RNA-binding proteins play key roles in regulation of gene expression via recognition of structural features in RNA molecules. Here we apply a quantitative RNA pull-down approach to 186 evolutionary conserved RNA structures and report 162 interacting proteins. Unlike global RNA interactome capture, we associate individual RNA structures within messenger RNA with their interacting proteins. Of our binders 69% are known RNA-binding proteins, whereas some are previously unrelated to RNA binding and do not harbor canonical RNA-binding domains. While current knowledge about RNA-binding proteins relates to their functions at 5′ or 3′-UTRs, we report a significant number of them binding to RNA folds in the coding regions of mRNAs. Using an in vivo reporter screen and pulsed SILAC, we characterize a subset of mRNA-RBP pairs and thus connect structural RNA features to functionality. Ultimately, we here present a generic, scalable approach to interrogate the increasing number of RNA structural motifs. Saccharomyces cerevisiae by dimethyl sulfate-sequencing. Here the authors use quantitative proteomics to identify protein interactors of 186 RNA folds in S. cerevisiae, providing functional links between RNA binding proteins and distinct mRNA fold.Previous study identified in vivo structured mRNA regions in  Although protein-centric methods are widely employed to study RNAs associated with preselected RBPs, RNA-centric methods coupled to mass spectrometry such as RNA pull-downs and RNA interactome capture (RIC) allow identification of protein interactors at a target RNA2. Previous polyA RIC studies have cataloged a comprehensive repertoire of RBPs in budding yeast, extending the known set of RBPs from previous low-throughput studies and in silico predictions based on similar RNA-binding domains (RBDs)5.RNA-binding proteins (RBPs) are key players in several aspects of co- and post-transcriptional gene expression regulation, namely RNA splicing, capping, polyadenylation, export, translation and turnover6. The latter was also employed to identify structured mRNA regions in budding yeast7. Subsequent phylogenetic analysis revealed that 188 of these in vivo structured mRNA regions were conserved among yeast species7. However, to further characterize these conserved putative protein-interacting structures a streamlined approach is required that can investigate dozens of short RNA fragments. Employing SILAC-based quantitative mass spectrometry, we map RBPs for this previous published set of evolutionary conserved RNA structures in yeast, extending the structural information to a functional context and thus providing a generic workflow to systematically study RNA–protein interactions at a large number of RNA folds harboring putative protein recognition elements.RNA can adopt complex structures critical for binding to proteins that can vary at different cellular environments. Thus, intensive efforts have been undertaken to investigate RNA folding. In this context, the in vivo identification of RNA structures has been facilitated by techniques such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate-sequencing (DMS-Seq)Comparison with previous RBP datasets shows that a set of our RNA fold-associated proteins comprises well-studied RBPs for which we are able to reveal target mRNAs. Furthermore, we also report proteins with unassigned roles in RNA biology. Integration of genetic interaction data and experimental approaches to evaluate translational control provide first hints into the functional consequences of the reported mRNA–RBP interactions.S. cerevisiae7. In order to select for candidate structures with possible biological implications, a cut-off criteria was applied to DMS-Seq signal based on previously known functional structures like HAC1, RPS28B and ASH1. In addition, phylogenetic analysis on these mRNA structures revealed a list of 188 structured regions under positive evolutionary selection, lending additional support for a physiological function. These 188 evolutionary conserved RNA regions are of similar length, but found in different regions of the transcript   were enriched at folds irrespective of the functional region of the mRNA , 50% of them showed positional binding preferences (n = 82)  Fig. . Notably82) Fig. . Irrespe82) Fig. . Some RN10. Consistent with this observation, we found a high enrichment of the basic amino acids lysine, and to some extend also for arginine, in the amino acid composition of our RNA-binding proteins   bind to a small, but highly overlapping set of RNA folds . To this end, we used Pfam annotations and also evaluated RNA-binding domains from a manual curated datasetain Fig. . The thr27. Proteins with PUA domains might represent a novel type of translation factors, since this domain was detected in proteins that also harbor domains homologous to the translation initiation factors eIF1/SUI129. Consistently, Tma20 is homologous to the human MCT-1 gene that functions as a translation initiation factor30. At 8 of our 11 RNA folds targeted by Tma20, we also enrich Tma22, a protein with unknown function, but similar to the human DRP1 protein and possibly linked to translation regulation via its SUI1 domain. Our observations, together with previous studies reporting a physical interaction for these two proteins7, suggest a possible joint function for Tma20/Tma22 at their target mRNAs. Further experiments are needed to unravel a possible coordinated