RNA interference (RNAi) is an established tool for functional genomics studies that is also showing great potential for medical applications. Currently, one of the main goals in RNAi technology is the design and discovery of potent small interfering RNAs (siRNAs). Using a secreted luciferase from Gaussia princeps (GLuc), we developed a reporter assay, which allows for rapid potency assessment of siRNAs, by measuring luminescence activity in cell culture supernatants. The method was applied in microtiter plate format and validated by comparison to quantitative reverse transcription PCR (RT-PCR) and Western blot analysis. This reporter assay was used to evaluate in HeLa cells the potency of different siRNA mixtures generated by RNase III, or several synthetic siRNAs, all directed against human p53. The results show that all four siRNA mixtures generated by RNase III induce 50%—75% decrease of the reporter activity at less than 10 nM transfected concentration. In contrast, only one out of the five commercially available synthetic siRNAs showed comparable potency. These results suggest that one advantage of using enzymatic complex siRNA mixtures for RNAi is that, unlike single synthetic siRNAs, selecting a target region is not important to ensure potency.
Once Fire and Mello described the phenomenon called RNA interference (RNAi) (1), several studies that revealed its molecular mechanisms followed (see References (2,3,4,5) for reviews). Several methods to apply RNAi in mammalian cells have been devised, including chemically synthesized small interfering RNAs (siRNAs) (6) and siRNA mixtures generated by in vitro enzymatic digestion (7,8). Although guidelines and computational tools for designing effective siRNAs exist, experiments reveal that a majority of the synthetic siRNAs designed against a gene target are not very effective (9,10). Many published siRNAs do not work at low concentrations and are commonly used at 100 nM or even higher (9,11), despite the fact that off-target effects are observed at those concentrations (12,13,14,15).
In vitro processing of doublestranded RNA (dsRNA) by enzymatic digestion generates a population of siRNAs that targets multiple sites on the messenger RNA (mRNA). These have been shown to be more effective than single siRNA in silencing the target (7,8). Although these results support the idea that effective siRNA mixtures are less subject to design rules than single siRNAs, we wanted to quantify differences in potency of different siRNA mixtures or siRNAs used at low concentrations.
Generally, RNAi efficiency is quantified either indirectly by assessing target protein concentrations by Western blot analysis or directly by measuring target mRNA levels by quantitative reverse transcription PCR (RT-PCR). Western blot analysis and quantitative RT-PCR have proven to be specific and accurate. However, they are costly in terms of experimental time, because assays have to be designed specifically for each new sample (i.e., quantitative PCR primers or antibodies). An alternative approach to assess knock-down potency is to link the target of RNAi to a reporter gene and quantify the knock-down effect by measuring the activity of the reporter. This principle has already been used successfully with intracellular fluorescent/luminescent reporters (16,17,18,19,20). Here, we applied this general approach using a secreted luciferase reporter gene.
The luciferase of Gaussia princeps (GLuc) has some unique properties among luciferases, such as high activity, stability, and the fact that it is efficiently secreted from mammalian cells by virtue of its native signal sequence (21,22). In addition, its activity can be easily measured by available bioluminescent assays. The secretion of GLuc outside the cell facilitates the analyses of the samples. Since no cell lysis is required, rapid assays at different time points from the same experimental set are possible. We evaluated a GLuc-based reporter and used it to assay different synthetic siRNAs and different RNase IIIderived siRNA mixtures against the human p53.Materials and Methods pCMV-GLuc_p53 Reporter Vector
A segment of the human p53 cDNA [National Center for Biotechnology Information (NCBI) accession no. NM_000546], from position 514 to 1581, to be used as the target, was amplified by PCR and cloned. The primers used contain an XhoI restriction site as well as 5′ extensions compatible with the USER™ technology for rapid cloning into linearized LITMUS™ U plasmid using the USER enzyme system from New England Biolabs (Ipswich, MA, USA). The primer used were: 5′-GGAGACAUAAGCTTACTCGAGCCC CCTCCTGGCCCCTGTCATCTT-3′ (forward primer) and 5′-GGGAAAGUCTCGAGAAAGCTTGAGCCCCGGGACAAAGCAAATGGAA-3′ (reverse primer) (USER ends are bold, XhoI sites are underlined, and specific p53 sequence is italic). The PCR fragment was first cloned into the LITMUS U vector. The resulting plasmid was digested by XhoI, and the p53 fragment was subsequently inserted in the 3′ untranslated region (UTR) of GLuc, at the XhoI site of the pCMVGLuc Control Plasmid (New England Biolabs) to produce the pCMV-GLuc_p53 reporter vector.p53 Transcription Templates for siRNA Mixtures
A segment of human p53 (coordinates 553–1430) was inserted into the LITMUS 28i Vector (New England Biolabs). The insert was amplified with a T7 primer and digested with AciI. The resulting mixture of DNA fragments obtained was ligated at the BstBI site of LITMUS 28i Vector. Individual clones containing p53 fragments were selected. The coverage of p53 sequence resulting from selected clones is shown in Figure 1B.