Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

Short interfering RNA (siRNA), the active agent of RNA interference, shows promise of becoming a valuable tool in both basic and clinical research. We explore the tolerance to mutations and chemical modi®cations in various parts of the two 21-nt strands of a siRNA targeting the blood clotting initiator Tissue Factor. The mutations were G/C transversions. The chemical modi®cations were 2¢-O-methylation, 2¢-O-allylation and phosphorothioates. We found that siRNA generally tolerated mutations in the 5¢ end, while the 3¢ end exhibited low tolerance. This observation may facilitate the design of siRNA for speci®c targeting of transcripts containing single nucleotide polymorphisms. We further demonstrate that in our system the single antisense strand of the wild-type siRNA is almost as effective as the siRNA duplex, while the corresponding methylated M2+4 version of the antisense had reduced activity. Most of the chemically modi®ed versions tested had near-wild-type initial activity, while the long-term activity was increased for certain siRNA species. Our results may improve the design of siRNAs for in vivo experiments.

To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to a-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes (1). Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3′-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3′-overhang with 3′-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3′-overhang resides on the antisense (AS) strand and dicing proceeds to the ‘right’ (‘R’) and one where the 3′-overhang resides on the sense (S) strand and dicing proceeds to the ‘left’ (‘L’). Interestingly, the ‘R’ version of the asymmetric 27mer is generally more potent in reducing target gene levels than the ‘L’ version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the ‘R’ form favoring S strand and the ‘L’ form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

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