Digestion of Distinct dsRNA Sequences. Using optimized digestion conditions we analyzed the ability of RNase III to digest a number of long dsRNA substrates. Human GAPDH, La, and c-FOS dsRNA (200 bp) was prepared by in vitro transcription (Silencer siRNA Cocktail Kit (RNase III); See Materials and Methods). The dsRNA was digested using 1 U RNase III per microgram of RNA for 1 hour at 37C, to generate siRNA cocktails for each target gene. After a 1 hour digestion with RNase III, the long dsRNAs were reduced to fragments <30 bp, with the majority between 1215 bp (Figure 1A). In addition, dsRNAs to Cyclophillin,