Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to their target box and determine the specificity of a TALE.All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box.The yield of many crop plants can be substantially reduced by plant-pathogenic To this end, we extend our previously published tool PrediTALE by considering two epigenetic features: (i) chromatin accessibility of potentially bound regions and (ii) DNA methylation of cytosines within target boxes. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS data in rice.We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq data of infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both.Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets.Xanthomonas TALEs.Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of The online version contains supplementary material available at (10.1186/s12864-021-08210-z). Xanthomonas bacteria. In many parts of the world, the crop yield of rice plays a key role in ensuring nutrition of the population. However, the yield of a rice field can be substantially reduced due to infection with Xanthomonas oryzae pv. oryzae (Xoo) or Xanthomonas oryzae pv. oryzicola (Xoc), which cause significant loss in many cultivation areas [The cultivation of crop plants can be severely impaired by the infestation with phytopathogenic on areas .Host plant infection depends on the bacterial type III secretion system. Specific bacterial effector proteins are secreted into the plant cell, where they modulate plant response. Of these, transcription activator-like effectors (TALEs) are sequence-specific DNA-binding proteins that bind to host promoters to activate the expression of downstream genes. If such genes promote disease, they are termed susceptibility genes.TALE proteins comprise a nuclear localization signal, a modular DNA-binding domain, and an activation domain. The DNA-binding domain of natural TALEs is composed of 1.5 to 33.5 consecutive repeats, where each repeat binds to one nucleotide of the target box , 3. EachThe target boxes of a TALE can be predicted based on the one-to-one correspondence between RVD and target base , 6. For As shown recently , 10, DNAFurthermore, it has been shown that the accessibility of chromatin in the region of the target box has an impact on the binding ability of TALEs , 14.http://tale-nt.cac.cornell.edu/) [http://bioinfo-web.mpl.ird.fr/cgi-bin2/talvez/talvez.cgi) [http://www.jstacs.de/index.php/TALgetter) [Several tools to identify potential target boxes based on the RVD sequence exists. These include the “Target Finder” of TALE-NT suite (ll.edu/) , 16, thevez.cgi)  and TALgLgetter) .http://www.jstacs.de/index.php/PrediTALE) [Our recently published tool PrediTALE (ediTALE)  models bediTALE) . PredictStill, the predictions of all tools suffer from many false positives. All existing tools neglect epigenetic features when predicting TALE target boxes, although DNA methylation and chromatin accessibility are known to be important determinants of TALE-DNA binding. Hence, we aim at improving the accuracy of TALE target prediction by incorporating such epigenetic features into an extended version of PrediTALE termed EpiTALE.Our new application suite EpiTALE contains all tools necessary for TALE target prediction incorporating epigenetic features of the target site. First, we extend the PrediTALE model to consider DNA methylation information when making predictions and, second, we filter predictions using accessibility data such as DNase-seq and ATAC-seq.We approximate the specificities of the different RVD types for methylated cytosine based on experimental data. Users of our new suite EpiTALE may then provide methylation data in addition to genomic sequence or extracted promoters, which will be considered in prediction scoring. EpiTALE is the first approach that accounts for methylated cytosine when predicting TALE target boxes.To incorporate chromatin accessibility, we annotate the chromatin accessibility of target sites predicted by EpiTALE using DNase-seq or ATAC-seq data and suggest criteria to filter putatively inaccessible target boxes.Xanthomonas infection of rice plants. Here, we consider infection studies for 3 Xoo and 10 Xoc strains, where each strain expresses a different repertoire of TALEs, with up to 27 TALEs per strain [We benchmark EpiTALE based on RNA-seq data after r strain –23.We further apply EpiTALE using both, methylation information and a filter based on chromatin accessibility, for genome-wide predictions and identify previously neglected putative TALE target boxes, which show a transcription response in infection experiments according to RNA-seq data.https://www.ebi.ac.uk/ena available under run accessions SRR3485276 (replicate 1) and SRR3485277 (replicate 2). These data have been collected as part of a study by Zheng et al. [sativa L. ssp. japonica) under normal conditions.We obtained publicly available whole genome bisulfite sequencing (WGBS) data of rice from the European Nucleotide Archive (ENA) g et al. , who invhttp://rice.uga.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/all.chrs.con) via Bismark (v0.20.0) [We clipped sequencing adapters and removed low-quality bases at the ends of these paired end reads using Trimmomatic (v0.33)  with parv0.20.0)  and Bowtv0.20.0) . We usedWe used the Bismark methylation extractor to determine the methylation levels and set the following parameters “–bedGraph –CX -p”, which reports methylation of cytosines in the three contexts ‘CpG’, ‘CpHpG’ and ‘CpHpH’ . The output contains a coverage file reporting count values for methylated and unmethylated cytosines. We finally merged the coverage files of both replicates by simply summing the  counts at identical positions and then computing the methylation level. To obtain conservative methylation calls, we introduced a bias towards unmethylated cytosines in sparsely covered regions by adding a pseudo count of 1 to the count values for unmethylated cytosines.Supplementary Figure Xoo strains PXO83, PXO142, ICMP 3125T and publicly available data from infection studies with Xoc strains BLS256, BLS279, CFBP2286, B8-12, L8, RS105, BXOR1, CFBP7331, CFBP7341, CFBP7342 [To benchmark EpiTALE, we used RNA-seq data as described previously . BrieflyTo identify accessible regions, we mapped publicly available DNase-seq and ATAC-seq data to the rice genome (MSU7). Neither of these libraries has been collected under conditions perfectly matching the RNA-seq data from the infection studies. However, chromatin accessibility might still be sufficiently similar to be informative for subsequent TALE target prediction.We downloaded DNase-seq reads of rice seedlings  from NCBFor the second study , ATAC-seFor both datasets we used Trimmomatic (v0.39) in paired end mode for clipping sequencing adapters and for removing low-quality bases from the ends of reads with parameters “ILLUMINACLIP:NexteraPE-PE.fa:2:30:10 SLIDINGWINDOW:4:20 MINLEN:20”, mapped the resulting reads with Bowtie2 to rice genome (MSU7) and removed duplicates with Samtools .% uniquely mapped reads. Hence, we decided to use only DNase and ATAC2 for benchmarking.The mapping statistics of all three datasets are summarized in Supplementary Table For both, the DNase and the ATAC2 dataset we used JAMM  for peakWe used the open-source library Jstacs , 36  the score m0 of the zero-th nucleotide x0 given the first RVD r1, ii) the score r1 and first nucleotide x1, and iii) the score p(ℓ|θp). In the EpiTALE model, the terms ii) and iii) further depend on the methylation probability qℓ of nucleotide xℓ: The total binding score of a putative target box In the following, we provide a detailed definition of each of the individual terms of the EpiTALE model.m0 is independent of methylation probabilities, since there are no appropriate activity or binding studies regarding methylation sensitivity available, yet.As in the original PrediTALE model, the term for binding to the zero-th nucleotide a-priori parameter of nucleotide zero r1 is in this set r1: As in PrediTALE, this term corresponds to the sum of the following parameter values: i) the πT= log(0.6),πC= log(0.3),πA=πG= log(0.05).Here, we define x1.The binding of the first RVD to the first nucleotide of the target box is modelled by the term q1) that the first position is unmethylated is multiplied by the term adopted from the original PrediTALE model, which always contains a parameter r1,13 of the RVD to nucleotide x1. For a subset Specifically, the probability , 5-hydroxymethylcytosine (5hmC), cytosine and thymine [A thorough study by Zhang  thymine . For thiet al. [et al. to match the range of parameter values of the original PrediTALE model. Specifically, we used the two reference points for cytosine and thymine also present in the data of Zhang et al. to scale the raw measured values To obtain fitted values for the parameters g et al. . To thisLet The specificity of the 13th AA of the RVD to bind 5mC is determined by the following scaling: Here, we consider only values measured for 5mC but not those measured for 5hmC for two reasons. First, the plant genome contains much less 5hmC than 5mC , 43. SecA visualization of the model parameters of EpiTALE including the parameters for 5mC is shown in Fig. Target boxes predicted by EpiTALE may further be filtered for chromatin accessibility. For each predicted target sequence, a window from 300 bp upstream to 50 bp downstream of the target box is checked for an overlapping peak within the peaks determined by JAMM  from chrn of positions that correspond to at least one 5’-end of a read within a defined region around the predicted target box is considered. For promoterome-wide predictions, this region corresponds to the complete promoter sequence  that isor the coverage filter criterion is fulfilled, we consider the target box as accessible.If there is an overlapping peak Xoo strains and 10 Xoc strains  including methylation information and the filter based on chromatin accessibility. For the resulting top 100 predictions of each TALE, we use above mentioned RNA-seq data to search for a differentially expressed region near the putative target box using DerTALE, as described previously [http://rice.uga.