Advantages of esiRNA

High on-target specificity and silencing efficacy

Off-targeting remains a challenge in RNAi knockdown experiments utilizing si or shRNAs. Off-targeting means that the RNAi mediator silences other transcripts in addition to the intended one in a sequence-dependent manner. Consequently, observed phenotype changes can be due to the knockdown of the intended target, the unintentionally silenced transcripts, or a combination of both. Especially in medium- to large-scale screens, off-targeting increases costs and efforts because the majority of primary hits are false positives.
Specificity of siRNAs has improved, e.g. by avoiding the 3’-untranslated regions or homologies to the seed region. However, a design algorithm that can faithfully predict and prevent off-target effects is presently not available.

In contrast, esiRNAs are inherently target specific due to the complex mixture of many different siRNAs: All siRNAs in the pool have the same on-target, but differ in their sequence-dependent off-target signatures. Since the concentration of each siRNA in the pool is very low, the silencing capacity of off-targets is very low, too. In summary, all siRNAs in the pool contribute to the silencing of the on-target , while their off-target effects are diluted out (Figure 1a).

This positive correlation between pool complexity and silencing specificity was confirmed experimentally. Microarray analysis of transcript changes after transfection of increasingly complex pools of siRNAs showed that the number of off-targets decreases drastically with the number of siRNA sequences in the pool from several hundred off-targets to just three (Figure 1b, c).

Figure 1a
Figure 1b
Figure 1c

Next, we compared the silencing specificity of DEQOR-optimized esiRNAs with chemically synthesized siRNAs using microarray expression analysis. esiRNAs showed more than 20-fold higher target specificity in comparison to individual siRNAs (figure 3a, 3b), while maintaining the same silencing efficacy as single-sequence siRNAs (figure 3b). These results make esiRNAs the silencing triggers of choice for efficient silencing with minimal off-target effects.

Figure 3a
Figure 3b: esiRNAs produce on average 33% fewer false positives than siRNAs.

High knockdown-efficacy

All esiRNAs are optimized by the design algorithm DEQOR, which ensures the presence of highly potent siRNAs in each esiRNA pool. Therefore, esiRNAs are highly efficient silencers as confirmed by qPCR (figure 4a) and Western blotting (figure 4b). esiRNAs relieve of the tedious and time-consuming search for efficient silencing triggers. Make esiRNAs your silencing trigger of choice for efficient and specific RNAi experiments!

 

figure 4a
figure 4b