Author response: MicroRNA 3′-compensatory pairing occurs through two binding modes, with affinity shaped by nucleotide identity and position

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract MicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2–8), preferences of the miRNA 3′ region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2–miRNA complexes to measure relative affinities of >1000 3′-pairing architectures for each miRNA. In some cases, optimal 3′ pairing increased affinity by >500 fold. Some miRNAs had two high-affinity 3′-pairing modes—one of which included additional nucleotides bridging seed and 3′ pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3′ pairing is determined for each miRNA. Editor's evaluation This manuscript will be of interest to readers in the field of microRNA (miRNA) biology, particularly those interested in miRNA targeting. The authors interrogated non-canonical miRNA target recognition to a depth vastly exceeding any study to date. The results revealed unexpected, sequence-specific diversity in miRNA-targeting modes, providing new insights relevant for improved target prediction. https://doi.org/10.7554/eLife.69803.sa0 Decision letter Reviews on Sciety eLife's review process Introduction MicroRNAs (miRNAs) are ~22-nt regulatory RNAs that are processed from hairpin precursors. Upon processing, miRNAs associate with an Argonaute (AGO) protein and base-pair to sites within mRNAs to direct the destabilization and/or translational repression of these mRNA targets (Jonas and Izaurralde, 2015; Bartel, 2018). For most sites that confer repression in mammalian cells, pairing to miRNA nucleotides 2–7, referred to as the miRNA seed, is critical for target recognition, with an additional pair to miRNA position 8 or an A across from miRNA position 1 often enhancing targeting efficacy (Lewis et al., 2005; Bartel, 2009). Such sites with a perfect 6–8-nucleotide (nt) match to the miRNA seed region (Figure 1A, left) are heuristically predictive of repression, with longer sites being more effective than shorter ones and more sites being more effective than fewer sites (Grimson et al., 2007; Agarwal et al., 2015). In addition, contextual features extrinsic to a site itself can influence targeting efficacy (Brown et al., 2005; Ameres et al., 2007; Grimson et al., 2007; Kedde et al., 2007; Nielsen et al., 2007; Saetrom et al., 2007; Tafer et al., 2008; Kedde et al., 2010; Wan et al., 2014; Agarwal et al., 2015; McGeary et al., 2019). Figure 1 Download asset Open asset Features of miRNA 3′-compensatory sites characterized using AGO-RBNS. (A) Pairing of typical canonical sites (left), 3′-supplementary, canonical sites (middle), and 3′-compensatory, noncanonical sites (right). Canonical sites contain contiguous complementarity (blue) to the seed (red). Sites with shifted complementarity (i.e., the 6mer-A1 and 6mer-m8 sites) are sometimes also classified as canonical sites. 3′-supplementary sites have pairing to the miRNA 3′ region, which supplements canonical seed pairing and is reported to be most effective if it centers on miRNA nucleotides 13–16 (green and orange). This 3′ pairing can supplement 8mer sites (as shown) as well as other canonical sites (not shown). 3′-compensatory sites resemble 3′-supplementary sites, except they lack perfect pairing to the seed and thus pairing to the 3′ region helps to compensate for this imperfect seed pairing. Vertical lines represent Watson–Crick pairing. (B) The architectures of 3′ sites. Three independent features define each architecture: (1) the length of 3′ pairing (left), measured as the number of contiguous base pairs to the miRNA 3′ region; (2) the position of 3′ pairing (middle-left), defined as the 5′-most miRNA nucleotide engaged in 3′ pairing; and (3) the offset between the seed pairing and 3′ pairing (middle), which specifies the number of unpaired nucleotides separating the seed- and 3′-paired segments in the target RNA relative to that in the miRNA. Mismatches to the seed pairing (middle-right) or within the 3′ pairing (right) can elaborate on these architectures, as can bulged nucleotides (not shown). (C) A programmed RNA library for using AGO-RBNS to examine 3′ pairing of let-7a. The library contains an 8-nt region with all 18 possible single-nucleotide mismatches (purple) to the let-7a seed (red), with 25 nt of random-sequence RNA upstream of this region and 5 nt of random-sequence RNA downstream. k-mer positions are numbered with respect to the programmed 8-nt mismatched site. B represents C, G, or U; D represents A, G, or U; V represents A, C, or G; N represents A, C, G, or U. The black vertical line depicts perfect pairing at position 8, and gray vertical lines indicate Watson–Crick matches at only five of the six seed positions. (D) The top 20 8-nt k-mers identified by AGO-RBNS performed with the highest concentration