RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types1,2,3. RNAi is mediated by ∼21-nucleotide small interfering RNAs (siRNAs)4,5,6,7,8, which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III–family enzyme9,10,11. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response12, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of ≤ 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.
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We thank Greg Hannon for providing a CMV-Dicer clone and synthetic siRNAs and for helpful discussions, Thomas Wehrman for help with luciferase assays, M.-C. Yee for help with Hi5 and Sf9 cultures, Jianbo Yue for help with chromatography, and members of the Ferrell lab for advice and critical reading of the manuscript. This work was supported by grants from the National Institutes of Health to J.E.F. (GM46383) and T.M. (CA83229).
J.W.M. and J.E.F. Jr. have filed a patent application related to using recombinant Dicer to generate siRNAs as described in this article.
We expressed full-length, His6- and T7-tagged-Dicer in Hi5 insect cells by baculovirus infection and purified it on a cobalt Sepharose column. Fractions were collected and elecrophoresed in a 6% (wt/vol) SDS-polyacrylamide gel and proteins were detected by either coomassie-staining or immunoblotting. The main protein eluted was a 225 kDa protein, which corresponds to the predicted molecular mass for Dicer (Supplementary Fig. 1A, top left panel). The main immunoreactive band on an anti-tag immunoblot also migrates at the predicted molecular mass for Dicer (Supplementary Fig. 1A, left middle panel), however the 225 kDa band is not present in the elution fractions collected from a mock purification of uninfected cellular lysates in either the coomassie stained gel (Supplementary Fig. 1A, top right panel) or on the immunoblot (Supplementary Fig. 1A, right middle panel). Taken together these results confirm the identity of the 225 kDa band as recombinant Dicer. We then determined whether the purified recombinant Dicer was able to process dsRNA into siRNAs. We incubated column fractions with a ∼500 bp dsRNA and analyzed the reaction products by native polyacrylamide gel electrophoresis. The purified Dicer efficiently converted the dsRNA to a form (Supplementary Fig. 1A, bottom left panel) that co-migrated with a synthetic 21 bp siRNA (data not shown). Because Drosophila extracts are capable of generating siRNAs4, it is likely that the Hi5 insect cells used to produce r-Dicer contained some endogenous dicing activity. Therefore, we addressed the question of whether the Dicer activity in our purified fractions was due to the r-Dicer or to some adventitiously co-purifying insect protein. We subjected lysates from uninfected Hi5 cells to the same purification procedure and assessed fractions for Dicer activity. A small amount of RNA processing activity could be detected in the total soluble Hi5 cell lysate and in the flow through from the cobalt affinity column (Supplementary Fig. 1A, bottom right panel). However this activity was not detected in the column eluates, indicating that it could not account for the high levels of dicing activity seen in the purified r-Dicer preparation. In addition, Dicer protein and activities co-chromatographed on Q-Sepharose (Supplementary Fig. 1B), again supporting the hypothesis that the r-Dicer is required for the dicing activity seen. r-Dicer was subjected to various reaction conditions to determine cofactor and substrate specificity as well as to ensure that the ∼21-mer was generated by an RNase IIIfamily enzyme (like r-Dicer) and not a nonspecific RNase or nuclease. dsRNA was efficiently processed by r-Dicer (Supplementary Fig. 1C, lane 1) but dsDNA was not (Supplementary Fig. 1C, lane 2), establishing that r-Dicer is RNA-specific. r-Dicer generated small amounts of ∼21 bp product from ssRNA, which may be due to cleavage of hairpins or contaminating dsRNA (Supplementary Fig. 1C, lane 3, and data not shown). r-Dicer was not inhibited by RNasin (Supplementary Fig. 1C, lane 4), distinguishing it from most ribonucleases. Mg2+ was required for r-Dicer activity (Supplementary Fig. 1C, lanes 6 and 7), but ATP was not (Supplementary Fig. 1C, lane 5). In contrast, other workers have found that ATP is important for RNAi and dicing in drosophila extracts4 and in Dicer immunoprecipitates9. However, since submission of this manuscript others have shown that in vitro dicing, at lest with recombinant human Dicer, does not require ATP19,21. High concentrations of manganese (2.5-5 mM) and calcium (10 mM) inhibited r-Dicer activity (data not shown). (PDF 118 kb)
Various ∼500-2300 bp dsRNAs were cleaved by r-Dicer to generate d-siRNAs. Large dsRNAs shown in Supplementary Figure 2A were 500-600 bps and those shown in Supplementary Figure 2B were 1400-2300 bps (see Supplementary Table 1 online for exact sizes). The contaminating reaction components and larger dsRNAs were separated from the d-siRNAs by a series of spin columns or by gel purification as inidicated. Shown are 15% native polyacrylamide gels after ethidium staining illustrating the quantity, quality and purity of siRNAs and d-siRNAs after purification as well as the constituents of the unpurified reaction. Electrophoresis verified that the d-siRNAs were not contaminated with large dsRNAs, the d-siRNAs remained double stranded, and the concentration estimates (estimated by A260) were accurate. The siRNAs/d-siRNAs shown in Supplementary Figure 2A were used in the experiments shown in Figure 2E-H and those shown in Supplementary Figure 2B were used in the experiments shown in Figure 3B. (PDF 406 kb)
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Myers, J., Jones, J., Meyer, T. et al. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat Biotechnol 21, 324–328 (2003). https://doi.org/10.1038/nbt792
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