To evaluate set screw fracture in the Trochanteric Femoral Nail Advanced implant  and to identify additional mechanisms of set screw failure in the TFNa.Patients who had experienced failure after open reduction and internal fixation (ORIF) with the TFNa were identified. TFNa implants were explanted and inspected following revision surgery. Medical device reports (MDRs) and manufacturer’s inspection reports describing similar failures for the TFNa in the United States Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) database were also reviewed.Four set screw fractures that occurred at a level II trauma center were observed. Sixty-seven reported failures were identified in the MAUDE database for review. Twenty-eight failed implants were returned to the manufacturer for inspection with a published inspection report available for analysis. Set screw fractures can occur in the TFNa when the set screw is deployed prematurely into the proximal screw aperture prior to blade/screw insertion. The set screw can also bend and deform if it is advanced against a helical blade/lag screw that is not fully seated into position, thereby potentially compromising its function.The TFNa set screw allows for potential fracture during implant insertion leading to uncontrolled collapse, early excessive proximal femoral shortening, and rotational instability of the helical blade/lag screw. Similar failures in the TFNa can be prevented by having the surgeon inspect the proximal screw aperture after attachment of the proximal aiming aim to ensure the set screw has not been deployed prematurely.Therapeutic Level III.The online version contains supplementary material available at 10.1186/s10195-021-00594-8. Mechanical failure after fixation of proximal femur fractures with cephalomedullary nails has been well described in terms of implant “cut out” from the femoral head and implant fracture –16. FeweThis study aimed to evaluate set screw/locking mechanism fracture in the TFNa implant to answer the following questions; What causes the implant to fracture, and how can failures be identified? Are there additional mechanisms by which set screw failure can occur? Due to the paucity of information available published on this topic, the FDA’s MAUDE database was employed for reports of similar failures in the TFNa that could assist the investigation into the mechanism. The FDA’s MAUDE database has previously been used to analyze implant failures provided that the data are not used to compare rates of failures between implants within the same category , 18, 19.An 86-year-old female sustained an unstable reverse obliquity intertrochanteric femur fracture (AO A3) following a ground-level fall. The patient was an independent ambulator prior to initial injury and was independent with functional tasks, ASA category 3. Operative treatment was performed by the on-call orthopedic surgeon the following day within 24 h of the injury. Fixation was achieved with a TFNa implant, 12 mm × 420 mm × 130°. Intraoperative fluoroscopic images from the procedure are included as part of Fig. While at a rehabilitation facility, the patient noted progressive pain level increases, wherein the pain levels spiked to 9 out of 10, particularly with transfers. Increasing difficulty with ambulation and functional mobility were noted, and the patient deteriorated to ambulating less than 5 ft with a walker and moderate assistance. Eventually, complete ambulation became impossible because of pain, and repeat imaging was ordered on postoperative day 12. The repeat radiographs revealed interval displacement of the fracture with loss of reduction and are included as part of Fig. Urgent revision surgery was performed the following day. The implant was revised to a rotationally stable construct with dual integrated screws, Intertan . The failed TFNa implant was fully disassembled and inspected on the back table. The set screw/proximal locking prong was found to be broken at its base Fig. . FollowiAfter the revision procedure, the patient immediately noted significant pain level improvement from 8–9 to 2–3. Ambulatory distance increased to 50 ft in the early postoperative period, and the patient returned to rehabilitation on postoperative day 3. Continued improvements in functional status were noted as the patient was able to ambulate independently over 150 ft with a walker upon discharge from rehabilitation. The patient eventually returned to baseline functional status with no complaints of hip pain. The X-rays obtained at 6 months after the revision procedure show full union, maintenance of reduction, and no appreciable proximal femoral shortening Fig. .Fig. 4, and Intertan implants. Among those three implants, only the TFNa set screw was reported to have fractured. Altogether, 117 reports of locking mechanism failure in the TFNa were obtained from a search of “break” events of the MAUDE database. Sixty-seven reports were available for review after eliminating duplicate reports. A summary of the categorical data is included in Table Few studies have described set screw/proximal locking mechanism failure because of its rare occurrence, and limited information exists to demonstrate the clinical presentation of those failures , 20, 21.One such failure, described in detail in the sample case report, illustrates the impact of TFNa set screw fracture on excessive shortening, or uncontrolled collapse. Although shortening is to be anticipated via a mechanism of controlled collapse by which most intertrochanteric femur fractures achieve union, the extent to which proximal femoral shortening is predictive of construct failure and “uncontrolled” collapse has yet to be fully determined . “ControThe locking mechanism for the TFNa is composed of two major components coupled by a spring, an upper portion or “lock drive” and a lower portion or “lock prong.” Per the manufacturer, the reported fractures occur when the lock prong is advanced into the proximal screw aperture prior to blade/screw insertion Fig. . Either Altogether, eight reported failures in the MAUDE database were related to medial migration of the blade/screw in the TFNa, although none of the manufacturer inspection reports attributed this complication to damage of the locking mechanism. For the TFNa, there is an edge along the lateral surface of the helical blade/lag screw that engages the lock prong, limiting how far the blade/screw can migrate medially or laterally. In one of the observed failures, medial migration of the lag screw was preceded by severe collapse that progressed up until the lock prong prevented further slide of the lag screw in the proximal aperture. Predictably, the lock prong eventually fractured, which ultimately permitted escape of the lag screw from the proximal implant as it then migrated medially into the abdomen.In addition to implant breakage, the locking prong can also bend or deform if it is advanced against a helical blade/lag screw that is not fully seated into position. The aiming arm of the TFNa is designed so that, when the blade is fully seated, the recess on the lateral side of the blade/screw will be oriented to allow passage of the advancing lock prong Fig. . If the Although some set screw failures/fractures can be attributed to user technique during insertion, design differences in the set screw themselves may also contribute to additional sources of failure Fig. . Key difSince the set screw is internal to the proximal aspect of the implant, intraoperative damage and compromise to the locking mechanism may potentially go unrecognized. Among the reported failures in the MAUDE database, premature deployment of the locking prong into the aperture was discovered when the helical blade was unable to pass through the proximal screw aperture owing to increased resistance or blockage. Concerning were reports in which the helical blade became incarcerated within the proximal screw aperture and was unable to be disimpacted from the cephalomedullary nail in the operative theater. The inspection report from the manufacturer pertaining to this event describes it as a “cold welding” that required the implant pieces be separated in their lab after their return en block. Per the technique guide, failures can also be detected by checking to see if blade/screw is able to be rotated within the proximal screw aperture via the insertion handle following locking.As the locking mechanism is internal to the implant and the lock prong is poorly visualized on fluoroscopy, failures may be difficult to detect. As per the author’s experience, use of CT scan to assist in detecting failure of the TFNa locking mechanism is limited because of the actual size of the locking prong. Artifact in the area of the locking mechanism is typically present on CT, preventing accurate imaging of deformed and broken pieces. CT scan can be successful in measuring the height of the lock drive in relation to the top of the nail, and when fully deployed, it averages 2.2 cm from the top of the implant. In cases of under-insertion of the blade/screw with deformity of the locking prong or in cases where the locking mechanism was not fully advanced, it would result in a heightened position of the lock drive, as discovered in a non-union case at our institution.There are several limitations to our study. As there is little information available on this important topic, fully understanding the multitudes of factors contributing to the mechanism of failure is challenging. The MAUDE database can be a valuable resource in these instances as it provides a large collection of reports pertaining to medical device failures in the USA.The benefits of this database are offset by several important limitations. Key information can often be missing in the reports, particularly when that information has not been deemed relevant to the device failure that affects both the quality and validity of the data .Protected patient information can be missing from the reports, which may limit the scope of the analysis that can accurately be performed , 18. EacTherefore, as opposed to other implants, the TFNa set screw allows for potential fracture during implant insertion leading to uncontrolled collapse, excessive proximal femoral shortening, and construct failure. Per the manufacturer, these reported failures occur when the lock prong is advanced into the proximal screw aperture prior to blade/screw insertion. In addition to breakage, we discovered several other scenarios where the lock prong can deform, compromising both control of rotation and shortening of the helical blade/lag screw. We recommend the surgeon inspect the proximal screw aperture of the TFNa implant after attachment of the proximal aiming arm to ensure that the locking prong has not been deployed prior to insertion. Since the set screw is internal to the proximal aspect of the nail, and proper inspection requires complete disassembly, many locking mechanism failures potentially are unrecognized and underreported.Additional file 1: Video S1. Depicted is the lock prong of the TFNa set screw rotating in the proximal implant as it is advanced. The dual component set screw of the TFNa is designed so that only the upper portion, or lock drive, should rotate as it is deployed. Accessory rotation of the lock prong outside of its intended path as it is advanced could result in jamming/bending of the lock prong against the implant wall prior to it being fully seated in its correct position next to the helical blade.