of AGO2–let-7a (840 pM) and the programmed library (100 nM). k-mers were ranked by the sum of their enrichments at the five positions of the library at which they were most enriched. Left, alignment of k-mers, indicating in pink nucleotides that were not Watson–Crick matches to the miRNA. Right, heat map showing k-mer enrichment at each position of the library, with pairing shown for the top 8-nt k-mer at the position of its greatest enrichment. Black vertical lines depict perfect Watson–Crick pairing, and gray vertical lines indicate Watson–Crick matches at only five of the six seed positions. Pairing to the miRNA 3′ region, particularly pairing that includes miRNA nucleotides 13–16, can supplement perfect seed pairing to enhance targeting efficacy beyond that of seed pairing alone, and extensive pairing to the 3′ region can compensate for imperfect seed pairing to enable consequential repression (Brennecke et al., 2005; Lewis et al., 2005; Grimson et al., 2007). These two bipartite site types are referred to as 3′-supplementary and 3′-compensatory sites, respectively (Figure 1A, middle and right). Although 3′-supplementary sites are less common than sites with only a seed match, comprising ~5% of all conserved sites observed in mammals, thousands of sites with preferentially conserved 3′-supplementary pairing are present in human 3′ UTRs (Grimson et al., 2007; Friedman et al., 2009). Conserved 3′-compensatory sites are even less common, comprising only ~1.5% of all preferentially conserved sites observed in human 3′ UTRs (Friedman et al., 2009). Nonetheless, two instances of this relatively rare site type within the 3′ UTR of lin-41 mediate the extreme morphological and developmental defects by which the let-7 miRNA was discovered in C. elegans (Pasquinelli et al., 2000; Reinhart et al., 2000; Ecsedi et al., 2015). Moreover, the use of these 3′-compensatory sites rather than canonical sites for lin-41 repression is consequential; site mutations that create perfect seed pairing while maintaining the 3′ pairing cause precocious repression of the mRNA by other members of the let-7 seed family expressed during earlier larval stages (Brancati and Großhans, 2018). These results support the notion that 3′-compensatory sites enable differential target specificity between miRNAs that share a common seed sequence but differ within their 3′ regions (Brennecke et al., 2005; Lewis et al., 2005). Although global analyses of site conservation and efficacy provide compelling evidence that pairing to the miRNA 3′ region is also utilized in mammalian cells (Friedman et al., 2009), these approaches have limitations for evaluating which 3′-pairing architectures are most effective, due to the vast number of 3′-pairing architectures that are possible for a single miRNA sequence. The pairing architecture of a 3′-compensatory site can be described by five characteristics: (1) the length of contiguous pairing between the site and the miRNA 3′ region, (2) the position of pairing to the miRNA 3′ region, as defined by the 5′-most miRNA nucleotide involved in 3′ pairing, (3) the difference between the number of unpaired target nucleotides and the number of unpaired miRNA nucleotides bridging the seed and 3′ pairing, hereafter referred to as the ‘3′-pairing offset,’ (4) the nature of the imperfect pairing to the seed, and (5) the nature of any imperfections in the 3′ pairing (Figure 1B). When considering only sites with perfect 3′ pairing with lengths ranging from 4 to 11 base pairs (bp) at all possible 3′ positions, offsets ranging from −4 to +16 nt, and seed pairing interrupted by one of 18 possible single mismatches (or wobbles) to the 6-nt seed, there are >16,000 possible variants to the site architecture. However, for each miRNA, most of these possibilities are not present even once in all the 3′ UTRs of a transcriptome. Thus, data from multiple miRNAs must be aggregated to observe a reliable signal of either efficacy or conservation, which prevents identification of miRNA-specific pairing preferences. Indeed, even when aggregating multiple miRNA-perturbation (e.g., transfection) datasets, which enables efficacy of 3′-supplementary sites to be detected (Grimson et al., 2007), a signal for the efficacy of 3′-compensatory sites has not been reported, underscoring the challenge of using global analyses of conservation or repression efficacy to determine which architectures are more effective than others. The observation that miRNA targeting efficacy observed in the cell is largely a function of the affinity between AGO–miRNA complexes and their sites (McGeary et al., 2019) indicates that contributions of 3′ pairing to affinities measured in vitro can provide insight into biological targeting efficacy. Early measurements showed that pairing to positions 13–16 of let-7a imparts only a twofold increase in binding affinity, which led