Energy Profile and Secondary Structure Impact shRNA Efficacy

Many characteristics of functional synthetic siRNAs have been identified. Our three-phase algorithm was developed to design siRNA on a whole-genome scale based on these characteristics. When this algorithm was applied to design shRNAs, the validated success rate was over 70%, which was almost double the rate reported for TRC library. This indicates that the designs of siRNA and shRNA may share the same concerns. Further analysis of the shRNA dataset of 444 designs reveals that the high free energy states of the two terminals have the largest positive impact on the shRNA efficacy. Enforcing this energy discrepancy of both terminals can further improve the shRNA design success rate to 83.1%. We also found that functional shRNAs have less probability for their 3´ terminals to be involved in mRNA secondary structure formation, which indicates that the accessibility of the 3´ terminal is another key factor to shRNA efficacy.