Short 5 '-phosphorylated double-stranded RNAs induce RNA interference in Drosophila

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)" [1-4]; this process is calle d "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA be comes processed by Dicer [14] or another RNase III-like enzyme to short dsR NA fragments of about 21-23 nucleotides (nt) [15], which are incorporated i n the RNA-induced silencing complex (RISC) [16] that directs target-specifi c RNA degradation [17, 18]. Here, we show that different synthetic dsRNA ca ssettes, consisting of two 5 ' -phosphorylated RNA strands of 22 mt each, c an initiate RNA! in Drosophila embryos. The cassettes were active at simila r quantities required to initiate RNAi by conventional dsRNA. Their sequenc e specificity was confirmed using synthetic dsRNA cassettes for two differe nt genes, Notch and hedgehog; each time, only the relevant embryonic phenot ype was observed. Introduction of point mutations had only a moderate effec t on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. W-phosphorylated synthetic RNA was m ore active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.