shRNA Sensor Analysis of 5'' U converted shRNAs reveals 3'' target bias exists in addition to 5'' guide bias

shRNAs selected with the shERWOOD algorithm were converted to have a U at the 5'' end of their guide. When endogenous 1U shRNAs were compared to artificial shRNA via the sensor algorithm, the endogenous shRNAs were found to be more efficacious. Overall design: Purpose: Structural studies have hinted that the 5'' end of shRNA guides is engulfed in the RISC complex. It has also been reported that shRNAs with a 5'' U are more efficacious than those with other 5'' caps. We wished to determine whether replacement of shRNA guide 5'' nucleotides with a U, regardless of the corresponding target base, would increase their efficacy. Method: For each gene in the "druggable genome" 10 shRNAs were selected with the shERWOOD algorithm. In each case the score was assessed as if the guide had a 5'' U. Sensor constructs were designed pairing 1U-guide shRNAs with their endogenous target. shRNAs were assessed for efficacy via the shRNA sensor assay (Fellmann et al. Mol Cell 2011). Results: shRNAs with artificial 5'' Us were found to be less efficacious than those with an endogenous 5'' U,