The infra-acetabular screw which is placed from the pubis to the ischium can be used as a special positional screw of the posterior column of the acetabulum. This study was performed to simulate the surgical procedure and obtain the ideal insertion point, diameter, length and angle of the screw through the method of axial perspective in Chinese patients.We randomly collected the pelvic computed tomography (CT) scans of 200 adults. DICOM-formatted CT-scan images were imported into Mimics software to establish the 3D digital model of the right semi-pelvic was established. A virtual cylinder representing the screw was placed from the pubis to the ischium to fix the posterior column. The largest secure diameter and length of the virtual screw were measured and the position of the insertion point and the directions of the screw were also researched.The screw insertion safe zone exhibits an irregular shape of “tear drop” in the reconstructed pelvic model. The mean maximum diameter of screws was 5.01 ± 1.28 mm, and the mean maximum length of screws was 93.99 ± 8.92 mm. The screw insertion corridor with the least diameter 3.5 mm was found in 94 of 100 males (94%) and 86 of 100 females (86%). We found gender-dependent differences for the mean maximum diameter and the maximum length of the screw. There was statistically significant difference between genders in the position of insertion point.In this study, we suggest an individual preoperative 3D reconstruction simulation to develop better screw placement plans, which provides a valuable guideline for seeking the largest secure corridor of infra-acetabular screw. Further biomechanical studies are needed to verify the function of the screw. The treatment of complex acetabular fractures involving quadrilateral displacement has been widely studied in recent years –6. The gEarly, Letournel and Judet suggested the ideal screw path is parallel of the quadrilateral wall, in which the screw could fix the quadrilateral fracture and prevent the medial subluxation . The plaAt present, there are multiple studies on the application of CT data utilizing various software for the fixation of periacetabular screws , 9–13. PWe retrospectively collected the pelvic CT scans of 200 adults who had undergone continuous slice CT scanning at the imaging research center of our hospital during August 2016 and June 2019. Patients were excluded if they had pelvic or acetabular fractures, tumors, severe deformities, or severe hip inflammation. This study was approved by the Institutional Review Board of Liaocheng people’s hospital. Due to the retrospective nature of the study, informed consent was waived. The mean age of the patients on whom the models were based was 46.31 ± 14.19 years (range 18–86 years).DICOM-formatted CT-scan images of each patient were imported into Mimics software . We removed the soft tissue, femoral head and sacroiliac joint by the function of image segmentation, region growth and multiple slice editing of Mimics software, respectively. A total of 200 right virtual semi-pelvic models were created.We reduced the transparency of the semi-pelvic models and turned it to the axial perspective view, which was parallel to the posterior of obturator formamen from top to bottom Fig . Observap < 0.05.The collected data were analyzed by SPSS 19.0 statistical software. The experimental data are represented as the mean ± SD. T tests were used to compare the data. Statistical significance was accepted at The study subjects included 100 males and 100 females aged between 18 and 86 years old, with a mean age of 46.31 ± 14.19 years. As shown in Fig P < 0.05).As shown in Tables P > 0.05).The mean angle α and β of different genders were also recorded in Table The screw insertion corridor with a diameter of at least 3.5 mm was found in 94 of 100 males (94%) and 86 of 100 females (86%). However, the corridor with a diameter of at least 4.5 mm was found in 77 of 100 males (77%) and 53 of 100 females (53%) from the Fig In recent years, the trend in treatment of acetabular fractures is achievement of less invasive single ilioinguinal approach, especially in elderly patients –18. Due Mimics software has been widely used in 3D reconstruction for the development of digital orthopedics technology. In our study, we applied the 3D method of axial perspective that described in previous studies were applied to simulate the surgical procedure , 11, 21.In our research, the diameter and length of the infra-acetabular screw were significantly larger in males compared with females. In addition, the vertical distance from the insertion point to the arcuate line or pecten pubis and the distances from the insertion point to eminelntia iliopectinea and pubic tubercle were all measured in this study, which exhibited significant difference between genders. This was due to the obvious anatomic differences in pelvic bones between female and male. This study showed that the mean angle α and β between male and female had no statistical inference. From the Table Gras et al found that 93% pelves contained an infra-acetabular corridor with a diameter of at least 5 mm . They alOn the basis of mastering the diameter and length of screw, the insertion point and direction are important factors affecting the safe placement of infra-acetabular screw. Unlike the common posterior column screw, the infra-acetabular screw needs to be placed through the middle window of ilioinguinal approach. Culemann et al reported that the entry point for the infra-acetabular screw was 1 cm caudal of the eminelntia iliopectinea and in the middle of the pubic ramus . Gras etThere are still some limitations to this study. We only analyzed the data based on genders, not different age groups. In addition, we did not collect data according to age, height, weight or body bone density. These factors may also affect the implantation of screws. We only studied the pelvises of Chinese people, who have different skeletal shapes with American and European populations. Thus, more biomechanical studies and related clinical research should be performed to compare the effect of the infra-acetabular screw with other acetabular screws.We indicate a valuable guideline for seeking the largest secure corridor of infra-acetabular screw, which is used as a special posterior column positional screw. The ideal screw position and the size of the screws can be determined in 3D-models by digital software. Further biomechanical studies are needed to verify the strength and effect of the screw.


Coptotermes formosanus (2-yr-old) to describe general foraging behavioral sequences and to find how foraging workers allocate time between the foraging site (food acquisition or processing) and non-foraging site (food transportation).Foraging in group living animals such as social insects, is collectively performed by individuals. However, our understanding on foraging behavior of subterranean termites is extremely limited, as the process of foraging in the field is mostly concealed. Because of this limitation, foraging behaviors of subterranean termites were indirectly investigated in the laboratory through tunnel geometry analysis and observations on tunneling behaviors. In this study, we tracked subsets of foraging workers from juvenile colonies of Once workers entered into the foraging site, they spent, on average, a significantly longer time at the foraging site than the non-foraging site. Our clustering analysis revealed two different types of foraging workers in the subterranean termite based on the duration of time they spent at the foraging site and their foraging frequency. After entering the foraging site, some workers (cluster 1) immediately initiated masticating wood fragments, which they transferred as food boluses to recipient workers at the foraging site. Conversely, the recipient workers (cluster 2) moved around after entering the foraging site and received food from donating workers.This study provides evidence of task specialization within foraging cohorts in subterranean termites.The online version contains supplementary material available at 10.1186/s12983-021-00446-5. Animals search for and consume food resources to survive and successfully reproduce, as food acquisition fundamentally supports their development and reproduction. Depending on the lifestyle of a species, foraging can be performed alone  or in groups, which often requires a collective coordination of actions to optimize foraging output . Among gIn social insect colonies, foraging is a collective process in which individual workers venture away from the safety of the nest, search for and collect food resources, and return to the nest to provision their nestmates with food. In many cases, foraging is performed by a subset of colony members, indicating that not all individuals in a colony equally participate in foraging –5. The pCoptotermes, have been largely neglected, as it is difficult to study termites in the field due their large colony size , unspecified nest location , long foraging distance, and invisible foraging pathways  [Although foraging behavior has extensively been studied in many social insects, most empirical data were obtained from species with readily visible access to foragers such as ants and bees –15. In cr tubes) , 16–19. r tubes) –26 and tr tubes) –30. DespIn subterranean termites, foraging is always initiated with the excavation of new underground tunnels toward putative foraging sites . TunneliCoptotermes formosanus Shiraki (Rhinotermitidae) workers from whole juvenile colonies (2 yr-old) were tracked at a foraging site. We determined how foragers allocate their time performing various behaviors involved in food acquisition or processing  and food transportation away from the foraging site  during a 12 h period. We also measured foraging frequencies of workers to find out how workers persistently participate in foraging tasks to determine if time allocation in foraging behaviors is homogenous among all foraging workers, or if workers would display distinct behavioral profiles.In this study, subsets of C. formosanus were established using alate (winged primary reproductives) pairs collected