(A, redrawn from Figure 6A) Sequences of native let-7a, native miR-155, a chimeric miRNA containing the seed region of let-7a appended to nucleotides 9–23 of miR-155 (let-7a–miR-155), and a chimeric miRNA containing the seed region of miR-155 appended to nucleotides 9–21 of let-7a (miR-155–let-7a). (B and C) Pairing and offset coefficients describing the 3′-pairing preferences of let-7a–miR-155 (B) and miR-155–let-7a (C). Orange cells indicate pairing coefficients or KD fold-change values between 700–1,200. Otherwise, this panel is as in Figure 5A. (D) Comparison of the pairing and offset coefficients determined for let-7a–miR-155 with those of miR-155. Left, comparison of pairing coefficients, after first dividing by the geometric mean of all pairing coefficients of the same length for that miRNA, which normalized the trivial effect of pairing length. Points are colored according to pairing length, as in Figure 2A; error bars indicate 95% confidence intervals. Right, comparison of offset coefficients, colored from light blue to dark blue, progressing from offsets of −4 to +16 nt; error bars indicate 95% confidence intervals. For each graph, the gray region indicates the 95% confidence interval for the linear least-squares fit to the data (r, Pearson correlation coefficient). (E) Comparison of the pairing and offset coefficients determined for miR-155–let-7a with those of let-7a. Otherwise, this panel is as in (D). (F) Comparison of the pairing and offset coefficients determined for miR-155–let-7a with those of miR-155. Otherwise, this panel is as in (D). (G) Comparison of the pairing and offset coefficients determined for let-7a–miR-155 with those of let-7a. Otherwise, this panel is as in (D). (H) Seed-mismatch coefficients of the let-7a–miR155 (left) and miR-155–let-7a (right) chimeric miRNAs. Otherwise, this panel is as in Figure 5F. (I) Correspondence between mismatch coefficients of chimeric miRNAs and those of their seed-native miRNAs. For let-7a–miR-155 (left) and miR-155–let-7a (right), the values from (H) are plotted against those of Figure 5F (r2, coefficient of determination). Colors and symbols indicate the position and nucleotide identity, respectively, of each seed mismatch coefficient (key).
The distinct pairing, offset, and seed-mismatch preferences of the three miRNAs measured using the programmed libraries raised the question of the extent to which these preferences depended on the sequence of the seed region, the sequence of the 3′ region (i.e., beginning at miRNA nucleotide 9), or a combination of the two. To answer this question, we generated two chimeric miRNAs, one fusing the seed of let-7a to the 3′ region of miR-155 (let-7a–miR-155) and the other fusing the seed of miR-155 to the 3′ region of let-7a (miR-155–let-7a) (Figure 5A), and then performed AGO-RBNS using their corresponding seed-mismatched programmed libraries. As done previously with the natural miRNAs, we first determined the pairing and offset preferences of both chimeric miRNAs by summing over all 18 seed-mismatch types, measuring the KD fold change for each range of pairing and offsets possible in the libraries, and fitting a multiplicative model of pairing and offset-preferences to the resultant 1,181 and 934 measured affinity values for let-7a–miR-155 (B) and miR-155–let-7a (C).
Both of the chimeric miRNAs had 3′-pairing and offset preferences that were remarkably similar to those of the natural miRNAs containing the same 3′ sequences (B) and (C), and (Figure 5A and C). Indeed, comparison for each chimeric miRNA to its 3′-native sequence revealed a high correspondence of pairing and offset coefficients (D and E), and comparison of each chimeric miRNA to its seed-native miRNAs showed much lower correspondence of the pairing and offset coefficients. (F and G). Furthermore, the fitted slopes for four 3′-native comparisons approached unity (range, 0.80–1.17), which showed that the effect sizes of these preferences were similar regardless of whether the coefficients were derived from chimeric or native miRNA datasets. When analyzing the influence of the 18 seed mismatches on the affinity of 3′ pairing, miRNAs with the same seed sequence but different 3′ sequences had largely similar preferences (H and I, and Figure 5F). The two most prominent exceptions were the increased affinity in the context of a mismatched A at position 7 of the let-7–miR-155 chimeric miRNA (I, left), and the decreased affinity in the context of a mismatched U at position 6 of the miR-155–let-7a chimeric miRNA (I, right). Despite these outliers, the influence of the seed mismatch on the magnitude of 3′-pairing affinity depended primarily on the seed-mismatch type and position, with relatively little dependence on the sequence of the 3′ region.