to the view that 3′-supplemental pairing contributes only modestly to affinity (Wee et al., 2012). Further measurements revealed some differences between miRNAs, with the observation that pairing to positions 13–16 of miR-21 increases affinity by 11-fold (Salomon et al., 2015), and a striking effect of longer pairing, with the observations that 10 bp of 3′-supplementary pairing to miR-122 and 9 bp of 3′-supplementary pairing (including a terminal G:U wobble) to miR-27a increases affinity by 20- and >400-fold, respectively (Sheu-Gruttadauria et al., 2019a). Other measurements illustrate the influence of the length of the target segment bridging the seed and 3′ pairing, with binding affinity varying ~10-fold as this length is varied over a range of 1–15 nt (Sheu-Gruttadauria et al., 2019b). Taken together, these reports demonstrate the potential for miRNA 3′ pairing to enable high-affinity binding, and also illustrate that the benefit of this pairing varies, depending on the miRNA sequence and 3′-pairing architecture. Understanding how these features together modulate the benefit of 3′ pairing will be possible only after acquiring many more measurements with multiple miRNA sequences. Imaging-based, high-throughput single-molecule biochemistry has recently been applied to acquire affinity measurements for ~23,000 sites for each of two miRNAs (let-7a and miR-21), including many sites with 3′ pairing (Becker et al., 2019). These measurements revealed that miR-21 relies more on 3′ pairing when binding to a fully complementary target than does let-7a, that homopolymeric insertions are the least disruptive to binding when inserted between nucleotides 8 and 11 within the context of fully complementary binding, and that mismatches near the miRNA 3′ terminus (after position 16) decrease binding affinity but increase target slicing. However, because the design of target libraries was based primarily on fully complementary RNA targets to which varying extents of mismatched, bulged, and deleted nucleotides were introduced, only a small minority of the possible 3′-pairing architectures were queried. RNA bind-n-seq (RBNS) enables unbiased, high-throughput assessment of binding sites embedded within a larger random-sequence context (Lambert et al., 2014; Dominguez et al., 2018). We recently adapted RBNS for the study of miRNA targeting, and we built an analysis pipeline enabling calculation of relative equilibrium dissociation constants (KD values) for many thousands of different RNA k-mers ≤12 nt in length (McGeary et al., 2019). Here, we further adapted the AGO-RBNS protocol to enable examination of sites >12 nt in length, thereby enabling the high-throughput investigation of bipartite sites containing near-perfect seed pairing and 4–11 additional pairs to the miRNA 3′ region. We applied this modified protocol to the systematic interrogation of the contribution of 3′ pairing for three natural miRNA sequences and four synthetic derivatives. We also performed a massively parallel reporter assay, which confirmed that key observations derived from affinities measured in vitro apply also to repression in cells. Results RBNS measures affinities for many 3′-compensatory sites of let-7a AGO-RBNS begins with a series of 4–6 binding reactions, each containing an RNA library at a fixed concentration and a purified AGO–miRNA complex at a variable concentration spanning a 100-fold range (McGeary et al., 2019). Each molecule of the RNA library has a central region of random-sequence nucleotides flanked by constant sequences on each side that enable preparation of sequencing libraries. Upon reaching binding equilibrium, each reaction is passed through a nitrocellulose membrane, which retains AGO–miRNA complexes and any library molecules that are bound to the complexes. These bound library molecules are isolated and subjected to high-throughput sequencing, along with the input RNA library. Binding of an individual k-mer can be detected as enrichment in the bound compared to input sequences, and relative KD values can be estimated simultaneously for hundreds of thousands of different k-mers by fitting a biochemical model to k-mer fractional abundances from each of the bound libraries. As originally implemented, AGO-RBNS cannot provide reliable information on sites with more than ~5 supplementary/compensatory pairs because such sites, which involve >12 bp of total pairing (Figure 1A, middle and right), are too rare in the sequences obtained from the input RNA library to enable accurate calculation of enrichment values. To overcome this constraint for sites to let-7a, a miRNA with physiologically relevant 3′ pairing (Pasquinelli et al., 2000; Reinhart et al., 2000; Brancati and Großhans, 2018), we used a library that contained a programmed region of imperfect seed pairing to let-7a, with 25 and 5 nt of random-sequence RNA separating the