Tma20/Tma22 function on translation regulation.Besides the common RBD domains  Fig. , we also26. In this line, a previous interactome capture study from mESCs reported enrichment of low-complexity and disordered regions on their RBP candidates and suggested it to be a general feature of RBPs31. Indeed, we also report significant enrichment of disordered region containing proteins on our interactor dataset, further substantiating this as a possible feature of RNA-binding proteins  . As shown above, our integration with physical interactomics data can provide hints towards a putative regulation mechanism of genes that share similar genetic interaction profiles.To further examine functional connections between our RBP and mRNA folds, we clustered them based on similarity in their large-scale genetic interaction profile and tested for enriched GO annotations in each group Fig. . Genes tRNA Fig. ; a funct 4) Fig. . In anotSFT27. Consistent with previous data, disruption of base-pairing interactions in the 5′-SFT2 hairpin resulted in an increase of GFP levels, demonstrating a repressive function of the SFT2 hairpin on mRNA expression  and has been reported to be involved in rRNA processing39. In our screen, we detected Nsr1 binding to 60 of our RNA folds  tail-binding protein Pab146.In line with our GFP reporter screen data, pulsed SILAC experiments showed a translational repression role of Nsr1 on its target TP1 Fig. , as no cTP1 Fig. . In agreWe also investigated Tma20 by pulsed SILAC, the protein we found in our screen to be binding to several RNA folds and that is homologous to the human MCT-1 translational regulator. We found proteins involved in different cellular metabolic processes related to carbohydrates , nucleotides (Pnc1), amides (YDL124W), acetyl-CoA (Sdh2) and amino acids (Met6) to be upregulated in its knock-out strain compared to wild-type. These data establish Tma20 is a positive translational regulator as suggested before based on our interactome data Fig. .S. cerevisiae. We showed that our interactor set was enriched for RBP features and fused our interactomics data with genome-wide genetic interaction data to suggest putative functional and mechanistic insights for the detected mRNA–RBP pairs. Finally, we explored translational regulation as a possible functionality of our mRNA–RBP interactions using a reporter screen system and pulsed SILAC.In this study, we have used a streamlined RNA interactomics strategy to map protein interactors to 186 evolutionary conserved RNA folds in As current approaches to study RNA–protein interactions such as RIC globally address RBPs binding to mRNA and polyadenylated lncRNA, we here map 162 RBP interactors to individual RNA folds. We show that our setup captures interactions at these RNA structures even for low abundant RBPs as judged by their low abundance in our measured proteome and that were missed by RIC experiments. Our approach does not only capture proteins in direct contact with RNAs but with the current washing conditions also enriches for RBP complexes. To quantify this ratio based on our analysis, 69% are known direct RBPs, whereas the remaining might be associated via protein-protein interactions.S. cerevisiae datasets to validate our detected RNA fold–RBP interactions. Although the number of PAR-CLIP datasets in yeast is extremely limited, the available studies show a very high overlap to our data attesting our ability to report physiologically relevant interactions. We furthermore validated experimentally more mRNA–RBP interactions by RNA immunoprecipitation ourselves.We used available PAR-CLIP YEF3 and RBG2 . These observations suggest that RNA-binding motifs may also form in the coding region, possibly allowed by the degenerated codons. Whether this binding results in functional consequences will need to be explored in the future.Current knowledge about RBPs involved in post-transcriptional regulation focuses on either the 5′ or 3′UTR sequences. Interestingly, a significant portion of the conserved RNA folds is found in the CDS of mRNAs. We here report a significant number of RBPs recognizing RNA folds in the CDS of mRNAs. These interactors are related to ribosomal biosynthesis, tRNA binding or are metabolic enzymes and kinases. In addition, the binding profile of some classical RNA-binding proteins can be surprising, such as the binding of Pab1 to CDS RNA folds such as 10. Using the resolution for individual RNA folds, we here showed that tRNA synthetases bind to a specific set of RNA folds. This observation immediately suggests that some tRNA synthetases harbor yet unknown RNA-binding domains and hint to a possible role as translation regulators.As noted in previous RIC experiments already, not all discovered RBPs harbor canonical RBDs, but also include known proteins with diverse cellular functions like kinases, metabolic enzymes and tRNA- and rRNA-metabolism factors47.Overall, we here outlined how smaller structural features within an RNA molecule can be investigated with a streamlined assay, resulting in the identification of 162 interactors to 186 evaluated RNA folds. Mutational studies should be the next step to characterize our set of protein–RNA fold interactions to decipher the required RNA-binding motifs in more detail. There will be a growing demand for such analyses as thousands of structures can nowadays be either obtained using in vivo RNA structure methods, like DMS-Seq or SHAPE, functional