Many characteristics of functional synthetic siRNAs have been identified. Our three-phase algorithm was developed to design siRNA on a wholegenome 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. While it has been reported that the free energy states of particular base pairs in siRNA sequences are critical for siRNA efficacy, our analysis found, however, that it is the free energy states of “the end groups” (the first 7 base pairs from the 3' terminal and the first 6 base pairs from the 5’ terminal) that are “collectively” responsible for the successful shRNA designs. Enforcing this ener...