during dispersal flights (April–May 2017) following the method used by Chouvenc et al. [Picea sp.) on top of the soil, and 3% agar solution poured into a plastic vial . Once the agar solution solidified, a pair of male and female reproductives was introduced and the rearing unit was closed with a perforated cap to allow air circulation. Hundreds of rearing units were prepared and kept at 28 ± 1 ℃. Colonies were processed 6 to 8 months after colony establishment, and surviving colonies were transferred to larger vials , containing organic soil (5 cm high), 6 pieces of wood , and 3% agar solution.Colonies of c et al. . HundredPicea sp.) was placed on top of the soil. Then, the vial that a colony was reared in for a year was uncapped and horizontally placed on the bottom of the container, allowing termites to forage out. Colonies were monitored weekly with replenishment of wood and water as needed until they were two years old, resulting in colonies with 3000 to 5000 individuals.One year after colony foundation, thriving colonies were transferred to container boxes . In these containers, moistened organic soil was placed at the bottom (3–4 cm high) and a piece of wood , soaked in water for 2 days prior to experiment, was placed in the arena (20 × 25 × 0.5 cm3) as food. Workers and soldiers were allowed to freely move between foraging site and central nest through the 1 m acryl tube for 3 days. For each colony, 13 termites were randomly collected at the foraging arena near the wood as a subsample of the active foraging population. These individuals were marked with different paint colors on the dorsal surface of their abdomen and re-introduced into the foraging arena. A camcorder  was mounted on top of the entry point of the arena, and video was recorded for 12 h.Two years after colony foundation, three visibly healthy colonies were selected for the experiment. A small hole (diameter: 0.5 cm) was drilled on the side of the container to connect it to a planar arena with an acryl tube  for observation  time spent at the foraging site for food acquisition, (2) time spent at the non-foraging site for food transportation or other non-foraging tasks, and (3) foraging frequencies (number of entry events in twelve hours)  since each individual worker had multiple observations. This allowed us to have a single value per termite for each variable. Then, average time spent was determined using this individual mean value. Average time spent during the observation period was compared with a Mann–Whitney U test. Comparisons were performed with pooled data and also separately for each colony to confirm variation among colonies.Next, three variables  were further analyzed with K-means clustering analysis in R v3.6.1 and library of tidyverse, cluster and factoextra were used for the clustering analysis . The numThe proportion of time spent at the foraging site and non-foraging site was calculated for the 12 h observation period and compared between clusters with Wilcoxson’s sum rank test and Fisher’s exact test. All statistical analyses except the clustering analysis were performed under SPSS V19.0 After workers entered the foraging site, they either directly moved to the wood piece to remove wood fragments or moved around the wood piece Fig. . WorkersDuring the observation period, time spent at the foraging site and at the non-foraging site varied depending on the individual    and foraging frequency    . Conversely, termites in cluster 2 spent proportionally longer time at the non-foraging site relative to termites in cluster 1 .The proportion of time spent at the foraging site and non-foraging site was significantly different within clusters 1 and 2, and termites from both clusters spent significantly longer time at the foraging site than the non-foraging site  Fig. . A compaForaging behaviors  in social insects are collectively performed by individuals , 38, andC. formosanus.In this study, two different types of foraging workers were identified Fig. . Both thOur results suggest that subterranean termites can be temporarily specialized in different aspects of foraging activity, such as food finding, acquisition, and transportation, to increase overall foraging efficiency similar to tunneling behaviors , 22, 23.C. formosanus molt every 45 days with a daily molting rate of about 2% [However, it is currently unknown how workers become specialized in a certain task in termites. One possibility of such specialization in food acquisition behavior is that it may inherently be imposed by the physiological constraints of termite workers. Although the mandibles of termites are hardened with metal components , chewingabout 2% . In thisabout 2% . Our obsabout 2% . These hC. formosanus ranged from 2 to 5 mm [Here, we provided preliminary evidence of food transportation specialization in workers. Some workers exited the foraging site toward the central nest with food boluses, but then returned to the foraging site after a short time without food visible in their mouthparts Fig. , suggest to 5 mm , 49. ConHodotermes mossambicus (Hagen) (Hodotermitidae), some workers in the foraging cohorts cut short grass pieces and deposit them along the foraging trail, where they are then later picked up by other workers, which eventually transport the food back to the nest [Macrotermes , foraging workers often process food at the foraging site and carry it to the nest following chemical trails [Coptotermes, but in other termite models with similar physiology and life traits.Task specialization within foraging cohorts was previously reported in various social insects, including some termite species, but not in subterranean termites. In a harvester termite (open forager), the nest . In anotl trails . Howeverl trails . The obsl trails . FurtherAdditional file 1: Table 1. Individual tracking data of marked foraging workers in C. formosanus. In total, 13 termites per colony were measured and 3 different colonies were used . Average and total time spent were calculated by averaging and summation all incidences respectively.Additional file 2: Figure 1. Comparison of foraging workers’ average time spent at the foraging site (gray) and non-foraging site (blank) based on each colony.Additional file 3: Figure 2. Average of time spent at the foraging site (A), at the non-foraging site (B) and foraging frequency (C) of cluster 1 and 2. Asterisk and NS denote significant and no differences respectively according to Mann-Whitney U test (P < 0.05).


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. 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 . For moslin-41 mediate the extreme morphological and developmental defects by which the let-7 miRNA was discovered in C. elegans , including many sites with 3′ pairing . These mKD values) for many thousands of different RNA k-mers ≤12 nt in length (RNA bind-n-seq (RBNS) enables unbiased, high-throughput assessment of binding sites embedded within a larger random-sequence context . We recen length . Here, wk-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.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 . Each moAs 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 , we usedk-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  or the 3′ end (adjacent columns) of the 3′ site, while maintaining the same offset. For example, in the heatmaps corresponding to offsets of +2 to +12 nt, the prominent contrast between the row corresponding to pairing beginning with nucleotide 11 and the row corresponding to pairing beginning with nucleotide 12 illustrated the strong benefit of pairing to G11 of let-7a , and accumulation of each variant in the presence of each co-transfected miRNA was monitored by high-throughput sequencing and compared to accumulation observed in the mock co-transfection . Most ofA). This site was more effective than the two possibilities with an offset of +1 formed by insertion of either a single A or U nucleotide , which were in turn more effective than the site that lacked an insertion (0) and thus had a 0-nt offset . Although this rank ordering was consistent with that predicted from relative affinities, some quantitative disagreement with the relative affinities was observed, with the three sites with positive offsets performing substantially better than expected from their relative affinities (U). Efficacy of this site was less than half of that of the other sites with positive offsets, and its greater efficacy over the site with a 0-nt offset was statistically significant only for the dual-site configuration . For the 3′-compensatory site with 9 nucleotides of complementarity starting at position 13, the efficacy of the +4U variant was also less than that of the +4A variant  were more repressive on average than the two sites found within lin-41 mRNA of C. elegans . The lin-41 sites, as well as the other effective sites, were all more effective when examined in the lin-41 local sequence context than they were in most of the other contexts . This correspondence for 3′ sites resembled that observed for seed-matched sites  of let-7, which is enhanced through the positive-offset binding mode to confer robust repression of the lin-41 mRNA, with repression further enhanced by favorable site context and some inter-site cooperativity.Overall, we found that affinity observed in vitro corresponded well to repression observed in cells . Moreover, these coefficients, which distilled the pairing preferences indicated by the 934, 1061, and 1180 relative KD values measured for let-7a, miR-1, and miR-155, respectively, quantitatively captured the qualitative observations made earlier from analysis of subsets of the data.To test this independence, we examined how well the affinities could be explained as the product of two coefficients, one representing the contribution of the pairing range, which was defined by pairing position and length  value was described as the product of the pairing, offset, and seed-mismatch coefficients corresponding to its 3′-pairing architecture , corresponding to an ~25% change in binding affinity , or between miRNAs (e.g. when comparing the effects of mismatches to the G at position 3 of miR-1 