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/all.gff3). For differentially expressed regions with no overlapping annotated gene, it might be that a true gene locus is missing from the existing annotation, even for high-quality annotations like MSU7. Especially the annotation of non-coding RNAs may be incomplete for non-model plants. For this reason, we use blastx and blastn to search for similar sequences in the non-redundant protein sequence (nr) database and the reference RNA sequences (refseq_rna) database using NCBI BLAST+ version 2.7.1 [ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). High-quality BLAST matches in other organisms may provide indication that the differentially expressed region is indeed a functional gene. Furthermore, functional annotations from these organisms may also give insights into the role of such genes in the infections process.With EpiTALE, we perform genome-wide predictions in the eviously . We visuon 2.7.1  , EpiTALE with consideration of methylation (P + Methyl), EpiTALE with filtering based on the accessibility filter criterion (P + Access) and EpiTALE with methylation and accessibility filtering (P + Methyl + Access).Xoo and Xoc strains based on the corresponding RNA-seq infection studies. For benchmarking based on differentially expressed genes, we consider a promoter region 300 bp upstream of the transcription start site to 200 bp downstream of the transcription start site or until the start codon as described previously [Xanthomonas strain. In case of multiple predictions per gene, only the prediction yielding the best prediction score is considered.We compare the performance of these 4 variants for the above mentioned eviously , 19. In eviously , 16, Taleviously , and TALeviously  using deXanthomonas strain, multiple TALEs lead to differential gene expression. Hence, it is not possible to clearly assign differentially expressed genes to a particular TALE. The RVD sequences of the TALEs of the Xanthomonas strains studied are given in Xanthomonas strain.The use of RNA-Seq data from inoculation studies to evaluate the predictions entails two problems: First, when plant tissue is inoculated with a t of predictions considered per TALE between 1 and 50, i.e., we only consider the t predictions with the largest prediction scores for each TALE. Given the list of predicted target genes corresponding to the top t predictions per TALE, we may check each of these target genes for up-regulation in the infection experiment.In order to compare previous tools and the 4 EpiTALE prediction variants mentioned above, we proceed in analogy to the previous comparison of PrediTALE with alternative approaches : We varyt on the number of predictions per TALE.Specifically, we define all genes as true positives (TPs) that are up-regulated in the RNA-seq data relative to control and have a predicted target box within the promoter. We define those genes as false positives (FPs) that are not up-regulated after inoculation, but have a predicted target box in their promoter. The definition of false negatives is not clearly possible, since up-regulated genes without a predicted target box could be indirect target genes. Following this procedure, we record the number of TPs given each cutoff http://jstacs.de/index.php/EpiTALE. A minimal example for testing is available from zenodo at https://www.doi.org/10.5281/zenodo.4749294. Source code is available from https://github.com/Jstacs/Jstacs in package projects.tals.epigenetic.The EpiTALE suite is available as a JavaFX-based stand-alone application with graphical user interface and as command line application under The EpiTALE suite contains tools, that (i) convert BedMethyl files to Bismark format, (ii) merge two Bismark files, (iii) compute a coverage pileup of 5’ ends of mapped reads from an DNase-seq or ATAC-seq experiment, (iv) normalize the coverage pileup relative to the mean of a 10000 bp sliding window, (v) convert methylation data (Bismark files) and chromatin accessibility data (coverage pileup and/or narrowPeak file) to promoter coordinates and (vi) predict TALE target boxes with optional epigenetic input within genomic or promoter sequences.It has been shown previously , 10 thatThe potential improvements achieved by these extensions may be twofold. First, introducing separate specificity parameters for methylated cytosines may yield more meaningful prediction scores, ideally improving the ranks of true positive predictions of target boxes. Second, an accessibility-based filter that predominantly excludes previous false positive predictions of target boxes may achieve a better enrichment of true positives among the top predictions of EpiTALE. In a practical scenario where only a limited number of predictions may be validated in wet-lab experiments, improved ranks of true positive predictions and exclusion of false positive predictions from the top ranks have the potential to increase the rate of validated and potential virulence targets.In the following sections, we first assess the stability of prediction performance regarding different filtering criteria on the accessibility data. We then present a comprehensive benchmarking experiment including different variants of EpiTALE considering epigenetic features, where we also include previously published tools for TALE target prediction as a reference. We further investigate to which extent epigenetic features help to improve the ranks of true positive predictions, and finally apply EpiTALE including methylation information and the accessibility-based filter to yield genome-wide predictions of TALE targets.Xoo strains, and we evaluate the chosen filter criteria on independent data for 10 Xoc strains. Here, we need to resort to a rather indirect assessment of predictions based on RNA-seq data, because currently ChIP-seq data for TALEs are lacking.In this section, we benchmark the effect of different filters based on chromatin accessibility applied to EpiTALE predictions – at this stage still without using methylation information. To this end, we test different filtering thresholds for predicting target boxes of the TALEs present in 3 Xoo and Xoc strains compared to mock control. Specifically, we consider a predicted target gene as true positive (TP) if its promoter contains a predicted TALE target box and this gene is up-regulated in the infection experiment. We consider a predicted target gene as false positive (FP) if its promoter contains a predicted TALE target box but is not up-regulated in the infection experiment  target boxes is plotted against the number of predictions allowed per TALE, with a rank cutoff from 1 to 50. To ensure comparability to the original PrediTALE publication [Xoo strains and are at least 2-fold up-regulated. Here, we consider these rather relaxed criteria, as we want to avoid predictions to be erroneously considered false positives due to the larger variation at the early time point after infection (24 h) for the Xoo strains.The performance of EpiTALE with a filter based on the DNase-seq dataset compared with the original PrediTALE neglecting chromatin accessibility is shown in Supplementary Figure lication , we use n=30 yields the largest area under the curve of TP predictions (AUC-TP) for the strains ICMP 3125T and PXO83. In case of ICMP 3125T, filtering with this threshold for any rank cutoff shows improved or at least identical performance as PrediTALE without filtering, and a larger improvement than any other filter threshold tested. For PXO83, the same effect can be observed, where only when considering the top 3 predictions per TALE, a threshold of n=35 results in one additional TP. For PXO142, accessibility filtering with a threshold of n=30 within a range of 1 to 30 total predictions per TALE increases or at least retains the number of TP predictions. For higher rank cutoffs, the filtering results in one TP less than for the unfiltered version. Thus, a threshold of n=30 leads to a reduction of TPs only in rare cases and mostly leads to an increase in the number of TPs within the top predictions. In complete analogy, Supplementary Figure Xoc strains. As described previously [q−value<0.01, log2 fold change > 2) for the Xoc strains, as these RNA-seq data have been collected 48 h after infection. For 8 of the 10 strains, the accessibility filtering based on the DNase dataset results in a higher AUC-TP for each of the 6 thresholds. However, for 7 of these 8 strains a threshold of n=25 results in the highest AUC-TP. The threshold of n=30 chosen from the Xoo datasets does not result in the optimal result for Xoc, but performs substantially better than the original PrediTALE version without filtering. Filtering based on the DNase-seq dataset works slightly worse only for strains CFBP7331 and CFBP7341.Using the accessibility filter, a threshold of eviously , we consXoo and Xoc strains considered, the proportion of TP and FP target boxes that pass the accessibility filter is shown in Supplementary Figure n=30. FP target boxes in turn are rather filtered out as they are occasionally inaccessible.For the predictions of the top 50 target boxes of each TALE of the In this section, we further investigate the effect of including methylation-specific parameters into the EpiTALE model, and its combination with the accessibility filter studied in the previous section. Specifically, we consider four modelling alternatives: i) the original PrediTALE model (P), ii) the EpiTALE model including specificities for methylated cytosines (P + Methyl), iii) the PrediTALE model combined with the accessibility filter (P + Access), and iv) the EpiTALE model combined with the accessibility filter (P + Methyl + Access). As a reference, we further include tools for TALE target prediction that have been published previously, namely Target Finder , 16, TalXoo TALEs are shown in Fig. T and PXO83, where the improvement due to the accessibility filter is more pronounced than the improvement due to including methylation levels into the EpiTALE model. For both strains, performance is further increased by