programmed region from the 5′ and 3′ constant sequences, respectively (Figure 1C). In each library molecule, this programmed region of imperfect seed pairing contained a let-7a 8mer site with a mismatch at one of its six seed nucleotides, such that each library molecule had one of 18 possible single-nucleotide seed mismatches (including wobbles) in approximately equal proportion. With this programmed region of imperfect seed pairing, each library contained 3′-compensatory sites at an ~250-fold greater frequency than expected for a fully randomized RNA library. AGO-RBNS was performed using this programmed library and purified AGO2–let-7a, choosing AGO2 from among the four human AGO paralogs because of its relatively high expression (Völler et al., 2016; Müller et al., 2019) and for comparison to previous biochemical and structural studies that use human or mouse AGO2 (Schirle and MacRae, 2012; Wee et al., 2012; Schirle et al., 2014; Schirle et al., 2015; Chandradoss et al., 2015; Salomon et al., 2015; Klum et al., 2018; Becker et al., 2019; McGeary et al., 2019; Sheu-Gruttadauria et al., 2019a; Sheu-Gruttadauria et al., 2019b). For our initial analysis, we calculated the enrichment of all 8-nt k-mers at each position between the programmed region and the 5′-constant region of the library, after first removing reads with any of the six canonical sites to let-7a. The enriched k-mers had substantial complementarity to the 3′ region of let-7a (Figure 1D). The most enriched was AUACAACC—the perfect Watson–Crick match to positions 11–18 of let-7a (Figure 1D). This 8-nt 3′ site was most strongly enriched when starting at position 15 of the library, which suggested that an internal loop with two miRNA nucleotides (9 and 10) and six target-site nucleotides (positions 9–14) separating seed pairing and 3′ pairing was optimal (Figure 1D, top). Using our nomenclature (Figure 1B), this 3′ site was classified as a position-11 site with pairing length of 8 bp and offset of +4 nt. Note that here and throughout this study we refer to contiguous complementarity as ‘pairing,’ even though constraints imposed by AGO2 might prevent physical pairing from occurring at some complementary positions. This 8-nt, position-11 site was also ≥5-fold enriched at seven other neighboring offsets (corresponding to library positions 8–15), indicating that looping out 3–10 unpaired library nucleotides opposite miRNA nucleotides 9 and 10 was tolerated, albeit to varying degrees (Figure 1D). The second-most enriched 8-nt k-mer was UACAACCU—the perfect Watson–Crick match to let-7a positions 10–17 (Figure 1D). This 3′ site had a maximal enrichment with five, rather than six, unpaired library nucleotides spanning the seed and 3′ pairing, with the distribution of enrichments shifted by 1 nt in comparison to that of the AUACAACC site. This 1-nt shift in the enrichment distribution corresponded with the 1-nt shift in site position (from 11 to 10 of the miRNA) to maintain an offset of +4 target nucleotides. Indeed, the next 18 most enriched 8-nt k-mers represented 3′ sites with pairing positions ranging from miRNA nucleotides 9–12, with enrichment distributions that correspondingly shifted to reflect an overall optimal offset of +4 target nucleotides (Figure 1D). Each had a contiguous stretch of 6–8 perfect Watson–Crick pairs to the let-7a 3′ region, usually including the ACAACC k-mer, which suggested that perfect pairing to let-7a positions 11–16, with a +4 nt offset, was particularly effective for enhancing site affinity. let-7a has two distinct 3′-pairing modes For a more comprehensive examination of 3′ sites of varied lengths, positions, and offsets (Figure 1B), we enumerated 3′ sites of lengths 4–11 nt that perfectly paired to the miRNA starting at any position downstream of nucleotide 8. For each length and position of 3′ pairing (e.g., for the 8mer-m11–18), we further enumerated all pairing offsets compatible with the 3′ site residing within the 25-nt random-sequence region upstream of the programmed site, converting each library position to an offset value based on the pairing position of each 3′ site (Figure 2A). For our initial KD estimation and analyses, we pooled the reads for the 18 possible seed-mismatch types. This pooling increased the read counts for each 3′-pairing architecture, which enabled examination of sites as long as 11 nt, which in turn enabled analysis of 1006 distinct 3′-pairing architectures. Figure 2 with 2 supplements see all Download asset Open asset Pairing to nucleotide 11 and a positive offset promote high-affinity binding to let-7a.in P. (A) Correspondence of enrichment and relative KD value of sites with the AUACAACC k-mer (the perfect match to miRNA positions 11–18) measured at each position in the programmed library. Each of these positions (upper x-axis) corresponds to the indicated offset (lower x-axis). For