Fig. 4: The 22-bulge enhances DICER activity and the knockdown efficiency of shRNAs.
From: Secondary structure RNA elements control the cleavage activity of DICER
a The 22-bulge (B) and nobulge-shRNAs (noB) contain different siRNA sequences and share the same USLs. b In vitro DICER cleavage assays for the 22-bulge and nobulge shRNAs. c The cleavage efficiency of DICER on the 22-bulge and nobulge shRNAs was shown in (b) and Supplementary Fig. 4b,n = 6 independent experiments. The p-values were calculated by two-sided Wilcoxon rank-sum tests. The center line is median, the lower and upper bounds of the box are the 25th and 75th percentiles, whiskers show 1.5x the interquartile extending from bounds of box, minima is the minimum value, and maxima is the maximum value. Individual data values are shown as dots. d The cleavage accuracy of DICER in (b) was calculated as a ratio of the cleaved products at a certain position to those at other positions. e Confirmation of DICER cleavages in human cells. The siRNAs resulting from cellular shRNAs were sequenced by NGS. f Construction of the firefly luciferase (FL) reporter gene and shRNA design. g The 22-bulge shRNAs (B) had higher gene-silencing activity than the nobulge shRNAs (noB) or 22-loop shRNAs (22-L) in reporter assays. These dual-luciferase reporter assays were repeated three times. A two-tailed t-test calculated the p-values. The error bars were presented with 95% confidence intervals. h The 22-bulge shRNAs silenced the expression of the TTR of endogenous genes more efficiently than did nobulge shRNAs. The western blot experiments were repeated three times. i Bar graphs showing the knockdown efficiency of the 22-bulge-shRNA and nobulge-shRNA on the expression of TTR protein, n = 3 independent experiments. A two-tailed t-test calculated the p-values. The error bars were presented with 95% confidence intervals. Source data are provided as a Source Data file.