combining both epigenetic features. For PXO142, the accessibility filter alone leads to slightly decreased prediction performance, whereas methylation information alone as well as the combination of both epigenetic features leads to a slight improvement compared with the original PrediTALE variant. All variants of EpiTALE yield a considerable improved compared with previous tools for TALE target prediction.The results of the performance evaluation of previous tools and these four EpiTALE variants for TALE target prediction of Xoc strains are shown in Fig. The results for the Xoo strains is presented in Supplementary Figure The performance based on the accessibility dataset ATAC2 for 3 Xoo and ten Xoc strains that also show upregulation after infection with the respective strains. The complete list of true positive predictions is given in Supplementary Table Xoo strains is provided in Table Xoo strains, all three EpiTALE variants including epigenetic features mostly yield an improvement of the rank of the true positive target gene compared with the original PrediTALE variant without epigenetic features. The strongest rank improvement is almost always achieved by the EpiTALE variant that considers methylation of the target box as well as its accessibility. However, an improvement can often be observed already when considering only one of the epigenetic features.In this section, we focus on the top 20 predictions of the four EpiTALE variants for three The gene Os09g07460, coding for a kelch repeat protein, is among the top 20 predictions for TalBA8 for all three EpiTALE variants considering epigenetic features. This gene has not been among the top 20 predictions of the original PrediTALE variant, but has been reported by Talvez , 19.Xoc strains  for 3 Xoo and 10 Xoc strains using the EpiTALE version with methylation and DNase accessibility data. As accessibility filter, we select regions around the binding site based on criteria that are similar to the promoter setting. Either a peak of chromatin accessibility must be present in a region from -300 bp to +50 bp relative to the target box or at least 30 positions within the window from -300 bp to +200 bp relative to the target box must be covered by the 5’-end of at least one DNase-seq read. The latter criterion implies that at least 30 DNase-seq reads must overlap this window around the target box, but might even require a larger number of DNase-seq reads if the 5’-ends of multiple reads map to the same location.Independently of existing gene annotations, we performed genome-wide predictions of TALE target boxes in To determine differentially expressed regions near predicted target boxes, we use our tool DerTALE  and the Xoo TALEs, differential expressed regions are close to at least one predicted target box. In total, we obtain 20 of such target boxes (complete list in Supplementary Table T) are identical and do not overlap with a gene annotation known from MSU7. We extracted the sequences under the differentially expressed regions, and first compared them against the NCBI protein database ‘nr’ using blastx but received no matching result. We additionally compared these sequences against the NCBI reference RNA sequences (‘refseq_rna’) using blastn, which resulted in a predicted mRNA, coding for a calcium-transporting ATPase (XM_015770644.2).Genome-wide prediction shows that for 16 However, one putative target box reported from genome-wide predictions in the original PrediTALE publication (Os04g05050) appears on a lower rank due to methylation of the target box, which might be caused by non-matching experimental conditions as discussed previously.Xoc strains is given in Supplementary Table Xoc strains. The corresponding class tree is shown in Supplementary Figure Xoc CFBP7342 infection. For the sequences under this differentially expressed region, database search using blastx and blastn against ’nr’ and ’refseq_rna’, respectively, did not result in a match.A complete list of the genome-wide predictions of TALEs from the ten Xoc strains. The corresponding class tree is shown in Supplementary Figure As a second example, we discuss a putative target box on chromosome 6 for members of the TalBN class present in 8 of 10 Xoo and Xoc target boxes, EpiTALE yielded improved prediction ranks of true positive targets compared with the original PrediTALE variant. Nevertheless, there are still false positive predictions and we suggest an experimental verification of novel targets.With the goal of improving the prediction of TALE targets, we present EpiTALE, an extended version of PrediTALE including epigenetic features. Both, methylation levels and the chromatin accessibility around putative target sites have a decisive impact on the likelihood of being bound by a TALE. Even if a putative target box matches the specificity of the RVDs of a TALE, inaccessibility of the respective chromatin may inhibit binding and thus inhibit activation of the transcription of the downstream gene , 48. We We perform promoter-wide and genome-wide predictions and find several predictions common to both approaches, but we also find target boxes upstream of differentially expressed regions in RNA-seq infection studies that do not overlap with a currently annotated gene.http://jstacs.de/index.php/EpiTALE.The use of the epigenetic features is optional for the user. Depending on the availability of data, only methylation and/or chromatin accessibility data can be provided to EpiTALE to improve target prediction. In our study, the strongest improvement in accuracy was achieved by considering both epigenetic features in EpiTALE. Our results suggest that collecting condition-matched WGBS and DNase-seq/ATAC-seq data may further improve the quality of computational TALE target predictions. The EpiTALE suite presented here provides the means necessary to integrate such data into TALE target prediction and is available from Additional file 1 PDF file integrating Supplementary Figures S1 – S12.Additional file 2 Mapping statistics of ATAC and DNase-seq data.Additional file 3 List of positive and negative targets for Xoo and Xoc.Additional file 4 Complete list of top 20 predictions for all four varaiants and Xoo and Xoc strains.Additional file 5 Genome-wide prediction for three Xoo strains.Additional file 6 Genome-wide prediction for ten Xoc strains.Additional file 7 RVD sequences of all Xoo and Xoc TALEs considered in this manuscript.


The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands. Viruses represent a universal infectious threat to all forms of life, from bacteria to fungi, plants and animals. In response, these organisms have evolved numerous mechanisms to fend off viral attack . One sucIn contrast to the progress made in re-engineering RNA-binding proteins, the design of small molecular scaffolds that recognize RNA structures has proven very challenging . This isFor the development of inhibitors of protein-protein interactions, peptide-derived ligands have been widely used . In thostomato aspermy virus 2b (TAV2b) protein, a viral suppressor of RNA silencing ; TFA: trifluoroacetic acid; ACN: acetonitrile; DMSO: dimethyl sulfoxide; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; FCS: fetal calf serum; qRT-PCR: quantitative reverse transcription polymerase chain reaction; PAGE: polyacrylamide gel electrophoresis.Fmoc: fluorenylmethyloxycarbonyl; SPPS: solid-phase peptide synthesis; EMSA: electrophoretic mobility shift assay; EDTA: ethylenediaminetetraacetic acid; PBS: phosphate buffered saline  from baker's yeast was purchased from Hoffmann La Roche. For quantification, the ultraviolet (UV) absorbance of the oligonucleotides was measured in the buffer of the corresponding experiment using a V-550 ultraviolet/visible (UV/Vis) spectrophotometer (Jasco). Respective concentrations were calculated with a molar extinction coefficient at λ = 260 nm, determined according to the nearest-neighbor model using published parameters for oligonucleotides  and fluorescein-labeled peptide (c = 6 μM) were incubated in binding buffer (10 mM Tris (pH 8.0), 0.5 mM EDTA, 12.5% glycerol and 150 mM KCl) at 4°C for 1 h. After incubation, bound RNA was resolved from peptide-free RNA using 10% non-denaturing polyacrylamide gels at 120 V in running buffer (25 mM Tris (pH 8.0), 200 mM glycine, 20 mM NaCl) at 4°C for 1 h. Visualization of fluorescent gel-bound species was conducted using a Typhoon Trio + fluorescence scanner . For nucleic acid visualization, gels were stained using 2 μl of SYBR™ gold nucleic acid gel dye (Molecular Probes) in 20 ml of TAE buffer  for 45 min at RT before being visualized using a FluorChem™ gel documentation system (Alpha Innotech). To assess B3 binding to different ds RNA sequences (c = 1 μM) and fluorescein-labeled B3 (c = 2 μM) were incubated in binding buffer (TAE and 10% glycerol) at RT for 1 h, and then resolved using 15% non-denaturing polyacrylamide gels (acrylamide/bis-acrylamide (19:1) in TAE) at 150 V in running buffer (TAE) at 4°C for 1.5 h. Visualization of fluorescent gel-bound species was conducted using a Bio-Rad ChemiDoc (Bio-Rad). Subsequently, gels were stained with SYBR™ gold nucleic acid gel dye (Invitrogen) as described above and visualized using the same imaging system. To assess B3 binding to pre–miR-21 (c = 1 μM) and incubated with varying concentrations of fluorescein-labeled B3 (c = 0–5 μM) in binding buffer (TAE and 10% glycerol) at RT for 1 h. After incubation, bound nucleic acid complexes were resolved using 15% non-denaturing polyacrylamide gels (acrylamide/bis-acrylamide (19:1) in TAE) at 150 V in running buffer (TAE) at 4°C for 1.5 h, followed by visualization and staining with SYBR™ gold nucleic acid gel dye (Invitrogen).Electrophoretic mobility shift assays (EMSAs) were performed using a Bio-Rad Mini-Protean gel system paired with a direct current (DC) power source . Typically, 6 μl solutions containing ds RNA  were then transferred into the sample cell and oligonucleotides (c = 12–72 μM) were transferred into the syringe. 