reporter assays or computationally predicted based on conformation energy7. DMS signals of the corresponding genomic loci for each RNA fold were retrieved and normalized proportionally to the most reactive base within a given structure. For each RNA fold, denatured DMS signal was subtracted to the in vivo and in vitro DMS signals and Spearman correlations between the resulting in vivo and in vitro DMS signals were calculated as a measure of similarity. The probably of each RNA fold to form structure was calculated based on the Gini coefficient. This coefficient defines inequality within a population, which in our case means that RNA folds with high probability for structure formation will have very unequal DMS signal distribution along the sequence (Gini coefficient ≅ 1), whereas folds with low structural formation capacity will have an even DMS signal distribution (Gini coefficient ≅ 0). The resulting Gini coefficients of the conserved RNA folds (n = 188) were compared to the Gini coefficients of 200 randomly picked regions of similar average length from different genomic locations .DMS-Seq datasets from three different experimental conditions  were retrieved from GSE45803PMA1 RNA fold sequences for S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus were obtained from UCSC (SacCer_Apr2011/SacCer3 assembly) and aligned with ClustalW version 1.8148. The resulting multiple sequence alignment was fed into RNAalifold 2.2.8 from the Vienna RNA package49 to predict the consensus secondary structure.g, 5 min) and resuspended in Lyticase buffer . After 1 h incubation at 37 °C with 2.5 μL lyticase (Zymolyase® 20 T (≥20 U mg−1)), samples were centrifuged  and pellets washed twice with 1 mL spheroblast wash buffer . Pellets were then resuspended in 500 μL TE 50/100  and incubated with 50 μL 10% SDS for 30 min at 70 °C. 250 μL of 5 M potassium acetate was added and mixed by pipetting, followed by 15 min incubation on ice. Samples were centrifuged at 14,000 rpm for 20 min and supernatants transferred to a clean tube before isopropanol precipitation . Pellets were cleaned with 70% ethanol before adding 500 μL TE 10/1 solution  followed by 30 min heating at 42 °C and gentle shaking. When DNA was completely dissolved, 50 μL of 3 M sodium acetate were added, followed by a second isopropanol precipitation round . Samples were cleaned in 70% ethanol and pellets dissolved in 50 μL TE 10/1  during incubation at 42 °C for 1 h. DNA concentration was assessed by A280 absorbance on a Nanodrop instrument (Peqlab).Yeast genomic DNA from BY4741 strain was extracted from a 2 mL saturated culture, centrifuged  + 2*(nA + nT). Extracted genomic DNA together with the respective primer pairs were used to amplify each RNA fold in a PCR reaction using OneTaq polymerase according to manufacturer’s protocol (New England Biolabs). Successful amplification products were monitored on an agarose gel and were subsequently TA cloned into the pcDNA 3.3 TOPO vector following the manufacturer’s protocol (Invitrogen). Alternatively, for RNA folds failing PCR amplification, a primer extension approach using long primer dimers was employed, followed by TA cloning into pcDNA 3.3 TOPO vector. Correct sequence of all amplicons or chemically synthetized baits was checked by Sanger sequencing. For the GFP reporter screen, a yeast centromeric plasmid backbone was modified to incorporate the fluorescent yeast enhanced GFP (yeGFP) reporter gene driven by the Adh1 promoter and terminator. RNA folds were amplified from the pcDNA 3.3 TOPO plasmid with generic primers that introduced the corresponding restriction enzyme cutting sites for subsequent cloning into the GFP plasmid. According to the RNA fold UTR location, RNA folds were cloned 5′ or 3′ of the GFP reporter with a short linker region; plasmids were transformed into BY4741 yeast strain and selected by the URA3 marker. For the SFT2 3′UTR experiments, only the hairpin sequence as previously described7 was inserted at the 3′-end of the GFP reporter.The chromosome coordinates of all conserved RNA folds were retrieved from −1 YNB without amino acids, 2 % Dextrose, 200 mg L−1l-adenine sulfate, 100 mg L−1l-tyrosine, 10 mg L−1l-histidine, 60 mg L−1l-leucine, 10 mg L−1l-methionine, 60 mg L−1l-phenylalanine, 40 mg L−1l-tryptophane, 20 mg L−1 Uracil, 20 mg L−1l-arginine  and 30 mg L−1 of either ‘light’  or ‘heavy’ [13C6]l-lysine (Euriso-top). Cells were grown for at least 10 doublings to allow complete labeling and harvested at exponential growth by centrifugation at 20,000 rpm for 45 min at 4 °C . Two-liter yeast pellets were resuspended in 30 mL lysis buffer  and lysed three times at 35,000 psi in a French press at 4 °C. Different batches of lysate preparation were pooled for homogenization and successful incorporation of labeled lysine was checked by mass spectrometry. Extracts for western blotting analysis were prepared in lysis buffer  by bead milling using 0.5-mm Zirconia/Silica beads in a FastPrep for three cycles of 30 s at 4 °C with 1 min rest in between. Extract protein concentration was determined by Bradford (BioRad).Yeast strain YAL6B auxotrophic for arginine and lysine was grown at 30 °C to stationary phase in YPD media and then inoculated 1:10,000 into self-made filter-sterilized SILAC media (6.7 g L9. PCR products were used for in vitro transcription was performed according to the manufacturer’s protocol (Fermentas) and successful transcription monitored by agarose gel electrophoresis. Tagged RNA oligonucleotides were purified with G-50 micro spin columns  and concentration assessed by A280 absorbance on a Nanodrop system (Peqlab). 