An RNAi reagent is said to be specific if it interferes with the intended target mRNA(s) only ('on-target') but with no other, unintended target mRNA(s) ('off-target').
E-RNAi tries to optimize the reagent's specificity the following way:
- For the design of long dsRNAs any perfect homologies of contained siRNAs (by default 19 bp long with an offset of 1) to other than the intended target mRNA(s) are avoided. For the design of single siRNAs perfect homologies of this sequence to unintended target mRNA(s) are avoided.
- 'Off-target' effects can also arise from imperfect homologies to unintended target mRNA(s). To address this E-RNAi maps the full reagent sequence (long dsRNAs or siRNA) to the transcriptome and reports homologies of all targets identified (with E-values below a selected cutoff). E-RNAi does not include this step during the design, but only as evaluation of the reagent specificity. RNAi reagents with high homology to others than the intended target mRNA(s) should be used with caution.
- Regions of low sequence complexity can lead to unwanted 'off-target' effects. E-RNAi avoids several kinds of low-complexity regions in the design process described here.