35 Injections per measurement were performed at 30°C  with an initial delay of 60 s and a stirring speed of 307 rpm. Measurements were performed in triplicate. Using the MicroCal LLC ITC software , the heat associated with each injection was calculated by integrating the area under the curve in microcalories per second [μca/s] versus time [min] for each injection and then normalized to concentration. A ‘single set of identical binding sites’ model was used to fit the binding curves from which thermodynamic binding parameters  were obtained.Isothermal titration calorimetry (ITC) was conducted using a MicroCal VP-ITC . Before measurement, oligonucleotide and peptide samples were dissolved in PBS and degassed using a ThermoVac  and heated to 30°C. N-terminally acetylated wt33 and ds RNA were recorded with a Jasco J-1500 spectropolarimeter (Jasco) equipped with a programmable Peltier thermostat in a stoppered quartz cuvette . Samples of pal-RNA (c = 2 μM) and wt33 (c = 4 μM) were prepared in buffer . For each sample, 10 CD spectra were measured between λ = 200 and 350 nm with continuous scan mode . Obtained spectra were averaged and then subtracted from a reference buffer spectrum. CD data were normalized to oligonucleotide strand concentration using Formula (θ = observed ellipticity (mdeg), c = DNA strand concentration (mol·l−1) and l = path length (cm).Circular dichroism (CD) spectra of Tm) determination was conducted using the same instrumentation and sample preparation, where ellipticity (θ = 267 nm) was measured by ramping the temperature from 15–90°C . Data points were recorded every 0.5°C. Raw data was normalized as described above and Tm-values were determined using the CDpal program (Melting temperature ( program  before bHellma). Peptide samples (c = 75 μM) were prepared in buffer: 10 mM disodium hydrogen phosphate, pH 7.4. For each sample, 10 CD spectra were measured between λ = 190 and 260 nm with continuous scan mode . Obtained spectra were averaged, then subtracted from a reference buffer spectrum and smoothed using a fast Fourier transform filter (FFT filter). The mean residue ellipticity (Θ) (deg·cm2·dmol−1·104) was calculated using Formula (2) and plotted against wavelength (λ) (nm). The ratio of secondary structure elements was calculated with CDNN using the PEPFIT set of reference spectra for secondary structure determination (Θ]m = mean molar ellipticity per peptide bond (degree·cm2·dmol−1·104), Θ = measured ellipticity (degree), d = optical path length (cm), cg = mass concentration (g·l−1), MRW = mean residue weight, M = molar mass of the peptide (g·mol−1) and N = number of peptide bonds.Spectra of fluorescein-labeled peptides were recorded with a J-715 circular dichroism spectrometer (Jasco) using a stoppered quartz cuvette \dc = 100 μg·ml−1). To begin each experiment, peptide and proteinase K stocks were mixed  and incubated at room temperature. At each time point, 50 μl of the reaction mixture was quenched by adding 100 μl of TFA/ACN/H2O  and subsequently incubated for 2 min on ice. Following centrifugation , samples (45 μl) were monitored by analytical HPLC using a linear gradient of 10–60% HPLC solvent B over 20 min. The amount of remaining peptide was quantified via absorbance at λ = 210 nm (intact peptide (%)) and plotted against time (t) (h). Half-life (t1/2) was calculated by fitting the data to an exponential decay using the software Prism 5.0 (GraphPad).Protease stability assays were performed in analogy to a previously published protocol . Stocks 6 K562 cells were plated per well in 48-well plates in presence of fluorescein-labeled peptides  in a total volume of 300 μl. After 90 min, cells were treated with proteinase K (2 μg·ml−1) for 10 min to remove extracellular peptides. Cells were extensively washed and total uptake of labeled peptide was measured by flow cytometry . For each peptide, 20 000 events per sample were collected with >85% viable cells (forward/side scatter) that were considered for analysis using a 525/40 nm filter. DMSO was used as a control to distinguish general DMSO-related and peptide-specific effects. Each experiment was performed in triplicate.2 × 10wt33 peptide was generated based on the crystal structure of the TAV2b/siRNA complex (PDB ID: 2ZI0)  . Models D: 2ZI0) , which uD: 2ZI0)  and an ewt33 peptide using the Amber 18 program suite  simulations were carried out for pre-miR-21 with complexes of three copies of am suite . The staam suite . A combiam suite  and the am suite ,59, was am suite ,61.6 K562 cells were incubated with N-terminally biotinylated peptides (c(peptide) = 10 μM, t = 2.5 h) for uptake on a rotating wheel at 37°C. The cells were extensively washed in PBS and lysed in lysis buffer  supplemented with 0.5 U/μl RNAse inhibitor . The lysate was cleared by centrifugation at 21 000 g at 4°C, an aliquot of the supernatant was removed and re-suspended in Trizol, while the remaining was incubated with streptavidin-coupled magnetic beads  on a rotating wheel . After magnetic separation and washing (3× in lysis buffer), the beads were re-suspended in 20 μl lysis buffer, re-suspended in Trizol (ThermoFisher), and 400 ng t-RNA was added per sample as a carrier. RNA was extracted from Trizol using the standard protocol and the qRT-PCR was performed according to a previously published protocol (D1.2 × 10c(peptide) = 1 μM, t = 30 min) in the Eppendorf tubes at 37°C with vertical rotation to avoid cell pelleting. After incubation, cells were washed once with phenol-red free media, re-suspended again in the same media, and transferred into the wells of an 8-chamber μ-slide (Ibidi). Cells were immediately imaged at 37°C with an SP5 Laser Scanning Confocal Microscope (Leica). Fluorescein was excited with an argon ion laser at wavelength of 488 nm and fluorescence was detected in the range 505–530 nm.Non-adherent K562 cells were counted and collected in 1.5 ml Eppendorf tubes at 30 000 cells per tube. After a wash, cells were re-suspended in 200 μl of phenol-red free media containing 5% FCS and fluorescein-labeled peptides  were prepared with ds pre–miR–21 (c = 40 nM) in buffer (20 mM Tris hydrochloride (Tris·HCl), pH 6.8, 12.5 mM NaCl, 2.5 mM MgCl2, 1 mM dithiothreitol, 2% DMSO, and 0.02% bovine serum albumin (BSA)). Next, RhDicer  was diluted in buffer  to a concentration of 40 U·ml−1 and then added to the peptide/RNA solutions . The reaction solution was incubated for 150 min at 37°C, then denatured for 5 min at 70°C, and stored at 4°C. Subsequently, a 1:10 dilution in H2O of the reaction solution was performed and added to a Poly(A) Tailing and reverse transcription solution as previously reported  with a final primer concentration of 125 nM in 20 μl per sample. Samples were initially denatured for 5 min at 95°C followed by 45 temperature cycles of 15 s at 95°C and 30 s at 66°C. The quantification was done using miR-21-5p standard curves. To visualize the inhibitory potential of each peptide the normalized abundance of mature miR-21 (%) is plotted against the peptide concentration (nM).To assess the inhibitory ability of reported . The samBromoviridae family of plant viruses, the Tomato aspermy virus (TAV) is a common infectious agent known to cause mosaic symptoms, necrosis and deformation in tomato and chrysanthemum plants. As a positive-sense, single stranded RNA virus, the TAV genome is tripartite, where RNA 1 encodes for proteins involved in replication, RNA 3 encodes for capsid proteins, and RNA 2 encodes for the protein TAV2b, a viral suppressor of RNA silencing (Belonging to the ilencing . TAV2b cilencing  37). Bo. BoBromoilencing . These tilencing  to avoidwt33, orange, Figure wt33 with ds pal–RNA, revealing a 2:1 binding stoichiometry i.e. two peptides recognizing one pal-RNA duplex and moderate binding affinity  . Subsequently, circular dichroism (CD) spectroscopy was used to characterize unbound peptide wt33 and pal-RNA. The spectra indicate the expected double-helical structure for pal-RNA . Co-incubation of pal-RNA and wt33  resulted in a pronounced negative ellipticity between λ = 208 and 222 nm. Notably, this is not the case when the individual spectra of wt33 and pal-RNA are simply added . From the truncation series, a 33-mer peptide (= 75 nM) . The repwt33-derived hydrocarbon-stapled peptides was designed. Hydrocarbon-stapling involves the introduction of two olefin-bearing, non-natural α-methylated amino acids  and two in the C-terminal helix (B and C). In addition to single-stapled peptides, crosslinks were combined resulting in six double-stapled peptides  = 5.0 versus ΔH(stapled peptides) = 3.9−4.1 kcal·mol−1). This decrease in binding enthalpy is compensated by a reduction in the entropic penalty upon binding (−T·ΔS(wt33) = 42 versus −T·ΔS(stapled peptides) = 30–32 kcal·mol–1) which likely stems from the helical pre-organization induced by stapling. Evidence of this structural pre-organization was also observed with CD spectroscopy. In solution, peptides B1, B3 and C1 display characteristic α-helical spectra. Unlike wt33, an enhancement in ellipticity between λ = 208 and 222 nm was not observed when these peptides were incubated with ds pal–RNA  with the double-stapled derivatives exhibiting a stronger helical character than the mono-stapled analogs  with their CD spectra showing minima at λ = 208 and 222 nm characteristic for α-helices  while stapling of the N-terminal helix did not improve protease resistance . A combination of both N- and C-terminal staples yielded the most stable peptides, with double-stapled peptides B1 and C1 experiencing the strongest increase in tolerance towards proteinase K  which was followed by a stringent wash protocol including tryptic digestion. Flow cytometry-based mean fluorescence intensities (mFI) were then determined  all stapled peptides caused increased fluorescence intensities indicative of increased cellular uptake. In line with trends observed for α-helicity  or the ds DNA analog of pal-RNA (B3 and C1 to a mixture of yeast transfer RNAs (t-RNAs) indicating that peptide binding indeed requires accessible RNA duplex structures. Although not reported for TAV2b, other viral suppressors of RNA silencing are known to bind microRNAs (miRNAs)  . Similar(miRNAs) . miRNAs (miRNAs) .B3 and C1 