25 μg of each S1-tagged RNA fold was coupled to paramagnetic streptavidin C1 beads  in RNA-binding buffer  and incubated on a rotating wheel for 30 min at 4 °C. RNA-bound beads were washed 3 times with RNA washing buffer , followed by incubation with 400 μg yeast extract for 30 min at 4 °C on a rotating wheel. At this point, 650 ng of a competitor mixture containing a pool of all RNA baits without the S1-aptamer tag was added to reduce the number of sticky protein binders. After mild washing, light and heavy fractions were combined and samples were boiled in 1× LDS buffer (Invitrogen) and separated on a 4–12% NuPAGE Novex Bis–Tris precast gel (Life Technologies) at 180 V in 1× MOPS buffer.The RNA fold baits were created by a PCR reaction with generic amplification primers using the pcDNA 3.3 TOPO plasmid as a template. The forward primer (5′- CGTTAATACGACTCACTATAGGGATCGAACCCTT-3′) incorporated the T7 promoter sequence at the 5′-end of the RNA fold amplicon and the reverse primer (5′- CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAAAAAAAGATCGAACCCTT-3′) added the S1 minimal aptamer sequence at the 3′-end20 μg of whole lysate were run on a 4–12% NuPAGE Novex Bis–Tris precast gel (Life Technologies), transferred to a Protran 85 membrane (Whatman) and probed with either a rabbit TAP antibody  or mouse GFP  as primary antibodies. Either rabbit or mouse HRP-conjugated antibodies  were used for detection. Chemiluminiscence detection was done using a SuperSignal West Pico solution (Pierce) and the SeeBlue Plus2 Pre-stained Protein Standard (Thermo Scientific) was used as a marker.52 using two layers of C18 material (Empore). Eluted peptides were injected via an autosampler into an uHPLC  and loaded on a 25 cm capillary  packed in-house with Reprosil C18-AQ 1.9 μm resin (Dr. Maisch) for reverse-phase chromatography. The EASY-nLC 1000 HPLC system was directly mounted to a Q Exactive Plus mass spectrometer (Thermo). Peptides were eluted from the column with a 90 min optimized gradient from 2 to 40% ACN with 0.1% formic acid at a flow rate of 200 nL min−1. Chromatography was stabilized with a column oven set-up operating at 40 °C (Sonation). The heated capillary temperature was set to 250 °C. Spray voltage ranged from 2.2 to 2.4 kV. The mass spectrometer was operated in data-dependent acquisition mode with one MS full scan and up to ten triggered MS/MS scans using HCD fragmentation53. MS full scans were obtained in the orbitrap at 70,000 resolution with a maximal injection time of 20 ms, while MS/MS scan resolution was set to 17,500 resolution and maximal injection for 120 ms. Unassigned and charge state 1 were excluded from MS/MS selection and peptide match was preferred.Coomassie stained gels were cut in one slice and destained with 50% EtOH/25 mM ammonium bicarbonate (ABC). The resulting gel pieces were dehydrated with 100% acetonitrile (ACN) and dried for 5 min in a concentrator (Eppendorf). Samples were incubated with reduction buffer (10 mM DTT/50 mM ABC) for 30 min at 56 °C and further alkylated for 30 min in the dark with iodoacetamide (50 mM IAA/50 mM ABC). Gel pieces were completely dehydrated with ACN and covered in LysC solution (1 μg LysC per sample). Proteins were digested overnight at 37 °C and peptides were extracted twice by incubation with extraction buffer (3% TFA and 30% ACN) for 15 min. The gel pieces were dehydrated with 100% ACN and the extracted volume reduced to ~150 μL in a concentrator (Eppendorf). Extracted peptides were desalted in StageTips54 and searched against Saccharomyces cerevisiae Ensembl annotated protein database R64-1-1.24 Oct 2014 (6692 entries) using the Andromeda search engine55. Carbamidomethylation was set as a fixed modification, whereas acetyl (N-term protein) and oxidation (Met) were considered as variable modifications. LysC (specific) was selected as enzyme specificity with maximal two miscleavages for MaxQuant analysis. Proteins were quantified with at least 2 ratio counts based on unmodified unique and razor peptides. Known contaminants and reverse hits were removed before plotting the protein ratios of the forward and reverse experiments in R (version 3.2.2). Protein interactors for each RNA fold were identified requiring an enrichment of two-fold in both forward and reverse experiment (log2 SILAC ratios > 1). The enrichment value for each mRNA-protein pair was calculated as the log2 of the Euclidean distance to the origin . Alternatively, another selection method is proposed to identify interaction partners 1. For yet unclassified interactors, their human homologs were classified with the same RNA binding and RNA-related annotation criteria. As a control, proteins from a whole cell lysate measurement were equally classified. For RNA-binding domain classification, a curated list of known PFAM RNA-binding domains1 was used  contained the string RNA binding. For those interactors that did not relate to the term RNA binding, a second classification was done as RNA-related based on previously described RNA-related GO-term annotationsWe performed GO enrichment analysis of binders using SGD Slim Mapper tool. Binders were classified in 3 groups according to the genomic position of the RNA fold they bind to .56. Significant differences of our RBP set against