Fig. 2: The bipartite pairing rule.

a Analysis of shRNA cleavage patterns with 21bp-stem to 23bp-stems according to the 2-nt loop counting rule or the −1mM inhibitory rule. Green and red arrowheads signify DC21 and DC22 cleavages, respectively. b Determination of DICER's double cleavage accuracy at positions 19–23 in shRNAs with the stem of 23 or 24 base pairs, featuring a base pair (bp) or mismatch (mM) at the −1 position relative to DC21 and DC22. Each line on the graph corresponds to a different shRNA variant. c Comparative analysis of double cleavage accuracy between DICER and DICERΔHelicase, in shRNAs with a base pair (bp) or mismatch (mM) at the −1 position relative to DC21 and DC22, with DC21 and DC22 denoting double cleavages at the 21 and 22 nucleotides from the 5p-end of shRNA, respectively. n indicated the sample size. The median value (center line), lower quartile and upper quartile (box edges), and whiskers extended to ±1.5 × IQR (interquartile range) were indicated in each boxplot. d Depiction of shRNA structures and sequences. e In vitro DICER cleavage assays for RNAs shown in (d). f Calculation of DICER-WT cleavage efficiency on various shRNAs from data obtained in three replicate assays as depicted in (e) normalized to 2122-mM RNA. g Evaluation of cleavage accuracy at DC21 and DC22 for DICER-WT based on three repetitive assays as shown in (e). h Assessment of DICER's double cleavage accuracy at positions 19–23 in shRNAs with 22bp-stem, including a base pair (bp) or mismatch (mM) at the −2 position relative to DC21 and DC22. i Comparison of double cleavage accuracy between DICER and DICERΔHelicase in shRNAs with a base pair (bp) or mismatch (mM) at the −2 position relative to DC21 and DC22, with DC21 and DC22 indicating double cleavage at the 21 and 22 nucleotides from the shRNA's 5p-end, respectively. n indicated the sample size. The median value (center line), lower quartile and upper quartile (box edges), and whiskers extended to ±1.5 × IQR (interquartile range) were indicated in each boxplot. j Depiction of shRNA structures and sequences. k In vitro DICER cleavage assays for RNAs shown in (j). l Evaluation of cleavage accuracy at DC21 and DC22 for DICER-WT based on three repetitive assays as shown in (k). m Depiction of pre-mir-339 structures and sequences, highlighting DC21, DC22, and DC23 cleavage sites with green, red, and blue arrowheads, respectively. n In vitro DICER cleavage assays for RNAs shown in (m). o Evaluation of cleavage accuracy at DC21, DC22, and DC23 for DICER-WT based on three repetitive assays as shown in (n). p A model highlighting the bipartite pairing rule, demonstrating the essential roles of the −1 and −2 base pair positions in both DC21 and DC22 cleavages. q The bipartite pairing rule elucidates the cleavage patterns of shRNAs with varying stem lengths or mismatches near the cleavage sites. In the bar plot representing quantitative data from the gel, data values are shown as dots, and the error bars are presented with 95% confidence intervals.