Fig. 2: The cleavage activity of DICER on different upper stem-loop structures.
From: Secondary structure RNA elements control the cleavage activity of DICER
a The positional structural profile of 58 shRNA structures, each had at least 10 variants identified in the HT cleavage assays. We predicted the secondary structure of 20,034 shRNA variants using RNAfold62. The dot-bracket structure, obtained from RNAfold, was converted into custom-designed structures in which each nt was assigned one of the following letters: L (loop), M (match), S (symmetric mismatch), A (asymmetric mismatch), B (bulge), and T (3’-overhang). The grey, red, green, and blue circles represent M, S, A, and B’s positional structure. The randomized regions are shown in red. b Two representative shRNA structures. c Scatter plot of 58 shRNA structures comparing the global cleavage efficiency and the cleavage accuracy of the major cleavage sites, as well as the SC/DC ratios. The log2(SC/DC) values were colored according to the color bar. The number inside each circle indicates the name of each structure as shown in (a, b) (Supplementary Data 1). Fifty-eight shRNA structures were further classified into I, II, and III groups. d The cleavage accuracy scores of DICER on shRNAs containing various distances between the major cleavage site and the loop. The cleavage accuracy score at the different cleavage sites on each variant was measured as described in the Methods. e The in vitro DICER cleavage assays of the variants containing structures 6, 15, or 54 (Supplementary Data 2, Supplementary Fig. 2b). The assays were repeated three times. f The DICER cleavage accuracy in (e) was calculated as the ratio of the cleaved products at a certain position to those at other positions. g The cleavage sites of DICER on human pre-miRNAs containing different stem lengths. h The cleavage accuracy score of DICER at DC21 on shRNAs having no mismatch or a single mismatch at 2 nt from DC21. i The cleavage accuracy score of DICER on shRNAs containing no mismatch, a single mismatch, or double mismatches at 2 nt from DC22. Source data are provided as a Source Data file.