to also bind miRNA structures. To test this hypothesis, we chose ds miRNA-21 (miR-21) as an example. miR-21 represents the first miRNA species which was shown to directly influence oncogenic progression. Upregulation of miR-21 is common to most types of cancer and is implicated in the suppression of genes associated with cell growth and differentiation such as PDC4, RECK, TPM1 and PTEN  with unlabeled peptides B3 or C1 (c = 4 μM) resulted in increased melting temperatures (ΔTm > 10°C), while D1 only showed a mild effect on duplex stability .,69.B3 anand PTEN  resultinand PTEN . Initial binding . It has  binding . Therefoy Figure .B3 and C1, we assessed their binding to endogenous miR-21 in a cellular context. For that purpose, pull-down experiments were performed in which K562 human leukemia cells were initially incubated with biotinylated analogs of B3, C1 or D1 . Subsequently, cells were rigorously washed, lysed and finally lysates were treated with streptavidin beads to capture the peptides in complex with RNA. After extraction of RNA from the beads, the amount of miR-21 was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In this experimental setup, peptide cell permeability, bio-stability and RNA-binding contribute to pull-down efficiency. For peptides B3 and C1, we observed enrichment when compared to control peptide D1 . Given the structural similarities between ds miR–21 and its hairpin precursor, pre-miR–21 , the high affinity binding of pal-RNA, miR-21 and pre-miR-21 suggests sequence-independent recognition of ds RNA structures. This is in line with the binding characteristics of the parent protein TAV2b  exceeding the binding affinity of B3 for the pal-RNA duplex . We also used ITC to investigate B3 binding to pre-miR-21, revealing a high affinity (Kd = 75 ± 8 nM) though with a changed binding stoichiometry indicative of 3:1 or 4:1 binding  was used to process pre-miR–21 (c = 40 nM) into mature miR-21. A qRT-PCR detection protocol  in this assay. This indicates efficient competition with Dicer under these conditions.Dicer binding of pre-miR-21 eventually results in strand cleavage at two sites  as the minimal sequence for ds RNA binding. Based on this peptide, a panel of hydrocarbon-stapled peptides was designed aiming at the stabilization of the RNA-bound α–helical conformation of TAV2b. The obtained stapled peptides show enhanced α-helicity, protease stability, and cell permeability, particularly pronounced for double-stapled analogs. From that panel, double-stapled peptide B3 was investigated in most detail. It binds palindromic siRNA with high affinity but not single-stranded and transfer RNA or ds DNA. Notably, B3 also shows high affinity for additional double-stranded RNA structures including miR-21 and its precursor pre-miR-21. Binding of B3 to pre-miR-21 was also shown to inhibit RNA maturation by the endoribonuclease Dicer in a biochemical assay.The design of RNA-targeting ligands remains an outstanding challenge. Antisense oligonucleotides represent the most established entities and while possessing high affinity and selectivity they tend to suffer from delivery issues . AlternaB3 represents a structural mimetic of the RNA-binding TAV2b protein. Recapitulating central features of TAV2b binding, B3 engages ds RNA in a sequence-independent and enthalpy-driven manner presumably featuring an analogous interaction pattern. In analogy to TAV2b, complex formation with ds RNA is strongly preferred over ss RNA, likely due to the absence of a defined binding groove in the unpaired and highly flexible ss RNA chain. B3 also exhibits a strong preference for ds RNA over ds DNA. This can be explained as ds DNA tends to exist in the B-form possessing wider and shallower major grooves than A-form RNA thereby preventing analogous interactions (wt33) is unstructured in its free form but shows high α-helicity upon binding. This can be expected to result in a considerable entropic penalty and likely contributes to wt33’s relatively low affinity. Introduction of chemical constraints to stabilize the α-helical conformation, as seen for B3, consequently increases binding affinity for ds RNA. Highest binding affinities were obtained when both α-helices had been stabilized. However, despite the judicious placement of hydrocarbon staples opposite to the RNA-binding interface, differences in binding affinities and therefore presumably in the structural pre-organization have been observed. These differences could arise from ‘over-constraint’, where the peptide is held in a helical or intermediary conformation which is restrictive to binding the dynamic RNA interface. We have previously observed such an effect on constrained peptides targeting a transcription factor interface, where the introduction of more flexible cross-links was required to fine-tune target affinity (Double-stapled peptide ractions . The α-haffinity .B3 is a prime example of a modular scaffold combining bioavailability and -stability with high affinity for ds RNA major grooves. A sequence-specific recognition of ds RNA would increase the applicability of such peptides and could be achieved, e.g., via the incorporation of base-recognizing residues or the addition of modalities that feature such properties. This may involve the conjugation of antisense nucleic acids or the introduction of minor groove-targeting motifs as well as the development of RNA degrader molecules (B3 represents an attractive starting point for the future design of ligands with altered RNA-recognition properties.Overall, this study highlights how RNA-binding proteins can serve as valuable starting points for the structure-based design of cell-permeable, high affinity RNA ligands, in particular when considering the growing number of available protein/RNA complex structures . Such deolecules . Overallgkab1149_Supplemental_FilesClick here for additional data file.


Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform–PAZ–Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.The online version contains supplementary material available at 10.1007/s00018-021-03795-w. Dicer belongs to the ribonuclease III (RNase III) family of double-stranded RNA (dsRNA)-specific endoribonucleases that are essential for the maturation and decay of coding and noncoding RNAs in both prokaryotes and eukaryotes . ProteinExtensive studies have deciphered the roles of the Dicer domains in binding and processing of its canonical substrates, i.e., dsRNAs and single-stranded hairpin precursors of microRNAs (pre-miRNAs). The putative helicase domain selectively interacts with the apical loop of pre-miRNA and helps to discriminate between substrates . The DUF+ or K+ . G-quadiews see . Since tiews see ). The foiews see –14.Previously, we characterized the inhibitory potential of structurally diverse short RNA molecules, 12- to 60-nt in length, able to influence processing of pre-miRNA by hDicer either by binding to the enzyme or by base pairing with the substrate (pre-miRNA) –17. We sOxytricha nova. Using molecular modeling and mutation studies, we demonstrated that the Platform and PAZ domains can bind both RNA and DNA G-quadruplexes. We showed that binding of RNA and DNA G-quadruplexes by hDicer precludes pre-miRNA cleavage, which suggests the existence of yet another mechanism regulating Dicer activity and miRNA biogenesis in vivo.Here, we demonstrate that a hDicer-binding guanine-rich 12-mer can form a G-quadruplex structure. This finding inspired us to investigate possible interactions between hDicer and nucleic acids adopting G-quadruplex structures. In our studies, we used a full-length hDicer, as well as the hDicer fragment responsible for canonical substrate binding, encompassing the Platform and PAZ domains. We also analyzed several structurally well-characterized short telomeric RNA and DNA G-quadruplexes from human and the ciliate The 12-nt guanine-rich RNA, found to bind to hDicer, was perfectly complementary to the apical loop of pre-mir-210, and consequently, it was named “AL-210” . We hypoN-methyl mesoporphyrin IX (NMM) fluorescent probe  called “TER22”, which is an extended version of TER10, or G4U4G4 (Table S1). PPC formed stable complexes with both RNA oligomers, with a Kd value of ~7 nM for the PPC and TER22 complex , 58-nt pre-mir-21, and 19-bp RNA duplexes having 2-nt 3′ overhanging ends  . The Kd values calculated for PPC and RNA substrates were ~46 nM for LIN21, ~205 nM for pre-mir-21 and ~50 nM for dsRNA_OV . The results indicate that the PPC cassette of hDicer binds RNA G-quadruplexes with higher affinity than it binds a pre-miRNA substrate, a miRNA-size RNA, or a miRNA-like duplex.According to the current knowledge about the mechanism of Dicer action, the initial recognition and anchoring of the substrate occur within the region spanning the Platform–PAZ–Connector helix cassette, called “PPC” . The PPC32P-labeled DNA oligomers with sequences identical to TER22 and G4U4G4, i.e., “TEL22” and “G4T4G4”, respectively  than G4U4G4 and G4T4G4, respectively. Together, these results indicate that both the type and length of nucleic acid adopting a G-quadruplex structure determine its binding affinity to the PPC cassette of hDicer.Given the fact that telomeric repeat-containing sequences are present not only in telomeric RNA but also within telomeric DNA at the chromosome ends, we asked whether PPC could interact with DNA G-quadruplexes as well. Accordingly, we used 5′-O. nova G4T4G4 (PDB entry 1JPQ) [To test whether oligonucleotides adopting G-quadruplex structures could, like canonical Dicer substrates, be bound in the 3′-pocket and/or 5′-pocket of the PPC cassette, we performed molecular docking and modeling. For this we used the structure of hDicer PPC cassette (PDB entry 4NGF)  or O. nory 1JPQ) . TER10 fry 1JPQ) , whereasry 1JPQ) .We inspected docked poses of both TER10 and G4T4G4 with hDicer protein for clashes with pre-miRNA binding . We selected eight best models (docked poses) for the TER10-PPC complex and six best models for the G4T4G4-PPC complex. In seven out of the eight models selected for the TER10-PPC complex, TER10 was positioned within the 3′-pocket