with those to the G at position 6 of miR-155) . Howevers lin-41 , furtherHaving found that the 3′-pairing affinities of each of the three miRNAs were largely a function of the miRNA pairing, offset, and seed-mismatch preferences, we investigated the extent to which different regions of each miRNA contributed to these preferences. To do so, we performed AGO-RBNS with synthetic miRNA variants. Two of these variants were chimeric miRNAs in which nucleotides 1–8 of let-7a and miR-155 were swapped . The othKD values for 38,108 let-7a sites, 44,190 miR-1 sites, and 52,166 miR-155 sites. Analysis of these variants in the context of the best sites at each length , four possible single-nucleotide bulges (occurring opposite the linkage of two miRNA positions and assigned to the more 3′ miRNA position), and one single-nucleotide deletion . Consideration of these variants together with the original sites with perfect contiguous pairing resulted in the measurement of h length  revealedh length , 12–15 oh length , and 15–h length , which cKD fold change for a mismatch at each position for all three miRNAs, averaging both over the three mismatches at each position and over each of the 10-nt sites that contained the position . For each miRNA, a small but significant benefit to terminal bulges was observed . Thus, an isolated complementary target nucleotide separated from a longer contiguous stretch of pairing can contribute modestly to site affinity.To examine if the benefit of bulged nucleotides over mismatched nucleotides applied to the very 5′ and 3′ ends of 3′ sites, we considered all possible 10-nt sites for all three miRNAs with programmed libraries, and calculated the fold difference in relative G of each mismatch in the context of all 10-nt 3′ sites of the three miRNAs. We first averaged these values over all the contiguous sites, and then over all positions with the same miRNA nucleotide, and then over the three miRNAs, resulting in one global average ∆∆G value for each of the 12 possible miRNA–target mismatch possibilities. Comparison of these values with those predicted using the nearest-neighbor parameters revealed that the effects of the mismatches were typically much lower than expected for RNA in solution, with no strong relationship between the observed and predicted ∆∆G values (r2 = 0.02). The outlier in this analysis was the miRNA–target U:G wobble, which was as disruptive as the typical mismatch but predicted to be much less so  the overall contribution to binding energy was far less than that predicted, (2) mismatched target G nucleotides were relatively more deleterious than predicted, and (3) wobble pairing was relatively less favorable than predicted. Indeed, the U:G possibility, which both contained a target G nucleotide and was a wobble, was the mismatch with the greatest deviation from expectation.To enable comparison of the observed effects of mismatches with those predicted by the nearest-neighbor model of RNA duplex stability, we calculated the ∆∆h values , right. G and the fractional reduction in ∆G for each type of mismatch for each of the six miRNAs for which there was random-sequence RBNS data  within the context of canonical 8mer pairing, calculating the average ∆∆BNS data . These abilities , left. Tredicted , right. An AGO-loaded miRNA can be divided into three regions: the seed region (nucleotides 2–8), the central region (nucleotides 9–10 or 9–11), and the 3′ region . BecauseAlso problematic for the notion of a short 3′-supplementary region common to all miRNAs was our observation that the positions most important for 3′ pairing differed between different miRNAs. For example, at their optimal offsets, both let-7a and miR-124 preferred pairing to nucleotides 11–14 over pairing to nucleotides 13–16 , and theCaenorhabditis elegans. C. elegans requires let-7 repression of lin-41 for viability, and this repression occurs through two 3′-compensatory sites that each have pairing to nucleotides 11–19 of the miRNA  revealed a similar polarity, with mismatches near position 11 tending to be most consequential and those near position 19 tending to be least consequential , which paired to nucleotides 10–15. Thus, our results explain why an overall preference for pairing to nucleotides 13–16 was detected in meta-analyses of both functional data for 11 miRNAs and evolutionary conservation of sites for 73 miRNA families . Our keyC. elegans lin-41, which both include pairing to nucleotide 11 and an offset of +1 nt, as well as the first lsy-6 site within the 3′ UTR of C. elegans cog-1, which includes pairing to nucleotide 11 and an offset of +2 nt. The discovery of these two binding modes required knowledge of the interplay between preferred pairing position and preferred pairing offset, which underscored the utility of obtaining affinity measurements for a large diversity of 3′ sites.Another key insight is evidence of two distinct 3′-binding modes, observed as different offset preferences of let-7a, miR-124, lsy-6, and miR-7 with and without pairing to nucleotide 11 . In the G and measured 3′-pairing affinities . Although lsy-6 did not have an oligo(G) stretch, it did have a well-positioned C13-G14-C15 trinucleotide, which together with G11 was critical for pairing affinity. When considering all four miRNAs together, as well as the lack of any GG, CG, or GC dinucleotides within the 3′ regions of miR-1 or miR-7, we suggest that miRNAs with GG, CG, or GC dinucleotides within positions 13–16 are the ones most likely to participate in productive 3′ pairing, and that pairing that extends to an oligo(G) sequence outside of positions 13–16 preferentially enhances affinity.The importance of pairing to miRNA G nucleotides, and not C nucleotides (other than the C13-G14-C15 of lsy-6), suggested that a miRNA–target G:C base pair is read out differently than a C:G base pair. Perhaps G nucleotides participate in base-stacking interactions that position or pre-organize the guide strand to favor nucleation of 3′ pairing. Alternatively, the explanation might involve target-site accessibility. Pairing to a C in the miRNA 3′ region would require a G in the vicinity of the seed match, which compared to a C would cause poorer target-site accessibility , therebyOur results also revealed a functional difference between 3′-supplementary and 3′-compensatory pairing. The added affinity of a 3′ site was relatively constant when it supplemented different sites that had seed matches , whereasPairing to the miRNA 3′ region not only increases site affinity and target repression, but it can also influence the stability of the miRNA itself, in a process called target-directed miRNA degradation (TDMD) . The hanThe crystal structures of several known TDMD substrates bound to their corresponding TDMD-inducing target sites reveal a distinct conformation for these AGO–miRNA–target RNA ternary complexes in comparison to ternary complexes that have supplementary pairing involving only nucleotides 13–16 . During 2. HEK293T cells  with 10% FBS  at 37°C and with 5% CO3T cells  were obtAGO2–miRNA complexes were generated and purified as described .For each miRNA, a programmed library was constructed by in vitro transcription of multiple chemically synthesized DNA libraries, and then mixing these RNA libraries after gel purification. Each library contained 25 nt of entirely randomized sequence, followed by an 8-nt programmed site, followed by either 5 nt of random sequence (let-7a and miR-1 libraries) or 4 nt of random sequence (miR-155 library). For every AGO-RBNS experiment other than that with native miR-155, the final library was made by mixing six different libraries, in which each of the six libraries had at its programmed site an 8mer containing a mismatch to one of the six seed nucleotides. For the experiment with native miR-155, the library was assembled by mixing the six 8mer-mismatch libraries with a seventh library that had a 6mer at the programed site, flanked by non-A residues at positions 1 and 8.Each individual library was transcribed from synthetic DNA (IDT) and purified as described . The fraAGO-RBNS was performed as described , using ck-mers were calculated by comparing the reads from RNA bound to 840 pM AGO2–let-7a complex to reads from the input library. For each of the two sets of reads, reads that contained one of the 18 possible 8mer-mismatch sites in the correct position (such that the CUACCUCA 8mer-consensus sequence spanned positions 26–33 of the read), but did not contain a canonical 8mer, 7mer-m8, 7mer-A1, 6mer, 6mer-m8, or 6mer-A1 site, were used to enumerate all possible 8-nt k-mers at each position within the library. Both count tables were normalized such that they summed to 1, and the normalized count table corresponding to the bound sample was divided by that of the input library to arrive at the enrichment of each k-mer at each position within the library. These k-mers were ranked according to the sum of the top five positional enrichments of each, considering each possible 8-nt window located entirely within the random sequence region 5′ of the programmed site. These 8-nt windows are labeled in k-mer position 9 of k-mer position 26 corresponds to library positions 1–8.Positional enrichments of 8-nt KD assignment, but their measured relative KD values were not used in any further analyses, owing to the ambiguity of whether the presence of these reads was the result of errors during chemical synthesis, in vitro RNA transcription, library preparation, or sequencing.When counting seed sites and contiguously paired 3′ sites within the random-sequence region of the programmed libraries, reads were first queried for whether they included a canonical or 8mer-mismatch site at the programmed region . Reads containing a canonical site  in the programmed region despite its design were still counted and used for relative Those reads containing a seed site  at the programmed region were further assigned to one of four categories based on whether the random region contained: (1) neither a seed site nor a 3′ site (defined as a site of 4–11 nt of contiguous complementarity to a region of the miRNA spanning position 9 to the 3′-most nucleotide), (2) at