FIGURE 1.
Transcription of siRNAs from convergent RNA Pol III promoters and their RNAi activity in cultured cells. (A) Construction of the duplex siRNA-expression plasmid, pGD-siC. Two complementary RNA strands with poly(U) at their 3′-ends are synthesized from the human H1 and U6 promoters. [(T)5] Transcription termination signals; (+1) the transcription initiation site. The restriction enzyme sites HindIII and BamHI were used for cloning oligonucleotides encoding siRNAs. (B) Sequence of H1/U6 promoter-driven siRNAs and tsiRNAs. (siC) Control, (siE) EGFP-specific, and (siR) Renilla luciferase-specific siRNAs. (s21) The 21-nt sense-strand transcription from the H1 promoter; (a21) the 21-nt antisense-strand transcription from the H1 promoter. tsiC and tsiER(s25s25) are longer duplex RNAs, fusing two different control siRNAs or the extended 25 nt siE(s25) and 25 nt siR(s25). Colored boxes show the position of the antisense sequences for (green) siE and (red) siR within the duplex RNAs. (Bold) The extra nucleotides required for efficient initiation of Pol III promoter-driven transcription from a purine sequence. The two U's at the 3′-end of each strand are from the transcription termination signal. (C) RNAi activity of the siE(s21)-, siE(a21)-, siR(s21)-, and siR(a21)-expression plasmids. Luciferase activity from cells transfected with the control siRNA construct (siC) was set at 100%. Mock-transfected cells were used as a negative control. Values represent means (+SD) from three replicate transfections. (D) RNAi activity of a tsiRNA, tsiER(s25s25), targeting EGFP and RLuc genes. Luciferase activity from cells transfected with the irrelevant tsiC-encoding plasmid was set at 100%. Transfections and measurements were performed as described above.