of the PPC cassette. In all these models, the 3′ end of one out of the four RNA strands was anchored in the 3′-pocket , and the other containing six substitutions in the 5′-pocket (R778A/R780A/R811A/H982A/R986A/R993A variant) . These changes significantly affect the binding of small interfering RNA (siRNA) by hDicer PPC cassette . Using E32P-labeled TER22 using T4 RNA ligase, as described previously [To investigate whether the free ends of the RNA adopting a G-quadruplex structure are necessary for its binding to the hDicer PPC cassette, we circularized 5′-eviously  (Supplemo-BMVC) covalently attached to its 5′-end . o-BMVC is a fluorescent light-up probe that selectively binds to G-quadruplex structures [o-BMVC in the same way as TER22. Each oligomer was incubated with PPC, and the reaction mixtures were analyzed in a polyacrylamide gel under native conditions. After electrophoresis, we exposed the gel first to 532 nm light to detect bands corresponding to the RNA species adopting G-quadruplex structures  carbazole diiodide  and DNA oligomers representing the corresponding telomeric repeats . As well as those mentioned above, we used three RNAs of 12 to 20-nt: TER12, TER18, TER18-2A. All of the chosen oligomers are known to adopt various G-quadruplex architectures (Table S1). To assess the effect of RNA and DNA G-quadruplexes on hDicer cleavage activity, we performed a set of assays involving hDicer, 5′-32P-labeled pre-mir-21 or pre-mir-33a, and a respective oligomer. The particular pre-miRNAs were chosen as they did not interact with tested oligomers , which ensures that the effects observed in the experiment does not stem from direct RNA–DNA or RNA–RNA binding, but rather, from protein–DNA or RNA interaction. Moreover, pre-mir-21 and pre-mir-33a represent structurally distinct substrates .In vitro, RNA oligonucleotides bound to hDicer inhibit the cleavage of pre-miRNAs by this enzyme , 16. To First, we carried out cleavage assays with RNA G-quadruplexes. The efficiency of pre-miRNA ~5 nM) cleavage in reactions with individual oligomers applied at one of three concentrations  was normalized to the cleavage efficiency in a control reaction with no oligomer added. Another set of control reactions involved in 12-nt RNA , which does not bind to hDicer, nor adopts a G-quadruplex structure. In all cases, upon the addition of an RNA G-quadruplex, we observed a dose-dependent inhibition of the cleavage of pre-mir-21  by hDicer can be affected by DNA counterparts of G4U4G4 and TER22, i.e., G4T4G4 and TEL22  or pre-mir-16-1 added as a competitor. The reactions were performed under the low-turnover conditions, i.e., a twofold molar excess of hDicer (10 nM) over a substrate was used. In addition, the G-quadruplexes and the competitor were in 50-fold molar excess to hDicer. As expected, in control reactions with either pre-mir-21 or pre-mir-33a and hDicer, but no other oligomer added, we observed a hyperbolic relation between the yield of miRNA and the incubation time  , RNA and DNA G-quadruplexes exerted a similar inhibition effect on the hDicer cleavage of both pre-miRNAs used Fig. e, f. G4UKd values calculated for the PPC and TEL22 complex (Kd ~ 318 nM), and the PPC and pre-miRNA complex (Kd ~ 205 nM). However, considering the binding affinities, we cannot explain why G4T4G4 was not outcompeted by the substrate over the incubation time. TEL22 represents the human telomeric sequence, while G4T4G4 corresponds to ciliate O. nova telomeric DNA. Given that the human enzyme was used in the studies, a species-specific regulatory mechanism for Dicer binding to telomeric DNA might be responsible for the effects we observed.Subsequently, we performed a time course assay under the high-turnover conditions using a 50-fold molar excess of a substrate to hDicer (0.5 nM), and a 100-fold molar excess of the G-quadruplexes or the competitor to the enzyme. We found that G4U4G4, G4T4G4 and TER22 retained their inhibitory potential after 12 h incubation , whereas the inhibition of either miR-21 or miR-33a production was overcome in reactions with TEL22 . Similar results as for TEL22 were obtained for pre-mir-16-1 competitor, therefore, we conclude that under the applied reaction conditions (1:2 molar ratio of a substrate and an inhibitor), TEL22 can act as a competitive inhibitor. This conclusion is supported by the Under the low-turnover conditions, we did observe a difference in the effect of pre-mir-16-1 competitor on pre-mir-21 and pre-mir-33a cleavage by hDicer Fig. e, f. DesCaenorhabditis elegans, a caspase-cleaved form of Dicer acts as a deoxyribonuclease that introduces breaks in chromosomal DNA during apoptosis [C. elegans and human cells, Dicer binds various RNAs, including mRNAs and long noncoding RNAs, passively, i.e., without further cleavage [Dicer homologs are widely known for their important role in the biogenesis of small regulatory RNAs: miRNAs and siRNAs. However, growing evidence shows that, apart from their canonical role, Dicer proteins serve a number of other functions (for review see ). As an poptosis . Moreovecleavage . Furthercleavage , 33. OurA variety of proteins bind G-quadruplex-forming RNAs . In mostKd ~ 7–10 nM)  , we conclude that an RNA G-quadruplex, once bound to hDicer, occupies the substrate binding pockets of PPC thereby blocking access for a pre-miRNA  Fig. b, c was RNA Fig. b. This iProteins binding RNA G-quadruplexes are important for the intracellular transport of mRNA for its local translation . MoreoveDicer has been detected in the nucleus , which tAltogether, we have demonstrated that G-quadruplexes can bind to Dicer and affect its activity. The results reported here support the notion that Dicer is a versatile protein, whose function is not limited to RNase III activity.Sequences of all oligonucleotides used in this study are listed in Supplementary Table S2.Guanine-rich oligonucleotides were denatured in 50 mM KCl for 3 min at 90 °C, immediately transferred to 75 °C and slowly cooled down to 10 °C.32P-labeled oligomers were hybridized in water with 10 pmol of complementary oligomers by heating and slowly cooling the mixtures from 90 to 4 °C. The reaction mixtures were PAGE-purified in 12% native polyacrylamide gels to obtain double-stranded fractions free of single-stranded species.For the RNA duplexes used in the binding assays, 5′-N-methyl mesoporphyrin IX , as described previously [200 pmol of nonlabeled oligomers were folded in 20 μL of 50 mM KCl, mixed with 20 μL of loading buffer, divided into two equal portions (20 μL) and separately loaded on a 12% polyacrylamide gel supplemented with 5% glycerol. The samples were arranged in duplicate so that after electrophoresis, the gel could be vertically cut into halves containing the same samples. One half was then stained in a solution of SYBR Gold (Thermo Fisher Scientific)/1× TBE for 20 min and scanned with Fujifilm FLA-5100 Fluorescent Image Analyzer at 473 nm. The other half was stained with eviously . Brieflyo-BMVC-C3-NHS ester in DMSO. The mixture was incubated at 37 °C in the dark overnight. The oligo-o-BMVC-C3 conjugate was separated from salt and free o-BMVC-C3-NHS by 2% NaClO4/acetone precipitation. The purity and homogeneity of the oligo-o-BMVC-C3 conjugate were verified by denaturing gel electrophoresis.In-house synthesized C6-amino-modified oligo dissolved in sodium phosphate buffer  was combined with fivefold molar excess of in-house made 32P-labeled TER22 was denatured in 100 mM KCl for 3 min at 90 °C immediately transferred to 75 °C and slowly cooled down to 10 °C to adopt its native structure. The ligation reaction was carried out using T4 RNA Ligase 1 (Thermo Fisher Scientific) overnight at 17 °C. RNA was purified using NucAway Spin Columns (Thermo Fisher Scientific) according to the manufacturers protocol and resuspended in water to the final concentration of approximately 10,000 cpm/µL (50 nM). To verify the efficiency of TER22 circularization, RNA  was treated with 0.5 U of alkaline phosphatase (Thermo Fisher Scientific) according to the manufacturers protocol and analyzed by denaturing PAGE.The 5′-32P-labeled RNA/DNA oligonucleotide  or o-BMVC-labeled oligonucleotide  and incubated in binding buffer  for 30 min (unless stated otherwise) on ice. The samples were separated in 5% or 10% nondenaturing polyacrylamide gels (as indicated) at 4 °C for approximately 10 h at 7 V/cm in 1× TBE. For radiolabeled oligomers, gels were exposed to a phosphorimager plate, which was subsequently scanned with Fujifilm FLA-5100 Fluorescent Image Analyzer to visualize the bands. For o-BMVC-labeled oligomers, gels were first scanned with Fujifilm FLA-5100 Fluorescent Image Analyzer at 532 nm, then stained in a solution of SYBR Gold (Thermo Fisher Scientific)/1× TBE for 20 min and scanned again at 473 nm.RNA/DNA–protein complex formation was analyzed using an electrophoretic mobility shift assay (EMSA). The reactions were carried out in 10 or 20-μL volumes. PPC or PPC variants  were mixed with a trace amount of the 5′-2 buffer, 10 nM hDicer, 5′-32P-labeled pre-miRNA  and the indicated oligonucleotide . In addition, two control reactions were carried out: (i) a negative control (C−) with no enzyme and no inhibitor, to test the integrity of the substrate during the incubation time, and (ii) a positive control (C +) with enzyme but no inhibitor added. All samples were incubated at 37 °C for 30 min. The reactions were halted by adding 1 volume of 8 M urea loading buffer and heating for 5 min at 95 °C, and then separated in a 15% polyacrylamide gel with 7 M urea and 1× TBE.Dicer cleavage inhibition assay was performed in 10 µL reactions containing 20 mM Tris–HCl, pH 7.5, 100 mM KCl, and 2.5 mM MgCl32P-labeled pre-miRNA , and the indicated oligonucleotide (0.5 µM). Samples were incubated at 37 °C for 30 min, 1.5 h, 3 h, 6 h, or 12 h. In addition, two negative control reactions were carried out to monitor the integrity of the substrate during the incubation time: (i) a sample frozen immediately after the mixture was prepared (C0) and (ii) a sample incubated at 37 °C for 12 h (C12). After the incubation time, all of the samples were