least one seed site but no 3′ sites, (3) at least one 3′ site but no seed sites, or (4) at least one seed site and at least one 3′ site. This categorization enabled read enrichment from category 3 to be compared with that of category 1 as an indication of the contribution of pairing to the 3′ end with minimal influence from seed binding outside the programmed region.The categorization of each read proceeded as follows: (1) any canonical or 8mer-mismatch sites other than the programmed site were identified, looking within the entire random-programmed-random region with three nucleotides of constant sequence appended to the 5′ and 3′ ends of the read, (2) the 28-nt segment spanning library positions 9–37 (which included three nucleotides of the 5′ constant sequence) was queried for the longest contiguous match to the miRNA 3′ end, retaining multiple sites in the case of ties. Any site(s) 4–11 nt in length that was not contained within a canonical or 8mer-mismach site found within this 28-nt segment was counted as a 3′ site, and (3) the read was then assigned to one of the four categories described above. In the case of a read with either a single 3′ site or a single seed site within the random region, the read was assigned to that site, recording the type of site, the identity of the programmed site, and the distance between the two. In the case of a read with either multiple 3′ sites or multiple seed sites within the random region, the read count was divided equally among the sites. In the case of a read with both a 3′ site(s) and a seed site(s), the read was split between all seed-and-3′-site pairs, recording the names of the seed, 3′, and programmed site for each.KD estimation, or first pooled such that the identity of their programmed site was not considered. When pooling, all counts corresponding to reads with identical site-and-positional information were summed into two categories: those for which the programmed site was an 8mer-mismatch site, and those for which the programmed site was one of the canonical sites.This categorization yielded tables of counts for reads with (1) only programmed sites, (2) seed-and-programmed site pairs with positional information, (3) 3′-and-programmed site pairs with positional information, (4) seed-and-3′-and-programmed site triples with no positional information, and (5) reads with neither a canonical site nor an 8mer-mismatch site at the programmed site position. These tables were either used directly for relative When counting seed sites (including 8mer-mismatch sites) and contiguously paired 3′ sites within the random-sequence libraries, the 37-nt random-sequence region of each read was appended with 3 nt of constant sequence at each end, except in the case of miR-1, for which the 5′-most 36 nt of the random-sequence region was appended with 3 nt of only the 5′ constant sequence, due to the sequence bias present at the very 3′ end of these libraries caused by erroneous lack of a TCG sequence in the 3′ constant region required for pairing to the Illumina reverse-primer sequence during bridge-amplification . The relIf one or more 3′ sites persisted for the read after accounting for any seed-site overlap, the 3′ site(s) of length equal to the longest 3′ site(s) was retained. All possible bipartite sites associated with that read were then enumerated, such that each seed site was considered to form a bipartite site with all 3′ sites that were 5′ of that seed site. The read was then split among any bipartite sites identified. In the event that no bipartite sites were identified, the read was split among all the seed and 3′ sites equally. While this procedure ensured equal partitioning of read counts in cases of multiple seed and 3′ sites within a given read, in practice only a small fraction of reads contained multiple seed sites and a 3′ site, or a seed site and multiple 3′ sites.KD estimation, or the bipartite sites were first pooled to combine data for 3′ sites with different canonical sites or 8mer-mismatch sites prior to relative KD estimation. When pooling, all counts corresponding to reads with the same 3′ site and distance from the miRNA position 8 of the seed site were summed into two categories: those for which the seed site was an 8mer-mismatch site, and those for which the seed site was one of the canonical sites.This categorization yielded tables of counts for reads with (1) only seed sites, (2) only 3′ sites, (3) seed-and-3′ bipartite sites with recorded inter-site spacing, and (4) neither a seed nor a 3′ site (referred to as ‘no site’). These tables were either used directly for relative KD values were calculated as described  and 8mer-mismatch sites , and if this complementarity was between 4 and 11 nt in length, the KD value of that target sequence was assigned to that bipartite site, specifying its seed site, 3′ site, and intervening nucleotide length. If multiple 3′ sites were tied for the longest length, both 3′ sites were ascribed to the target sequence. Upon using the sequences to define the bipartite site information for each target RNA, only those target RNAs with a single bipartite site, or a single seed site and no 3′ pairing between 4 and 11 nt in length, were included in the downstream analyses. From these sites, we calculated the KD fold change for each available 3′-site, offset, and seed-site combination . For example, in the dual-site configuration of the lin-41 sequence context, the site under investigation replaced each of the two 3′-compensatory sites within the segment spanning nucleotides 650–798 of the lin-41 3′ UTR. Likewise, in each of the single-site configurations, the site replaced one of the two endogenous sites, whereas a no-site sequence replaced the other endogenous site. When in the other 13 de novo sequence contexts, sites were located at the same position as they were in the lin-41 sequence context, but the lin-41 sequence was replaced with arbitrary sequence of equal length. Each of the arbitrary sequence segments, as well as the no-site sequence segments, was generated by weighted sampling of dinucleotides according to the frequencies observed human 3′-UTR sequences, except at positions opposite let-7a nucleotides 9 and 10, where only the dinucleotides that would place an A or U at these two positions were used. For the lin-41 3′-UTR context, each of the central U nucleotides within the linking segments of the upstream (GUU) and downstream (AUU) sites was either removed to generate the 0-offset possibility, or replaced to generate the +1 and +4 possibilities, and the 9-nt 8mer-bA5 of the upstream site was replaced with 8 nt of sequence. The de novo 3′-UTR contexts and the no-site replacement sequences were also designed to exclude any (1) ≥6-nt contiguous match to the first eight positions of miR-1, (2) ≥6-nt contiguous match to the first 11 positions of let-7a, (3) ≥4-nt contiguous match to positions 9–21 of let-7a, (4) PUMILIO response element (UGUANAUA), (5) splice-donor sequence (GGGURAGU), (6) BstXI restriction site (CCAN6UGG), (7) BsrGI restriction site (UGUACA), or (8) poly(A) signal (AAUAAA). In total 952 variants were designed [(((15 3′-compensatory sites + 5 sites with only seed pairing) × (three configurations)) + (7 no sites + 1 lin-14 site)) × 14 contexts = 952 variants].The placement and spacing of the sites were based on the placement and spacing of the dual 3′-compensatory sites within the 3′ UTR of E. coli  in two separate reactions with 100 µL of cells each, and bacteria from both electroporations were incubated in SOC media for 90 min at 37°C to recover, and then plated onto 19 10-cm LB agar plates containing ampicillin. In parallel, a small fraction of the cells were dilution-plated, which indicated that approximately five million independent transformants were obtained. After 16 hr of bacterial growth under ampicillin selection at 37°C, bacteria were harvested, and the reporter-plasmid library was purified by MAXI-prep .The sequences of these 952 variants were inserted into an expression vector  at a posThe assay was performed in duplicate for each of the four conditions (three miRNA duplex transfections and a mock miRNA transfection), generating eight samples. For each condition, F9 cells were plated at 3 million cells in one 10-cm dish supplied with 10 mL media (DMEM +10% FBS). After 24 hr of culture, the cells were supplied with fresh media and were transfected with 5.8 µg of the plasmid library diluted with 28.9 µg of pUC19 carrier plasmid using Lipofectamine 2000  and Opti-MEM (Thermo Fisher 31985062), and either let-7a duplex, let-7a-21nt duplex, miR-1 duplex, or no duplex (mock) at a final duplex concentration of 25 nM, according to the manufacturer’s protocol. After 24 hr, cells lysis, RNA extraction, reverse transcription, PCR, and gel purification were performed to generate amplicons for RNA-seq, as described , with thi and two positions For analysis, reads were first filtered for quality, as described . Reads plin-41 sites only when within the lin-41 3′-UTR context). This yielded four values for each site in the let-7a arm and two for each site in the miR-1 control arm. The p values testing the significance of differences in mean repression were generated by performing Tukey’s HSD test to calculate the significance of all pairwise differences between the means of the 42 categories shown in where KD of each site possibility i, The model relating the measured relative where−3 and −1. Additionally, relative KD values for 3′-compensatory sites with pairing to positions 13–16 and nonzero offsets or for sites with 9 nt of 3′ pairing and a +1-nt offset were calculated using only reads compatible with that site . However, for sites with 9 nt of 3′ pairing and +4-nt offsets, read numbers were not sufficient for this approach, and relative KD values were calculated using reads with either a W (A or U) at each of the insertion positions, with the +4-nt offset 9mer-m11–19 possibilities further corrected by first calculating, for each of the +4A-nt and +4U-nt possibilities, the sum of the fractional abundance of that site across all five binding samples (when averaging the two 12.65%-binding samples) divided by