two control groups containing all known proteins of the yeast genome (predicted proteome) and our measured proteome were calculated with a Student’s t-test .A peptide properties table containing information about Molecular Weight, Isoelectric Point, Protein Length, Hydropathicity GRAVY Scores, Aromaticity Score , Codon Adaptation Index, Codon Bias, FOP Score , Instability Index and Aliphatic Index, was downloaded from the Saccharomyces Genome Database57, defined as a lack of known tertiary structure under native conditions. The default prediction type for long disorder region was used and a score based on the percentage of bases with disorder probability higher than 50% was calculated for each protein interactor.Complete peptide sequences for our interactor set were retrieved from Ensembl version 86 (Oct 2016) and used for disordered region probability calculation with IUPred2A34 was used. For genetic interaction scores (|E|) higher than 0.08 between our RNA folds and protein interactors, an annotation matrix was calculated depicting the reported |E|scores. Pearson correlation coefficients (PCC) of genetic interaction profiles for all relevant genes (including protein interactors and their mRNA targets) were calculated. Genes with similar genetic interaction landscape were identified using a simple network analysis: each gene was a vertex, and an edge between 2 vertices was defined if the PCC between these two proteins was higher than 0.2, as described in Costanzo et al. Community structure on the network was inferred using a method implemented in the igraph package58 based on propagating labels, which works by assigning vertices to unique communities and then updates those communities by doing majority voting around a vertex.For genetic interaction data integration, a recent large-scale genetic interaction study59. Reads were preprocessed with the Fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) to remove adapter sequences, keep sequences longer than 15 nucleotides, filter artifacts and remove low quality reads (Phread scores < 23). Preprocessed reads were then aligned onto the sacCer3 reference genome with Bowtie60 version 1.1.2 and options -q -p 8 -S -v 2 -m 10–best–strata to allow up to 2 mismatches and reads mapping to up to 10 loci, keeping only alignments in the best stratum. Scaled BigWig tracks were generated from the alignment files. To call peaks, the Piranha61 1.2.1 peak caller was used with options -b 50 -d Poisson, which bins reads into bins of 50 bp and uses the Poisson distribution to model the counts. Peaks called with Benjamin and Hochberg (BH) corrected p-value below 0.05 were considered true peaks.PAR-CLIP raw data for Nab2 (GSM1442550), Pab1 (GSM1442553) and Yra1 (GSM1442559) were downloaded from GSE59676Yeast knock-out and TAP-tagged strains used for pSILAC, WB and RIP experiments are listed in Supplementary Data −1 FastPrep . Samples were diluted in FA buffer supplemented with 0.1% sodium-deoxycholate (SOD). Soluble and chromatin extracts were separated by centrifugation (7 min at 17949 rcf). Subsequently, 2 mg of soluble extracts were incubated overnight at 4 °C with 75 µL of pre-washed IgG Beads  with 5% BSA. 50 µL of extracts was separated as an input control. Beads were washed with 1 mL of FA buffer, Buffer 500 , Buffer III  and TE buffer  at 4 °C with 5 min incubation times between washes. Proteins were eluted with Elution Buffer  twice for 8 min at 65 °C. Samples were de-crosslinked for 2 h at 65 °C and subsequently digested with 3 units of DNase I (QIAGEN) for 2 h at 37 °C. After digestion, eluted samples were digested with proteinase K (0.75 mg mL−1) for 2 h at 65 °C. RNA samples were purified using the RNeasy MinElute Cleanup kit (QIAGEN). Purified RNA samples were digested once more with 3 units of DNase I (QIAGEN) and purified. RNA samples were subjected to reverse transcription before quantification by qPCR for different loci. RNA samples were split into 2 fractions. One fraction was used to measure the RNA levels and the other fraction was used as a negative control of reverse transcription (no reverse transcriptase added). The RNA was incubated at 90 °C for 1 min with 0.4 µL 25 mM dNTPs, 0.8 µL 5 µM of different primer pairs in 10 µL final volume. The RNA was then cooled down to 55 °C at a 0.8 °C s−1 temperature rate. A mix of 1 µL 100 mM DTT, 1 µL SuperScript III in 1× FS-buffer (Invitrogen) was added to the reactions. Negative control sample did not contain SuperScript III reverse transcriptase. The RNA was reverse transcribed for 60 min at 55 °C. The enzyme was inactivated at 70 °C for 15 min. RNA samples were diluted with 30 µL H2O and used in qPCR for quantification.TAP-tagged strains from Dharmacon collection were used for TAP-RIP experiments. 