example, because this k-mer paired to miRNA positions 11–18, pairing beginning at k-mer position 11 had a 0-nt offset. The k-mer enrichments and their associated colors (top) correspond to those of the top row of Figure 1D. For details on how relative KD values were calculated for each site possibility, see McGeary et al., 2019, Figure 1C–E and Materials and methods section 11. (B) Relative KD values of let-7a 3′-compensatory sites that had optimally positioned 3′ pairing of 4 (orange) to 11 (dark blue) bp. For each length of 3′ pairing, the optimal position is shown in terms of its complementarity to let-7a (right). For each of the 3′-compensatory sites, the relative KD value is plotted as a function of its offset (left), as done for sites with 8 bp of optimally positioned 3′ pairing in (A). Vertical lines indicate 95% confidence intervals. The dashed horizontal line indicates the geometric mean of the 18 relative KD values of the seed mismatch sites, each calculated from reads with <4 nt of contiguous complementarity to the miRNA 3′ region. The horizontal blue and purple lines indicate the relative KD values of the canonical 6mer and 8mer sites, respectively. The arrows at +2 and +4 nt mark a shift in the optimal offset observed with increasing 3′-pairing length. The asterisk denotes the anomalously low binding affinity measured for 3′ sites that pair contiguously with seed pairing (i.e., sites with pairing at position 9 with an offset of 0 nt). (C) The dependency of let-7a 3′-pairing affinity on pairing length, position, and offset. Each panel shows the relative KD values of 3′-compensatory sites with 3′ pairing of a specified length over a range of positions and offsets. Each trend line is colored according to pairing position, spanning positions 9 (light violet) to 18 (red) when possible. The arrows between 0 and 1 nt and at +3 nt mark a shift in the optimal offset as the position of 3′ pairing shifted to include nucleotide 11 of let-7a. Otherwise, these panels are as in (B, left). (D) Schematics of the two 3′-binding modes. In the zero-offset binding mode (top), miRNA nucleotide 11 is inaccessible due to occlusion by the central region of the AGO protein. In the positive-offset binding mode (bottom), the longer stretch of bridging target nucleotides enables a conformation in which nucleotide 11 is available for pairing to the target RNA. Although not intended to accurately reflect the conformation of either binding mode, these schematics illustrate how a larger offset might enable pairing to a more centrally located miRNA nucleotide. (E) Affinity profile of the let-7a 3′ region. Each cell indicates the fold change in relative KD attributed to a 3′ site with indicated length, position, and offset of pairing. Each row within a heat map corresponds to a different miRNA nucleotide at the start of the 3′ pairing, and each column corresponds to a different miRNA nucleotide at the end of the 3′ pairing. Each heat map shows the results for a different offset. The three diagrams indicate the fold-change values and architectures for 3′ sites pairing to miRNA nucleotides 13–16 with an offset of 0 nt (left), pairing to miRNA nucleotides 13–21 with an offset of 0 nt (middle), and pairing to miRNA nucleotides 10–20 with an offset of +4 nt (right). Gray boxes indicate pairing ranges that were either too short (<4 bp) or too long (>11 bp) for relative KD values to be reliably calculated. Black vertical lines depict perfect Watson–Crick pairing, and gray vertical lines indicate Watson–Crick matches at only five of the six seed positions. Simultaneous estimation of the fractional abundance of these sites in each of the AGO2–let-7a-bound libraries in comparison to that of the input library enabled calculation of their relative KD values. As illustrated for the 8-nt k-mer identified as most enriched in the previous analysis (Figure 1D, top row), variation in KD values qualitatively tracked with that of enrichment values but quantitatively differed due to the attenuating effects of background binding and site saturation on enrichment values (McGeary et al., 2019; Figure 2A). Relative KD values corresponding to a broad spectrum of 3′-pairing architectures spanned a several hundred–fold range, with strong agreement observed between the results of replicate experiments performed independently with different preparations of both AGO2–let-7a and the let-7a programmed library (r2 = 0.96, n = 1477; Figure 2—figure supplement 1A, left). Agreement between the two replicates was maintained, albeit to a lesser degree, when read counts for each 3′-pairing architecture were not pooled over the 18 seed-mismatched sites in the programmed region (r2 = 0.78, n = 23,912; Figure 2—figure supplement 1A, right). Furthermore, for shorter 3′ sites, which could be analyzed using data from a standard AGO-RBNS experiment that used a non-programmed