the number of reads in the input sequencing library with either AAAA or UUUU within the 25-nt random region, and then dividing and multiplying respectively the relative KD values of the +4A-nt and +4U-nt possibilities by the square root of the ratio of the two values. The +4-nt offset 9mer-m13–21 possibilities could not be corrected in this way because of the lack of any reads with these sites within the bound samples.with KD fold-change values measured when summing the counts from each of the 18 8mer-mismatch sites. The model was constructed to produce a log10-tranformed KD fold-change value, denoted here using i, the 3′ terminus of pairing  i and In order to separate the intrinsic pairing preferences of each miRNA 3′ end from the effects of varying the offset of pairing, we fit a thermodynamic model of 3′-end binding efficacy to the Because the nature of the relationship between the pairing range and the offset preferences could not be known a priori, we constructed three variants of the model function where 10-transformed KD fold-change values i, Each of the models described by optim function in R using the L-BFGS-B method, supplying the cost function and its gradient and setting the ‘maxit’ parameter to 1 × 107. When optimizing all three models, all where r2 = 0.92, 0.86, and 0.96 for let-7a, miR-1, and miR-155, respectively, r2 = 0.81, 0.81, and 0.94), and because the multiplicative-plus-constant model r2 = 0.93, 0.86, and 0.96) while decreasing model interpretability (as the constant term corresponded to a benefit to binding irrespective of the manner of the pairing to the miRNA 3′ end), we selected the multiplicative model. For the purposes of interpretation of the model coefficients, we re-scaled the coefficients as follows:Because the multiplicative model k. Each re-scaled pairing range coefficient KD fold change that could be obtained by contiguous pairing to nucleotides i through which, because none of the coefficients were negative, caused each offset coefficient We estimated the model error by calculating the asymptotic covariance matrix where ves ∂κ∂θ . From tht statistic for 97.5% confidence with where KD fold-change values , we derived their theoretical relationship by comparison of the apparent KD for a biochemical model in which only seed pairing was allowed (model A) with that in which both seed and 3′ pairing were allowed (model B). Model A is described byBecause we were interested in the expected relationship between the measured where where and when substituted for KD.in which Model B is described bywhere, in addition to the terms defined for model A, which mechanistically corresponds to the AGO–miRNA complex engaging in 3′ pairing with a target to which it is already seed-paired. We note that this model does not allow formation of The fractional occupancy of the target RNA in model B is given bywhich can be substituted for and then further substituted for This can be simplified and rearranged in an analogous form to KD value is KD is KD fold change itself, would be expected to relate to the ∆G through the equation 10[(KD fold change) – 1] instead of log10(KD fold change), but ultimately chose not to include this conversion in our analyses, because it caused many of the lowest-affinity pairing coefficients to be fit to anomalously large negative values. This occurred because those pairing coefficients were fit using KD fold-change values that in some cases closely approached 1 , which is a regime in which small changes in measured KD fold change will lead to much larger changes in the modeled pairing coefficient. For example, a KD fold change of 1.1 would correspond to an apparent pairing coefficient of log10(1.1–1) = −1, whereas 1.01 would correspond to an apparent pairing coefficient of log10(1.01–1) = −2, or 10-fold worse binding.from which it is clear that the apparent i and ending at position j, the predicted ∆G of duplex formation between the sequence of the miRNA beginning at position 9 and the sequence reverse-complementary to miRNA positions i–j, with no non-complementary nucleotides appended to either terminus, was calculated using RNAduplex, as part of the ViennaRNA package, through its Python interface (For comparison with each pairing-range coefficient beginning at position nterface .10(KD fold-change) values measured for each of the 18 8mer-mismatch sites separately. This model took the form of:We extended the thermodynamic model of 3′-end binding efficacy to include seed-mismatch effects, by using as input data the logwhere where which preserved the same interpretation of each The 95% confidence intervals of this model also used KD fold change in the random-library experiments, we first attempted to apply the modeling approach as used when analyzing the data from the programmed-library experiments. However, we found that the read counts assigned to each possible pairing, offset, and seed-mismatch combination were too sparse, such that modeling using these data could not be reliably performed. We therefore analyzed the differences in the benefit of 3′-supplementary pairing between each of the six canonical sites  as well as a representative 3′-compensatory site given by summing the read counts of all 18 8mer-mismatch sites.When analyzing the effects of seed type on 10(KD fold change), thereby constructing a 7  × 20 (pairing range) matrix of log10(KD fold change) values. This matrix was then sorted by the average log10(KD fold change) of each pairing range, and then this value was subtracted from each column, such that the values within each column reported on the deviation of each site type from the average, for that pairing-and-offset possibility. These deviations were then averaged for each of the seven site types over the top five (i.e. the top quartile) of pairing-and-offset possibilities, to give the empirical contribution of each site type to 3′-binding affinity, in comparison to that of the average. These values are plotted for all six miRNAs in To compare these sites, we first took, for each miRNA, all 3′ pairing possibilities with lengths of 4 or 5 bp and pairing to miRNA positions 9–18, and determined for each of these possibilities the offset that imparted the maximum average logKD values, and as well to reduce assignment problems owing to any imperfect 3′ site from one region of the miRNA being sequence-identical to that from any other region of the miRNA.To calculate the effect of a mismatch, bulge, or deletion on the binding affinity of a 3′ site, the site counting was repeated for each fully paired 3′ site while also counting each of its derivatives containing either a single mismatched, bulged, or deleted nucleotide (hereafter referred to as ‘imperfect 3′ sites’). This was done to reduce the total number of sites being simultaneously counted and used to calculate relative The site counting was performed similarly to that of the fully paired 3′ sites, with some differences: those reads containing a seed site at the programmed region were still assigned to one of four categories, with the definition of a 3′ site expanded to include any fully paired 3′ site of length 4–11 nt in addition to any imperfect derivatives of that site. The categorization of each read proceeded through the following steps: (1) Any seed sites were identified, looking within the entire random-programmed-random region with 3 nt of constant sequence appended to the 5′ and 3′ ends of the read. (2) The 28-nt segment comprising 3 nt of the 5′-constant sequence and 25 nt of random sequence was queried for any instances of any imperfect 3′ sites, with any deleted- or mismatched-nucleotide sites that were contained with another mismatched- or bulged-nucleotide site not counted . Any imperfect 3′ sites were also queried to make sure that the nucleotides on each side of the site were not complementary to the next corresponding positions of the miRNA guide. We note that if such an imperfect 3′ site was found that failed these criteria, any identified fully paired 3′ sites were not counted toward that read. (3) The 28-nt segment comprising 3 nt of the 5′-constant sequence and 25 nt of random sequence was queried for the longest fully paired 3′ site(s), retaining multiple 3′ sites if more than one was of the longest length. If at least one fully paired 3′ site >4 nt in length and not contained within a seed site (if such sites were present) was identified, and if at least one imperfect 3′ sites was identified, the length of the fully paired 3′ site(s) was compared to that of the imperfect 3′ site(s), and the 3′ site(s) that was longer was retained. Lastly, if the fully paired 3′ site(s) was ≥11 nt in length, neither the fully paired nor imperfect 3′ site(s) were counted. (4) The read was then assigned one of the four categories, and the read count split as described when assigning contiguous 3′ sites.KD values used in KD fold-change values of each fully paired site and each of its imperfect sites at particular offset values were substituted with the geometric mean of the KD fold change of those sites over the three contiguous offset values centered on that offset value. The offset value used for reporting of the imperfect KD fold-change values was the offset value between −2 and +6 nt for which the KD fold-change of the fully paired site was the greatest.The relative G values corresponding to sites in which the position of a particular bulged nucleotide was ambiguous were used to calculate the average ∆∆G value for each of the inter-nucleotide positions at which it might occur. Conversely, these sites were omitted from comparisons of terminal bulges and mismatches of When performing the positional analyses of KD fitting for each fully paired site and its imperfect derivatives. Individual reads were queried for all seed sites and fully paired 3′ sites as described for the fully paired site counting with the random-library experiments, in addition to being queried for any imperfect 3′ sites derived from a fully paired site. If a read contained at least one seed site and at least one fully paired or imperfect 3′ site, each 3′ site was checked for any amount of overlap with any seed sites. If a fully paired 3′ site