100–150 mL of exponentially growing cultures were crosslinked for 10 min with 1.2% formaldehyde (Applichem) after cell number normalization. Samples were quenched with glycine  for 5 min at room temperature. After cooling down to 4 °C on ice for 15 min, cells were pelleted at 4 °C by centrifugation , washed twice with ice-cold PBS and stored at −80 °C until processing. Cell pellets were lysed in FA buffer  via two 30 s rounds of 6.5 M s−1 temperature rate. A mix of 1 µL 100 mM DTT, 1 µL SuperScript III in 1× FS-buffer (Invitrogen) was added to the reactions. Negative control sample did not contain SuperScript III reverse transcriptase. The RNA was reverse transcribed for 60 min at 55 °C. The enzyme was inactivated at 70 °C for 15 min. RNA samples were diluted with 30 µL H2O and subjected to qPCR. Data were processed according to the 2(−ΔΔCt) method and expressed as %input. For both ±RT conditions, %input was calculated normalized to the +RT input (5%) values. Finally, +RT %input were normalized to the respective −RT %input. Error bars show SEM, calculated as SEM = (SEM12 + SEM22)1/2.Exponentially growing cells were collected and resuspended in 400 µL AE Buffer (50 mM sodium citrate in 10 mM EDTA pH5.3) and lysed with 500 µL calibrated phenol (with AE buffer) at 65 °C for 5 min. The aqueous phase was separated by centrifugation and mixed with 500 µL phenol–chloroform–isoamyl alcohol for 5 min at room temperature. The aqueous phase was again separated by centrifugation and collected. RNA was precipitated with 40 µL 3 M sodium acetate and 1 mL 100% ethanol. RNA was pelleted by centrifugation and washed with 80% ethanol. Air-dried pellet was subsequently resuspended in a solution containing 3 µL DNase I (QIAGEN) in RDD buffer to digest genomic DNA. DNA was digested for 45 min at 37 °C and remaining RNA was purified with RNeasy MinElute Cleanup kit (QIAGEN). The RNA was incubated at 90 °C for 1 min with 0.4 µL 25 mM dNTPs, 0.8 µL 5 µM of different primer pairs in 10 µL final volume reaction. The RNA was then cooled down to 55 °C at a 0.8 °C sg, 5 min) and gently resuspended in 3 mL SORB buffer . 50 μg of sheared salmon sperm (Ambion) carrier DNA was boiled 5 min at 95 °C prior to the addition of 100 ng of plasmid DNA. Cells were resuspended in 100 μL LiT solution , plasmid-carrier DNA was added and followed by the addition of 500 μL of PEG/LiT (polyethylene glycol 3350 (Sigma-Aldrich)). Samples were vortexed and incubated in a rotation wheel for 30 min at room temperature. 50 μL of DMSO (Sigma-Aldrich) were added and incubated for 15 min at 42 °C. Cells were pelleted (500×g for 30 s) and resuspended in 200 μL of SD-URA medium, incubated at 30 °C for 30 min and plated on SD-URA plates for 2–3 days.Exponentially growing BY4742 yeast cells in YPD medium were pelleted . Doublets were excluded via SSC-W signal and dead cells were excluded by DAPI staining. 20,000 events were measured per experiment and median values used for data analysis with FlowJo software (v10.5.3). GFP mean fluorescence intensities for the knock-out experiments were normalized to the corresponding wild-type condition and the mean value of three experimental replicates was plotted. Error bars represent the standard deviation of the GFP knock-out/wild-type values.The relevant RNA folds were cloned into the corresponding UTR location of a centromeric GFP reporter plasmid. The resulting plasmids were used in the respective yeast knock-out strains (Dharmacon). The BY4741 yeast knock-out strains transformed with the GFP reporter plasmid were grown to saturation in SD-URA selection media at 30 °C, diluted 1:100 and further grown to ODl-adenine sulfate, 0.1 g l-tyrosine, 0.01 g l-histidine, 0.06 g l-leucine, 0.01 g l-methionine, 0.06 g l-phenylalanine, 0.04 g l-tryptophane, 0.02 g uracil and 0.02 g l-arginine per liter) was supplemented with 30 mg L−1 either lysine-0, lysine-4 and lysine-8 (Eurisotop) and sterile filtered through a 0.22-μm filter (Fisher Science). A preculture of 5 mL in lysine-0 medium was grown overnight at 30 °C. The culture was diluted and grown to OD600 = 0.4 in lysine-0 medium, prior to two washes with PBS (cells pelleted by centrifugation at 500 g). When cells were resuspended in 15 mL lysine-8 medium (knock-out strain) and lysine-4 medium (wild-type strain), actinomycin D was added at a final concentration of 1 μg mL−1. Cells were grown with gentle agitation for 2 h at 30 °C. Cells from both cultures were mixed at OD600 = 0.5 and harvested by centrifugation at 14,000×g for 2 min at 4 °C. The pellet was washed with PBS and transferred to a clean tube. The cells were again centrifuged at 14,000×g for 15 s at 4 °C and resupended in 50 μL 1× NuPAGE LDS buffer (Thermo), sonicated for 10 cycles (30 s ON/OFF), spun down at 14.000×g and 20 μL loaded in a 10% NuPage NOVEX precast gel (Thermo). Subsequent protein separation, staining and in-gel digest was done as described (see MS sample preparation and measurement section). Data were analyzed with MaxQuant and further processed with in-house scripts .SILAC medium (6.7 g YNB w/o amino acids and with ammonium sulfate, 20 g Dextrose, 0.2 g Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7


Hordeum vulgare L.) is an economically important crop cultivated in temperate climates all over the world. Adverse environmental factors negatively affect its survival and productivity. RNA silencing is a conserved pathway involved in the regulation of growth, development and stress responses. The key components of RNA silencing are the Dicer-like proteins (DCLs), Argonautes (AGOs) and RNA-dependent RNA polymerases (RDRs). Despite its economic importance, there is no available comprehensive report on barley RNA silencing machinery and its regulation. In this study, we in silico identified five DCL (HvDCL), eleven AGO (HvAGO) and seven RDR (HvRDR) genes in the barley genome. Genomic localization, phylogenetic analysis, domain organization and functional/catalytic motif identification were also performed. