Defining the optimal parameters for hairpin-based knockdown constructs.

Induction of gene silencing using intracellularly expressed silencing triggers has been explored for large-scale loss-of-function screening, creation of knockdown cell lines or knockdown animals, and disease intervention. In all of these applications, the use of highly potent silencing constructs can maximize the possibility of obtaining target knockdown and thereby is intrinsically important for the chance of success. Several attempts have been made to improve the potency of a silencing construct. Results published in high profile journals such as Nature Biotechnology and Nature Genetics suggest that shRNAs with a 29-nucleotide (nt) stem is much more potent than shRNAs with a 19-nt stem, and miR30-based silencing constructs are much more potent than shRNA-based constructs. In this study, we systematically investigated several parameters, including the use of shRNA- or miR30-based scaffolds, the length of shRNA, and the selection of shRNA sequences for their impact on the knockdown efficiency of a silencing construct. Our studies revealed that the optimal configurations for a potent silencing trigger could be an shRNA with a 19-nt stem and a 9-nt loop. By comparing properties that favor the functional shRNAs and siRNAs using a set of 190 shRNAs against 19 targets and 360 siRNAs against four targets, we found that the functional shRNAs and siRNAs displayed similar but not identical nucleotide preferences. Based on the characteristic nucleotide preferences in the functional versus the nonfunctional shRNAs, we developed a computer program that outperforms an advanced siRNA selection algorithm for the enrichment of highly functional shRNAs.