processed as described above.In the time-course assay performed under the low-turnover conditions, a 10 µL reaction contained reaction buffer as described above, 10 nM hDicer, 5′-32P-labeled pre-miRNA, and 50 nM oligonucleotide.For the high-turnover conditions, the time-course assay was performed similarly, except that a 10 µL reaction contained 0.5 nM hDicer, 25 nM 5′-Y = Bmax*X/(Kd + X) and Bmax = 100% set as a constraint; Kd values together with the associated standard errors are reported in the figures. Diagrams presenting data from cleavage assays were prepared in Excel 2016. In the case of all diagrams, error bars represent SD values calculated based on three independent experiments.For binding and cleavage inhibition assays, the data were collected using Fujifilm FLA-5100 Fluorescent Image Analyzer and quantified using MultiGauge 3.0 software (Fujifilm). Data from binding experiments were fit to a saturation isotherm using Prism 8.2.1 (GraphPad). Dissociation constants were calculated using the equation for one site, specific binding: Homo sapiens Dicer1 ribonuclease type III sequence  (GeneCopoeia). In the case of PPC variants, we used plasmids encoding hDicer 3′-pocket double mutant , and the 5′-pocket sextuple mutant  [E. coli strain BL21Star (Thermo Fisher Scientific), in standard Luria–Bertani (LB) medium. Gene expression was induced with 0.4 mM IPTG and bacteria were cultured for 18 h at 18 °C with shaking. The cell pellets were lysed and purified with Ni2+–Sepharose High-Performance beads  with imidazole gradient (0.02–1 M) in 25 mM HEPES (pH 8.0) (in the case of PPC), or 25 mM Tris (pH 8.0) (in the case of PPC variants), supplemented with 300 mM NaCl, 0.1% Triton X-100, and 5% glycerol. The protein purity was assessed by SDS-PAGE. Selected fractions containing homogeneous protein were concentrated using Amicon filters (Merck) in respective buffers (as described above) enriched with 40% glycerol, and stored at −20 °C.The PPC cDNA, which corresponds to the 316-amino acid (aa) sequence located between 753 and 1068 aa of hDicer, was amplified by PCR using a purchased plasmid encoding a complete , R993A)  Supplementary file2 (PDB 2461 KB)Supplementary file3 (PDF 1102 KB)Below is the link to the electronic supplementary material.


In a pre-purchase situation, consumer perception of mandarin quality is almost exclusively based on fruit appearance. Determination of consumer requirements in this regard is needed to preserve the current success of this crop in markets worldwide. In this study, the effect on consumer choice of different appearance characteristics that can occur mainly in early-season mandarins was quantified. Two independent Choice-Based Conjoint Analyses were performed to evaluate the effect of different external mandarin factors: (1) two factors linked mainly with harvesting practices: ‘Leaf’  and ‘Rind Colour’ (orange colour/rind with slightly greenish areas); (2) three factors related to postharvest handling: ‘Calyx Condition’ (sound/blackening/detached),’Waxing’ (absence/presence of wax), and ‘Rind Condition’ (dehydrated/turgid). A total of 280 consumers participated in each study. The evaluation of the factors linked with harvesting revealed four different choice patterns. Leaf presence was appreciated only by a small group of consumers (20%), and the presence of greenish areas on the rind was perceived mostly as a negative characteristic. Among the evaluated postharvest-related factors, ‘Waxing’ and ‘Calyx condition’ had the strongest effect on consumer choice. Consumers showed marked preferences for mandarins that had been waxed and presented shine and gloss. Calyx blackening and detachment had a negative effect mainly on waxed fruit, while rind dehydration more negatively affected the fruit that had not been waxed. Consumer perception of mandarin quality is importantly affected by external mandarin characteristics. The information herein reported can be very useful for the citrus industry for adapting quality control criteria that respond to consumer demands. The global citrus fruit production has been estimated to be close to 158 million tons, and represents the major fruit tree crop in the world [In order to maintain and enhance the current success of mandarins in markets around the world, it is necessary for all supply chain stakeholders  to be aware of how to meet consumer quality requirements.Flavour, taste, texture, and internal colour are intrinsic characteristics to mandarins that have been reported to influence consumer preferences ,3,4,5. HCommercial standards on this regard have been traditionally based on technical experts’ criteria. However, as stated by Kader , this imThe influence of aspects such as price, colour, size, seediness, skin blemishes, production region label, and organic production has been evaluated by Campbell et al.  in varieMoreover, in the last few years, some distributors have started commercialising early-season mandarins with still greenish rind areas as a sign of freshness linked with a recent harvest or to a sense of being more “natural” (as no postharvest degreening treatment is applied). In line with enhancing consumer perception of freshness and naturalness linked with local food, the commercialisation of mandarins with leaves is increasing. In the same line, different studies have reported consumer voicing more concerns about the use of fruit waxes ,10, and Recently, Tarancón et al.  confirmeTherefore, while there is no doubt about the positive effect of certain factors such as size (larger fruit), seedlessness, and absence of blemishes on consumer mandarin choice, a gap in information about the impact of factors such as leaf presence, wax absence and calyx condition still exists. Moreover, there is still some controversy about the influence of rind colouration. Besides scientific interest in obtaining this information, it is crucial to set quality controls that ensure that consumer requirements are met.In this context, our study aimed to bridge this gap by quantifying the effect on consumer mandarin preferences of different external appearance factors that are associated with harvesting (Rind Colour and Leaf Presence) and postharvest  practices. To this end, we used the choice-based conjoint analysis, which allowed us to determine the impact of different factor combinations and to identify consumer segments based on their choice profiles.There are two main types of conjoint analysis: rating-based conjoint, which is based on consumer evaluations of products individually using hedonic or willingness to buy scales, and choice-based conjoint (CBC), which relies on the selection of the preferred product among several alternatives . The CBCBased on these considerations, the CBC analysis was used in this study to examine the influence of different mandarin appearance factors, and their combination, on consumer choice.Mandarins cv. Clemenules are one of the most important early-season mandarin variety in Spain. Therefore, this variety was selected as representative of early-season varieties. Fruits were collected from a packing house in Valencia (Spain), and whenever necessary, they underwent different postharvest conditions  to obtain the external aspect required for evaluation.Two independent choice-based conjoint analyses were performed: one to evaluate the effect of harvesting-related factors and another to evaluate the effect of postharvest handling-related factors. The two harvesting-related factors and their levels were: ‘Leaf’ ; ‘Rind colour’   . The thrIn both cases, the “sound calyx-wax-orange rind-turgid rind” combination was considered the “standard mandarin” as it is the most widely commercialised one. Thus, the effect of the different attribute levels on consumer choice was studied comparatively to the standard mandarin choice. The two factors associated with harvesting practices, ‘Leaf’ and ‘Rind Colour’, were initially hypothesised to have a positive effect on consumer choice, as we expected consumers to associate the presence of both fresh leaves and slightly greenish areas on the rind with freshness. The three postharvest-related factors were hypothesised to have a negative effect on consumer choice as we evaluated the effect of detached/blackened calyx, absence of wax, and dehydrated rind compared to the standard mandarin. High-quality colour images of mandarins displaying the previously explained combination of factors  were takConsumer evaluations were made separately for conjoint study 1 (harvesting factors) and conjoint study 2 (postharvest factors). Consumers completed an online questionnaire that was implemented using Compusense Cloud . A total of 280 Spanish consumers  participated in study 1 and 280 consumers  in study 2. The participants took part in only one of the two studies. In each study, participants were presented with eight choice sets, each composed of three mandarin images. For each choice set, consumers were asked to observe the mandarin images and to indicate the mandarin that they would choose to eat. An incomplete block design was employed to generate the eight comparison sets to be presented to the different participants. XLSTAT software was used to this end.After evaluating the different images, participants answered some demographic questions, such as their consumption frequency, gender, and age.Analysis of choice data was made by multinomial logit model regression using the XLSTAT statistical software. The model included the main effects  and binary interaction between them. This enabled a deeper understanding of how the experimental factors determined consumer responses. The utility value for each level or interaction was obtained. In the analysis, one of the levels of each factor was considered as reference. Additionally, in conjoint study 1, individual utilities were obtained for each consumer by logit model regression considering only the main effects. A hierarchical cluster analysis (HCA) was applied to the individual utilities to identify groups of consumers who had similar choice patterns. Euclidean distances and Ward’s aggregation method were considered. p > 0.05). Accordingly, neither the two factors, nor their interaction had a significant effect on consumer choice (data not shown). In view of this result, hierarchical cluster analysis was performed to gain a better understanding of consumer mandarin choice. To this end, the utilities for each factor level were obtained for each participant and a cluster analysis was applied.