Defining the optimal parameters for hairpin-based knockdown constructs - PubMed (original) (raw)
Defining the optimal parameters for hairpin-based knockdown constructs
Leiming Li et al. RNA. 2007 Oct.
Abstract
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.
Figures
FIGURE 1.
shRNA-based constructs outperform miR30-based constructs in target knockdown. (A) The schematic depiction of a siRNA and the corresponding shRNA or miR30-based silencing triggers. The bold characters represent the double-stranded part of a siRNA sequence that each set of silencing triggers is designed to produce in cells. The solid and broken arrows represent the main cleavage sites in miR30-based silencing triggers by Drosha and Dicer, respectively. (B) H1299 cells were transfected with 0.05 μg of each shRNA- or miR30-based construct, 0.15 μg of the firefly luciferase reporter, pGL3-control, and 0.015 μg of the renilla luciferase reporter, pRL-TK. The luciferase activities in transfected cells were determined, and the results were normalized to the luciferase activities in cells that were transfected with pGL3-control, pRL-TK, and a control vector (control). The Y-axis represents the normalized luciferase activities of each sample. The right panel listed the siRNAs against luciferase that were expected to be produced from the miR30- or shRNA-based constructs. (C) H1299 cells were transfected with 1 μg of shRNA- or miR30-based constructs and 1 μg of an expression plasmid encoding mouse tyrosinase. The tyrosinase activities in each transfected cells were determined, and the results were normalized to the tyrosinase activities in cells that were transfected with the tyrosinase expression plasmid and a control plasmid (Control). The Y-axis represents the normalized tyrosinase activities from each sample. The right panel lists the siRNAs against mouse tyrosinase that were expected to be produced from the miR30- or shRNA-based constructs.
FIGURE 2.
shRNAs with a 19-nt stem outperform shRNAs with a 29-nt stem in the context of a 9-nt loop. (A) The schematic depiction of a siRNA and the corresponding shRNAs with a 19-nt or 29-nt stem and 4-nt or 9-nt loop. The bold characters represent the double-stranded part of a siRNA against luciferase. (B) Different amounts of synthetic shRNAs with a 19-nt or 29-nt stem and a 4-nt or 9-nt loop were cotransfected with the luciferase reporters pGL3-control and pRL-TK into H1299 cells. (C) Different amounts of vectors that express the luciferase-targeting shRNAs with a 19-nt or 29-nt stem and a 4-nt or 9-nt loop were cotransfected with the luciferase reporter pGL3-control and pRL-TK into H1299 cells. (D) Vectors that express the luciferease or HIF-1α-targeting shRNAs with a 19-nt or 29-nt stem and a 4-nt (CCAA) or 9-nt (UUCAAGAGA) loop were cotransfected with the pGL3-control (Fluc) or HIF-1 reporter (pHRE) together with the pRL-TK (Rluc) reporter into H1299 cells. In B, C, and D the luciferase activities in transfected cells were determined using the Dual-Glo assay and normalized to the luciferase activities in cells that were transfected with the luciferase reporters and a control plasmid (control). The Y-axis represents the normalized luciferase activities (Fluc/Rluc) in each transfected cells. The X-axis in B and C represents the doses (Log(μg)) of the shRNA expression constructs.
FIGURE 3.
The functional shRNAs exhibit similar but not identical sequence preference compared with the functional siRNAs. (A) The internal thermodynamic stability of the functional (>80% knockdown) vs. the nonfunctional (<50% knockdown) shRNAs at each position of the shRNA duplex (sense strand) was calculated as described in the literature (Khvorova et al. 2003). (_B_) The differential %GC in the functional vs. nonfunctional shRNA or siRNAs at each position of the siRNA duplex was calculated using the formula: %GC of the functional shRNAs (or siRNAs) − %GC of the nonfunctional shRNAs (or siRNAs). One hundred and fifty shRNAs against 14 targets were used for the analysis. shRNAs that induced >75% silencing were considered functional (n = 75), and shRNAs that induced <50% silencing were considered nonfunctional (_n_ = 40). shRNAs that mediated 50%–75% knockdown were excluded from analysis. A proprietary data set of 360 siRNAs for four genes (Luc, Cyclo, GADPH, and DBI) from Dharmacon was analyzed in parallel. siRNAs that induced >80% silencing were considered functional (n = 136), and siRNAs that induced <50% knockdown were considered nonfunctional (n = 118). siRNAs that mediated 50%–80% target knockdown were excluded from analysis. The Y-axis represents the differential %GC of the functional shRNAs (or siRNAs) vs. the nonfunctional shRNAs (or siRNAs). The higher the value, the stronger preference of GC is in the functional shRNAs or siRNAs. Negative values indicate a preference for AU at a particular position. The numbers 1–19 on the X-axis correspond to the nucleotide positions in the sense strand of the shRNA or siRNA duplex.
FIGURE 4.
A computer program enriches the functional shRNAs. (A) The shRNA prediction program (left panel) or an advanced siRNA prediction program (right panel) was used to generate prediction scores on a set of 40 shRNAs against five targets. The prediction scores generated from each program were plotted against the degrees of target knockdown produced by these shRNAs. (B) The differential %GC of the functional vs. nonfunctional shRNA from the 40 shRNAs in the testing data set was analyzed using the same method as in Fig. 3B. The differential %GC in functional vs. nonfunctional siRNAs was generated using the same set of 360 siRNAs as in Fig. 3B.
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