overlapped a seed site with the 3′ site being the 5′-most site, the 3′ site was trimmed to exclude the region overlapping the seed site, and retained if the site was still ≥4 nt in length. As before, any fully paired 3′ sites that either overlapped any seed sites from the 3′ end, contained a seed site within them, or were entirely contained within a seed site, were discarded. Any imperfect 3′ sites identified were examined to ensure that read positions just outside their limits were not complementary to the corresponding miRNA positions, and as well that they did not overlap any of the seed sites within the read.When counting imperfect 3′ sites within the random-library reads, we similarly preformed the read counting and relative If one or more 3′ site (either fully paired or imperfect) remained, all of the fully paired 3′ sites of length equal to that of the longest fully paired 3′ site in the read were retained. If at least one fully paired and at least one imperfect 3′ site remained, the length of the fully paired 3′ site(s) was compared to that of the fully paired site from which the imperfect site(s) was derived, and if it was shorter, the imperfect 3′ site(s) was retained, and the fully paired site(s) was discarded. If the fully paired 3′ site(s) was longer than the fully paired site from which the imperfect site(s) was derived, the fully paired site(s) was retained, and the imperfect site(s) was discarded. All possible bipartite sites associated with that read were then enumerated, whereby each seed site was considered to form a bipartite site with each 3′ sites that was fully 5′ of that seed site. The read was then split among any bipartite sites identified. In the event that no bipartite sites were identified, the read was split among all the seed and 3′ sites equally. While this procedure ensured equal partitioning of read counts in the case of multiple seed or 3′ sites within a read, in practice only a small fraction of reads contained either multiple seed sites and a 3′ site or a seed site and multiple 3′ sites.KD fold-change values associated with each imperfect site were calculated from the ratio of the relative KD value of the site when summing the counts for all 18 8mer-mismatch sites to the geometric mean of the relative KD values of each of the 18 8mer mismatch sites calculated individually. As with the relative KD values corresponding to the imperfect sites derived from the programmed libraries, the values reported for each of the perfect and imperfect sites reported in KD fold change of the fully paired site was the greatest.The  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. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andrea Ventura (Reviewer #2); Javier Martinez (Reviewer #3).Thank you for submitting your article "Pairing to the microRNA 3′ region occurs through two binding modes, with affinity shaped by nucleotide identity and position" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:eLife and not just those who appreciate all the details.All three reviewers and the reviewing editor found this to be a strong and significant manuscript that addresses important questions relevant to miRNA targeting. Nevertheless, each reviewer has made a few comments that if addressed would improve the manuscript. Specifically, it was agreed that some reporter assays as mentioned in two of the reviews would be important. Secondly, it was also agreed that the paper was quite dense and in some places difficult to read. Please consider this aspect during revision. It is important that the work be comprehensible by the general reader of Reviewer #1 (Recommendations for the authors):1. The study is an impressive computational feat with many interesting discoveries. However, the current manuscript is so long and time-consuming to absorb that the majority of the broader audience may lack the will to invest in reading the work and appreciating its full implications. We therefore recommend the authors shorten the manuscript with an emphasis on distilling and crystallizing the most salient points.2. Main text descriptions of the coefficients proposed in Figure 4 and used in Figure 5 and 6 are difficult understand. We recommend endeavoring to create more illustrative and/or succinct descriptions.3. Conclusions in this manuscript will be strengthened if the authors test key findings in different systems. For example, whether a let-7 3' compensatory site with +4nt offset shows a stronger repressive effect than the target with 0 or +1nt offset in cell culture; or, whether miR-155 target site with 3' paring to g15-g22 is indeed more repressed than target site with 3' paring to g12-g19 in the cells; or, measure the KD of Ago2-let-7 binding the 8mer canonical site and compare to the KD of endogenous let-7 site in lin-41. Such data would strengthen connections to findings in previous studies in the field.4. The high affinity +4 offset let-7 data are intriguing. Are there bridging sequences that are especially good for binding? i.e. is there over-representation of specific sequences in the bridging region of the let-7 +4 binding-mode? Why is +4 offset not very often observed in nature? Why does only let-7 show two binding modes, but not miR-1 and miR-155?Reviewer #2 (Recommendations for the authors):I have only a few comments/suggestions:a) The authors correctly note in the manuscript how some extensive pairings including position 9-10 are depleted as a result of Ago2 slicing activity, but the full impact of Ago2 endonuclease activity on the results of these experiments is not entirely clear. An informative additional experiment to address this point could have included either AGO1-miRNA complexes or a catalytically dead AGO2 mutant. A related point is that including other Argonaute proteins could uncover unexpected differences in the impact of compensatory sites on binding affinities. Finally, I wonder whether the impact of slicing activity can be inferred by sequencing the input library before and after incubation with the Ago2-miRNA complexes (of course bypassing the nitrocellulose purification step).b) A key difference between the experimental setting used by the authors and in vivo miRNA targeting is that in cells Argonaute is post-translationally modified and acts a part of a much larger RNP complex (the RISC). It would be useful to include some simple reporter assays to test whether the different affinities for different 3'-compensatory sites observed in the AGO-RBNS experiments translates to different repressive activity in cells. For example, the authors show that a 10-20 compensatory site with a +4 offset for let-7a results in an affinity comparable to the affinity of a perfect 8-mer seed match, while the same compensatory site with -1 offset leads to only a modest increase in affinity (comparable to a 6-mer match). This particular prediction could be easily tested with appropriately-designed reporter assays.c) On a related note, I also wonder whether some of the predictions on the efficacy of compensatory 3'-sites can be tested by looking at existing CLEAR-CLIP or CLASH datasets.Reviewer #3 (Recommendations for the authors):I found myself evaluating a paper with "invisible" results. There is a unique, technologically powerful, well-thought and most likely well-executed approach that is repeatedly applied to extract a large amount of data and derive conclusions; however, such data remains "hidden". As mentioned above, please include representative RNA bind-n-seq experiments and show how do you quantify the signals.Unfortunately, I found the text very dense and tiresome. The data is presented in a very homogenous way and the reader has to make sense of a myriad of tiny, colored squares and other, very complex and extremely data-rich graphics. After spending (a long) time trying to understand the first two Results sections, seeing that almost the same type of figures continue until the end will tempt readers – as I was tempted – to jump to the Discussion and just get the conclusions.I guess this is not the type of paper one feels excited to read; it is "too much". I urge the authors to re-think the format of the paper. Essential revisions:All three reviewers and the reviewing editor found this to be a strong and significant manuscript that addresses important questions relevant to miRNA targeting. Nevertheless, each reviewer has made a few comments that if addressed would improve the manuscript. Specifically, it was agreed that some reporter assays as mentioned in two of the reviews would be important. Secondly, it was also agreed that the paper was quite dense and in some places difficult to read. Please consider this aspect during revision. It is important that the work be comprehensible by the general reader of eLife and not just those who appreciate all the details.C. elegans lin-41 mRNA. Overall, these new results, which are presented in Figure 3 of the revised manuscript, showed that key conclusions made from in vitro affinity measurements also apply to repression in cells.As requested, we performed reporter assays. To avoid limitations and pitfalls of standard reporter assays, we implemented a massively parallel reporter assay. With this assay, we measured repression mediated by 952 different 3′-UTR variants, which allowed us to examine with rigor the interplay between the length, position, and offset of 3′ pairing in a variety of sequence contexts. For comparison, we also measured the efficacy of sites without 3′ pairing and the efficacy of the dual sites that mediate repression of To improve readability, we have condensed the portion of the paper describing experiments with chimeric and permuted miRNAs , such that only a summary of the findings is presented in the main text , with the details moved into Figure 6—figure supplements 1 and 2. In addition, we have shortened the introduction and removed some of the less pertinent details throughout the results and Discussion sections.Reviewer #1 (Recommendations for the authors):1. The study is an impressive computational feat with many interesting discoveries. However, the current manuscript is so long and time-consuming to absorb that the majority of the broader