‘Rind colour’  and ‘Leaf’  were the fruit harvesting-related attributes evaluated in this study. The results were expressed as the impact of the different factors on consumer choice compared to the “standard mandarin” choice , which was equalled to zero to this end. The statistics for the likelihood ratio and Score and Wald indicated that the model was not significant  preferred the “standard mandarin”, i.e., orange-coloured fruit with a sound calyx. For this consumer segment, the presence of either leaf or greenish areas on the rind negatively impacted their choice. A very different pattern was detected in Cluster 3 (13% of the participants), which grouped those participants for whom the presence of both leaf and greenish areas on the rind had a positive effect. It is worth mentioning that while fresh leaf presence was positive when fruit was orange-coloured, this effect was almost imperceptible in the fruit with rind areas that were slightly green. Finally, 10% of the consumers included in Cluster 4 were characterised by the negative effect that green colour had on their mandarin selection decision making. For this group, leaf state was decisive, but only when choosing orange-coloured mandarins, when fresh leaf had a positive impact, while dehydrated leaf barely affected consumers’ decision.p > 0.05). This indicates that the four identified consumers’ choice patterns were not linked with demographic factors. An analysis of the demographic characteristics of the participants in each cluster was performed by means of the z-test for multiple proportions (data not shown). This analysis revealed no significant effect for age, gender, or consumption frequency (p < 0.001). Accordingly, the study conducted in detail of the effect of the different factors revealed that the three individual attributes significantly affected consumers’ choice of their preferred mandarin (p < 0.05). The group of attributes directly related to postharvest handling included ‘Waxing’, ‘Rind condition’, and ‘Calyx condition’, which were analysed together. As in the evaluation of the harvesting factors, the “standard mandarin” choice  was equalled to zero. In this case, the chi-squared estimated values for the likelihood ratio, and Score and Wald statistics indicated that the model was highly significant  had a negative impact on consumer perception of quality and, consequently, in their choice, as indicated by the negative values of their utilities . RegardiAs observed in Of the waxed samples, rind dehydration negatively affected the selection of the fruit with calyx, but had no effect on the fruit with a black or detached calyx. However, consumer selection among the non-waxed mandarins was negatively affected by rind dehydration irrespectively of Calyx Condition. The non-waxed fruit with a dehydrated rind were the least likely to be chosen by consumers. Finally, consumer choice was more negatively affected by the absence of calyx than by calyx blackening on waxed fruit. However, of the non-waxed fruit, consumers preferred fruit with no calyx over blackened-calyx fruit.To summarise, of the evaluated postharvest factors, calyx detachment most negatively affected consumer choice, mainly when they had to select among waxed fruit. Absence of wax was the second most impacting factor, of which the negative impact was observed mainly when consumers had to choose among mandarins with a sound or black calyx. The five appearance attributes herein evaluated were divided into harvesting-related attributes (Leaf and Rind Colour) and postharvest handling-related attributes , and two individual conjoint analyses were performed. The division of the factors into these two groups was motivated mainly by our initial hypothesis: compared to the “standard mandarin”, the harvesting-related attributes (leaf presence and rind with slightly greenish areas) were expected to have a positive effect in choice linked to a higher quality perception due to perceived freshness, while the evaluated postharvest attributes  were expected to negatively affect consumer choice. About the harvesting-related factors, it is important to explain one point. In this study, ‘Rind colour’ was taken as a harvesting-related factor because early varieties usually reach internal maturity while the skin is still green in citrus-growing areas with tropical and subtropical climate conditions . Thus, rThe conjoint analysis, which was performed by taking into account the harvesting factors data from all the participants, showed a very low impact for ‘Leaf’ and ‘Rind Colour’ on consumer choice, which was an unexpected result. Therefore, we studied the data evaluation in-depth by performing HCA, as previously described by Lee et al. . This meWhen the utilities of the individual factors and their interactions were added to analyse the effect of the factor combinations, for most participants , the presence of greenish areas on the rind negatively affected their mandarin choices. This result disagrees with Campbell et al. , but conFreshness has been reported to be one of the main drivers for consumer choice of different fruit, including mandarins ,17, and Moreover, for the 20% of consumers for whom leaf presence was a positive factor (Clusters 3 and 4), leaf state was also a determinant. Thus, leaf freshness was crucial for the consumers in Cluster 4 when selecting among orange-coloured mandarins. A noteworthy pattern was also detected for the consumers in Cluster 3. This group of consumers preferred mandarins with fresh leaf presence compared to those with dehydrated leaves among orange-coloured fruit, which is probably related to their perception of freshness. However, for the mandarins with greenish rind areas, those with fresh leaf presence were less appreciated. Our hypothesis is that for this group of consumers, fresh leaf presence may enhance their expectations of immature fruit linked with greenish areas on the rind. With regard to the postharvest-related factors herein evaluated, our results confirmed our initial hypothesis of them having a negative effect on perceived quality. Thus, the “standard mandarin” was that preferred by consumers. The ‘Calyx Condition’ and ‘Waxing’ factors had a strong impact on consumer choice. Waxing fruit is a long-standing practice that helps to prolong the shelf life of fruit ,23,24. MHowever, the study conducted by Cliff et al. , in whicThe CBC design used in this study allowed us to visualise the effect of combining different factors on consumer decision, which coincides with the proposal of Yu et al. . These aAfter evaluating the effect of the factor combinations, we can affirm that the least attractive mandarins for consumers were characterised by having no shine in combination with dehydrated rind. It was also interesting that calyx state had an effect when consumers evaluated waxed fruit, when mandarins with a blackened calyx were preferred to those with no calyx. However, as the general fruit aspect worsened (no waxed fruit with dehydrated rind), the impact of calyx on consumer choice became secondary.This study quantifies the impact of different factors on consumer choice for the first time, which is valuable information for the citrus industry. However, some limitations of this study should be mentioned. In the choice task presented herein, consumers were shown images of individual fruit. This helps consumers to pay attention to the factors being evaluated, and we assume that they made their decisions based on them. However, in a real purchase situation, consumers have to base their decisions on the appearance of a fruit lot, and not only on one. Therefore, certain variability among fruit as regards the evaluated factors is expected. A clear example of this is the calyx condition because it may differ among fruit, even if they have undergone the same postharvest treatments. Determining the minimum percentage of the fruit showing a specific characteristic in a commercial lot that leads to changes in consumer decisions would be an interesting topic for future studies. Such information would supplement the results herein reported and would help to optimise quality controls. In the same line, the use of observational techniques, such as eye-tracking technology to investigate consumers’ visual behaviour, would be very useful for determining to what extent fruit characteristics capture consumers’ attention, and our group is already working on this.The evaluation of the effect of harvesting practices-related factors (Leaf and Rind Colour) revealed four different consumer segments based on their choice profiles. Most consumers (Clusters 1 and 2) indicated preferences for the “standard mandarin” , and the presence of greenish areas on the rind negatively impacted their choice. The main difference between these two consumer segments was that leaf presence had no effect on Cluster 1 consumers, but it negatively affected the choice of the Cluster 2 consumers. Leaf presence was only appreciated by 23% of the participants (Clusters 3 and 4), mainly on orange-coloured fruit. The Cluster 3 consumers (13%) were the only ones for whom greenish areas on the rind had a positive impact.A marked effect on consumer choice was also detected when the appearance factors linked to postharvest handling were evaluated. In this case, consumers’ response was more homogeneous and a general pattern was identified. The shine and gloss that waxing confers mandarins markedly and positively affected consumer mandarin selection. The extent of calyx deterioration  was a determinant for consumer selection for waxed fruit, while rind turgor was more relevant for choosing non-waxed fruit. The information reported herein is very useful for the citrus industry in order to set quality standards that meet consumer requirements.