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to moderate, persistent or gradient heat stress treatments: transcriptional accumulation of siRNA- but not miRNA-based silencing factor was consistently detected. These results suggest that RNA silencing is dynamically regulated and may be involved in the coordination of development and environmental adaptation in barley. In summary, our work provides information about barley RNA silencing components and will be a ground for the selection of candidate factors and in-depth functional/mechanistic analyses.Barley ( Hordeum vulgare L.) is the fourth most important crop in the world in terms of cultivated area (50 MHa) and grain yield produced (140 Mt) [Barley (produced 40 Mt 1](140 Mt)  and is u(140 Mt) (140 Mt) ,4. Barleted area 0 MHa and(140 Mt) ,7. Barle(140 Mt) . GermplaRNA silencing is an evolutionary conserved sequence-specific gene-inactivation pathway. Originally evolved as an immunity system ,10, RNA Arabidopsis thaliana genome encodes four DCLs (AtDCL1–4), each having specialised functions. AtDCL1 generates micro RNAs (miRNAs) that function primarily in development and environmental adaptation, AtDCL2 and AtDCL4 are required for vegetative phase change and disease resistance through phased small interfering RNAs (pha-siRNAs) and/or trans-acting siRNAs (ta-siRNAs) generation, while AtDCL3 produces 24-nt heterochromatic siRNAs (hc-siRNAs) that directs RNA-directed DNA methylation (RdDM)-mediated epigenetic regulation  (DEDH or DEDD) tetrad and the additional histidine  residue within PIWI, directly involved in enzyme catalysis A. thaliaencode 9 , and 16 encode 9  AGO gened so far . We descmstances .HvAGO1 and HvAGO5 have been duplicated. Grasses in general exhibit an expanded AGO1/5/10 clade [OsAGO1 family possess the catalytic residues and have slicer activity [HvAGO1a, HvAGO1d and HvAGO5a but not in HvAGO5b, where the histidine residue  was replaced by a proline  . Although tyrosine (Y) is similar to histidine (H), as both having aromatic and hydrophobic side chains, the absence of the charge  may alter the activity of HvAGO10. Overall, we postulate that within HvAGO1/5/10 clade, HvAGO1a and 1b may act canonically and redundantly, while HvAGO5a, HvAGO5b and HvAGO10 probably perform differentiated/ specialised functions.In barley, we have found five members of AGO1/5/10 clade. 10 clade . OsAGO1 activity . We haveeficient . At 798 his idea . AGO10 he in SAM ,126. WheHvAGO18 placed it close to Z. mays and O. sativa AGO18 ortholog proteins, implying similar functions.Grasses have evolved a further subgroup of AGO1/5/10, the AGO18 clade , that seAtAGO2 of the AGO2/3/7 clade binds 21-nt sRNAs and was shown to have roles during pathogen defence and double-stranded DNA repair [AtAGO2 and AtAGO3 are very similar, surprisingly, AtAGO3 binds 24-nt and targets transposable elements through TGS [HvAGO2 also contained the distinctive DEDD + H motif specific to AtAGO2/3 proteins , with partial redundancy and specificity [HvAGO4a, 4b and 6. The DEDH + P catalytic core of HvAGO4a, 4b and 6 is identical to AtAGO6  is needed for non-CG methylation of centromere region and knot-repeat DNA backs this theory [At/ZmAGO9 proteins are predominantly expressed in ovules and regulate cell fate [The modifier AGO clade (AGO4/6/8/9) contains three functional members in cificity ,135,136.cificity ,137,138.o AtAGO6 B. Domains theory . We coulell fate . Which oHvRDR genes were identified previously [OsRDR1 is induced and required for antiviral defence [HvRDR1 and HvRDR6 homologues suggests an evolutionary selection for specialisation either during development or under stress responses. Indeed, HvRDR1a/b and HvRDR6a/b transcription were selectively induced in different organs, in response to different pathogens or under elevated temperature [HvRDR3, 4) informs about a need for their activity. Absence of one or multiple RDR3, 4, and 5 proteins from different plants, however, suggests non-essential roles [Initially, RDR proteins were studied for their antiviral roles but later it became evident that they are also required during gene expression control and chromatin regulation. Several barley eviously , however defence ; maize R defence ; SHL2, 4 defence . The cloperature . On the al roles .AGO1s self-regulation occurs in monocots as well [HvAGO1a and 1d transcripts, implying that miR168-mediated regulation potentially occur in barley  were both activated by heat [AtAGO1 is required for heat-stress memory [trans factor regulation and epigenetic/chromatin reprogramming.Heat stress is a major threat to barley crop yield and quality. Previous studies demonstrated the heat-regulated changes of specific miRNAs, ta-siRNAs, hc-siRNAs despite the rather stable global level of sRNAs ,147,148. by heat . AtAGO1 s memory . Prolongs memory . These dtrans factors in barley, we studied RNA-seq data available [HvDCL3, HvAGO2, HvAGO6, HvRDR2 and HvRDR6a mRNA was confirmed (trans factors of miRNA pathway, HvDCL1 and HvAGO1a seem to be much stable under the investigated circumstances. In summary, data from RNA-seq, semi-quantitative and RT-qPCR measurements all converge and points towards the transcriptional accumulation of factors enrolled primarily in siRNA-based silencing, including hc-siRNA, pha-siRNA and RDR6-dependent sRNA pathways. Importantly, our gradient heat treatment mimics natural situations, e.g., temperature changes during a summer day, therefore may be relevant in field conditions. As DCL3, AGO2, AGO6, RDR2 and RDR6 factors were all involved in TGS or double-stranded DNA break repair [To unravel heat-induced transcriptional regulation of silencing vailable  (Figure onfirmed . The higonfirmed  suggestik repair ,137,149,We have identified members of gene families having key roles in RNA silencing of barley and provided basic data on their genomic location, clade, phylogenetic relations, domain and motif organisation, and catalytic core build-up. Our data firmly suggests that these players are potentially functional and likely required at some point during barley’s lifecycle. Transcriptional accumulation of siRNA pathway factors hints to a probable role in environmental adaptation. This work will be a stepping-stone to ask further fundamental and exciting questions that remained pending in barley and monocot RNA silencing field.