audience may lack the will to invest in reading the work and appreciating its full implications. We therefore recommend the authors shorten the manuscript with an emphasis on distilling and crystallizing the most salient points.We have shortened the manuscript, as described above, with hopes of increasing its readability.2. Main text descriptions of the coefficients proposed in Figure 4 and used in Figure 5 and 6 are difficult understand. We recommend endeavoring to create more illustrative and/or succinct descriptions.We have updated the associated text with improved descriptions of the coefficients.3. Conclusions in this manuscript will be strengthened if the authors test key findings in different systems. For example, whether a let-7 3' compensatory site with +4nt offset shows a stronger repressive effect than the target with 0 or +1nt offset in cell culture; or, whether miR-155 target site with 3' paring to g15-g22 is indeed more repressed than target site with 3' paring to g12-g19 in the cells; or, measure the KD of Ago2-let-7 binding the 8mer canonical site and compare to the KD of endogenous let-7 site in lin-41. Such data would strengthen connections to findings in previous studies in the field.r2 = 0.71). With respect to the examples mentioned by the referee, we compared the effects of the different offsets for let-7 3′-compensatory sites, and we compared the repression imparted by 8mer canonical sites to that of the endogenous let-7 sites in lin-41.As requested, we tested some of our key findings using reporters in cell culture and included these results in our revision . Overall, we found that affinity in vitro corresponded well to repression in cells  imparted significantly less repression than those with four A nucleotides (+4A-nt offset) . Perhaps these +4U bridge sequences promoted binding to RBPs, which often bind to short oligo(U) tracts , thereby interfering with AGO–let-7a binding.A and U were generally favored in the bridge region, and G was generally disfavored, which mirrored the nucleotide preferences observed flanking seed sites . Because of these preferences observed in our binding experiments, the sites for our reporter experiment were designed to have internal loop sequences formed by inserting either all-A or all-U sequences. Interestingly, 3′ sites with four U nucleotides in the bridge sequence , compared to that required to acquire and maintain a high-affinity canonical site without consequential 3′ pairing (7–8 nt of seed complementarity). Likewise, among canonical sites, 3′-supplementary sites are much less common than sites with no consequential 3′ pairing, presumably because a strong 3′-supplementary site has similar information content as dual 8mer sites, yet mediates less repression than the dual sites. Thus, sites with critical 3′ pairing will be favored only in special circumstances, such as the use of a 3′-compensatory site when repression must be specific to only one member of a seed family, or the use of a 3′-supplementary site when affinity to single site must be as great as possible. The second reason that the +4 offset sites are not more common is because the optimal offset is not always +4 nt. For example, miR-124, lsy-6, and miR-7 also appear to have two different binding modes , but for miR-124, the optimal offset of the second binding mode is +2 nt. Indeed, our reporter-assay results suggest that even for let-7, sites with a +1-nt offset confer much greater repression than those with a 0-nt offset, which is consistent with the idea that any  number of positive-offset nucleotides is sufficient to enable efficient repression by 3′ sites that include pairing to more centrally located nucleotides. By these more permissive criteria, more sites can be classified as exploiting this second, positive-offset binding mode, including the let-7 sites in We propose that let-7a preferentially benefits from the positive-offset binding mode because it has a GG dinucleotide at positions 11 and 12, and the positive-offset binding mode provides the most favorable access for pairing to this high-affinity G11-G12 dinucleotide. Supporting this proposal, re-analyses of results from random-sequence libraries  indicate that miR-124, lsy-6, and miR-7 each show two different binding modes , which we attribute to the G11-G12 dinucleotide of miR-124, and to the G11 nucleotide of both lsy-6 and miR-7.Reviewer #2 (Recommendations for the authors):I have only a few comments/suggestions:a) The authors correctly note in the manuscript how some extensive pairings including position 9-10 are depleted as a result of Ago2 slicing activity, but the full impact of Ago2 endonuclease activity on the results of these experiments is not entirely clear. An informative additional experiment to address this point could have included either AGO1-miRNA complexes or a catalytically dead AGO2 mutant. A related point is that including other Argonaute proteins could uncover unexpected differences in the impact of compensatory sites on binding affinities. Finally, I wonder whether the impact of slicing activity can be inferred by sequencing the input library before and after incubation with the Ago2-miRNA complexes (of course bypassing the nitrocellulose purification step).The proposed additional experiments would indeed be helpful for better understanding the quantitative contribution of slicing to the depletion observed for 3′-compensatory sites with contiguous central pairing . However, because these sites constitute only a small minority of the analyzed sites, and because our paper is already quite lengthy, we have chosen not to pursue these experiments for the current manuscript. Instead, we have revised our manuscript to make it clearer that our evidence of slicing is indirect. With respect to other Argonaute proteins, it would indeed be interesting if they might impart different 3′-binding preferences, but again because our paper is already quite lengthy, we have chosen not to pursue these experiments for the current manuscript.b) A key difference between the experimental setting used by the authors and in vivo miRNA targeting is that in cells Argonaute is post-translationally modified and acts a part of a much larger RNP complex (the RISC). It would be useful to include some simple reporter assays to test whether the different affinities for different 3'-compensatory sites observed in the AGO-RBNS experiments translates to different repressive activity in cells. For example, the authors show that a 10-20 compensatory site with a +4 offset for let-7a results in an affinity comparable to the affinity of a perfect 8-mer seed match, while the same compensatory site with -1 offset leads to only a modest increase in affinity (comparable to a 6-mer match). This particular prediction could be easily tested with appropriately-designed reporter assays.r2 = 0.71). Regarding the example mentioned by the referee, we found that a compensatory site with complementarity to positions 11–19 and a positive offset typically was as efficacious as a perfect 8mer seed match, whereas the same compensatory site with a 0-nt offset was substantially less efficacious.As requested, we performed reporter assays for let-7a sites and included these results in our revision . Overall, we found that affinity in vitro corresponded well to repression in cells (c) On a related note, I also wonder whether some of the predictions on the efficacy of compensatory 3'-sites can be tested by looking at existing CLEAR-CLIP or CLASH datasets.Although CLIP/CLASH data do identify sites of endogenously engaged miRNAs, quantitative analysis can be confounded by the differential crosslinking efficiencies observed in different sequence contexts. Thus, we opted instead to use a massively parallel reporter assay to test predictions on the efficacy of compensatory 3′ sites.Reviewer #3 (Recommendations for the authors):I found myself evaluating a paper with "invisible" results. There is a unique, technologically powerful, well-thought and most likely well-executed approach that is repeatedly applied to extract a large amount of data and derive conclusions; however, such data remains "hidden". As mentioned above, please include representative RNA bind-n-seq experiments and show how do you quantify the signals.KD values are extracted using the counts from all of the binding samples, as we had done in Figure 1C–E of McGeary, Lin, et al., (2019). However, we were concerned that this would bog down the beginning of the manuscript. We now cite these figure panels within the Figure 2A legend, so that interested readers can more easily access visuals that might help them better understand the computational workflow.First of all, we appreciate this sincere desire to help us improve our paper. The difficultly stems from the fact that our primary data are the sequences of millions of reads, not labeled molecules on a nitrocellulose filter, and thus these data are a step removed from what we can show in a representative experiment. We considered illustrating in more detail how the relative Unfortunately, I found the text very dense and tiresome. The data is presented in a very homogenous way and the reader has to make sense of a myriad of tiny, colored squares and other, very complex and extremely data-rich graphics. After spending (a long) time trying to understand the first two Results sections, seeing that almost the same type of figures continue until the end will tempt readers – as I was tempted – to jump to the Discussion and just get the conclusions.I guess this is not the type of paper one feels excited to read; it is "too much". I urge the authors to re-think the format of the paper.We greatly appreciate the effort expended to understand our results, and recognize that a typical reader will not have this level of commitment. We have attempted to condense the narrative and remove less pertinent details, moving the results from the experiments using chimeric and permuted miRNAs into figure supplements. We also hope that the added reporter experiments, which are somewhat more conventional, will be a welcome interlude from the RBNS analyses and help make the manuscript more readable.