random-sequence library (McGeary et al., 2019), the relative KD values determined from the programmed library correlated well with those determined from a random-sequence library (r2 = 0.83, Figure 2—figure supplement 1B). Despite the overall correlation, a minor systematic difference in the values for the same sites determined from the two types of libraries was observed. This distortion was presumed to be due to the absence of library RNA molecules containing no site and was corrected accordingly (Figure 2—figure supplement 1B). To investigate the interplay of pairing position, length, and offset, we identified the optimal 3′ sites of lengths 4–11 nt and, as in Figure 2A, examined the effect of varying offset on the affinity of each of these sites (Figure 2B). Nearly all possibilities examined had values readily distinguished from the log-averaged value for seed-mismatched sites alone, with compensatory pairing to miRNA nucleotides 11–16 at optimal offsets yielding binding affinities comparable to that of the canonical 6mer (Figure 2B, left). Further inspection of longer 3′ sites underscored the conclusion that pairing to the GGUUGU segment spanning positions 11–16 of let-7a is the most consequential for 3′-compensatory pairing, as all optimal pairing positions for 3′ sites ≥6 nt in length paired to this segment. Moreover, inspection of the optimal positions for shorter sites showed that pairing to the 5′ end of this segment (containing the sequence GGUU) was more impactful than pairing to its 3′ end (Figure 2B, right). In addition, increasing the length of pairing from 4 to 11 bp led not only to increased binding affinity at almost all offsets, as might have been expected, but also to a shift in the optimal offset, with a preferred offset of +2 nt when pairing with 4 bp compared to a preferred offset of +4 nt when pairing with 9–11 bp (Figure 2B, left). To investigate further the interplay between affinity, pairing position, and pairing offset, we plotted the relative affinities of all possible positions and offsets for let-7a 3′ pairing of lengths ranging from 4 to 9 bp (Figure 2C). These plots revealed a striking change in the affinities and preferred offsets as pairing shifted from position 12 to position 11. For 3′ sites of each length, those that began at let-7a position 12 (dark blue points of Figure 2C) had intermediate affinity and optimal offsets of 0 or +1 nt, with clearly reduced affinity as offsets increased beyond +1 nt. At these offsets of 0 or +1 nt, 3′ sites that began at position 11 (dark purple points) had affinities similar to those that began at position 12. However, in stark contrast to the sites beginning at position 12, sites beginning at 11 had strikingly increased affinity at more positive offsets, with affinity peaking at offsets of +2 or +3 nt. Sites beginning at +10 were similar, with affinity peaking at an offset of +4 nt. These results suggested that pairing to position 11 in the central region of the miRNA is less accessible than pairing to position 12, and therefore a longer loop in the target sequence is required to bridge seed pairing with 3′ pairing that includes position 11 (Figure 2D). Nonetheless, when the increased offset enables pairing to position 11, substantially greater affinity can be achieved. We call this newly defined binding mode, which includes pairing to miRNA position 11 and greatly benefits from short positive offsets, the ‘positive-offset’ binding mode. Accordingly, the more conventional binding mode, which lacks pairing to position 11 and does not benefit from offsets greater than +1 nt, we call the ‘zero-offset’ binding mode. Some of the weakest relative affinities were observed for extended 3′-pairing possibilities that began at position 9 with an offset of 0 nt (Figure 2B and C, asterisks). These weak values were attributable to AGO2-catalzyed slicing of molecules with extensive contiguous pairing, which would have depleted these molecules from our bound library. Supporting this idea, analogous sites with offsets of either −1 or +1 nt, which were expected to disrupt slicing due to single-nucleotide bulges in either the miRNA or the site, respectively, did not have aberrantly low relative affinities. This idea was also consistent with reports that AGO2 can slice sites that have a seed mismatch but are otherwise extensively paired to the guide RNA (Wee et al., 2012; Chen et al., 2017; Becker et al., 2019). We next used heat maps to visualize the interplay between 3′-site position and pairing length at different offsets (Figure 2E). Within each heat map, a difference between adjacent cells corresponded to the difference in KD fold change caused by the addition or removal of a pair at either the 5′ end (adjacent rows) or the 3′ end (adjacent columns) of the 3′ site, while maintaining the same offset. For example, in the heatmaps corresponding to off