The assay took 30 min, including the RT-RPA stage and the LFA stage. The testing of healthy and infected potato samples showed full concordance between the developed RT-RPA-LFA and quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the commercial kit. The obtained results proved the feasibility of using the developed assay to detect PSTVd from a natural source.An assay was developed to detect the potato spindle tuber viroid (PSTVd), a dangerous plant pathogen that causes crop damage resulting in economic losses in the potato agriculture sector. The assay was based on the reverse transcription and recombinase polymerase amplification (RT-RPA) of PSTVd RNA coupled with amplicon detection via lateral flow assay (LFA). Primers labeled with fluorescein and biotin were designed for RT-RPA for effective recognition of the loop regions in the high-structured circular RNA of PSTVd. The labeled DNA amplicon was detected using lateral flow test strips consisting of a conjugate of gold nanoparticles with antibodies specific to fluorescein and streptavidin in the test zone. The RT-RPA-LFA detected 10 The potato spindle tuber viroid (PSTVd) is the most primitive potato plant pathogen that causes perceptible crop damage . PSTVd cPSTVd can spread throughout hosts vertically—through seeds and vegetative propagation—and horizontally through pollen. The infected pollen can transmit the viroids to the same host species as well as other host species . When po6 [4 [7 copies) for infected samples after specific RNA extraction [The infection cannot be treated, so a PSTVd diagnostic is the only way to stop it from spreading and decrease economic losses. The nature of PSTVd restricts its detection based on antigen–antibody recognition. To date, no antibodies against the viroid have been found. Only nucleic acid detection methods can be applied, with the first methodological group being the hybridization of PSTVd with labeled oligonucleotides ,16,17,186  and 1:106 [4 ,25. The 6 [4 . While t6 [4 , only ontraction . RT-qPCR3 dilution of PSTVd (100–1000 copies in the sample) and was verified by a large-scale field trial of more than 400 samples [4 times diluted potato samples [4 copies of the PSTVd DNA template. Different isothermal tests have been developed for other viroids. Isothermal and chimeric primer-initiated amplification of nucleic acids was used to test for the chrysanthemum chlorotic mottle viroid [5–5·106 copies) of pure in vitro transcribed RNA [An alternative way of detecting nucleic acid is isothermal amplification. This method does not require complex or expensive equipment. Thus, one isothermal test, nucleic acid sequence-based amplification (NASBA), was developed for detecting PSTVd; it has a sensitivity equal to 250–2500 copies of the RNA . Another samples . The LAM samples . Another samples . The tese viroid . One pose viroid . RPA proibed RNA .For the research study discussed in this paper, the first RPA-LFA for detecting PSTVd was developed. This study included selecting optimal primers for effective recognition of the loop regions in high-structured circular RNA. The presented comparison demonstrates tools for further work on peculiar analytes, such as viroids.The linear PSTVd RNA was synthesized with in vitro transcription and obtained as a uniform product of 357 nt according to gel-electrophoresis  without the RNA template to register the sticking primers and the non-specific signals. Strong positive signals  with 1010 copies of the target RNA, but without Moloney murine leukemia virus (MMLV) reverse transcriptase. This stage had RNA-guided non-specific effects, which can be detected upon a high concentration of total RNA in an analyzed sample. Notably, a false-positive signal in four pairs of the chosen primers was observed. Only the F1 + R5 combination demonstrated no visible signal in the test zone of the test strips  was able to detect 106 copies of PSTVd RNA in a tube (5–5·106 copies of in vitro transcribed RNA [7 copies) previously demonstrated by PCR [First, the LOD of RT-RPA-LFA was determined using samples of serial diluted linear PSTVd RNA (in water) from 10n a tube . This reibed RNA . Moreoved by PCR ,27. The 7 fold dilution  and infected in vitro plants (n = 6). The results were interpreted as positive if the means of the duplicated RT-RPA-LFA were above 2 arbitrary units. The RT-RPA-LFA showed 10 negative  and 6 positive (infected) samples. The results were fully confirmed using the commercial RT-qPCR kit. The potato samples were tested using healthy in vitro plants . The PCR product of the gene was used as a DNA template for in vitro transcription. PCR was performed using M13 reverse (AGCGGATAACAATTTCACACAGGA) and PSTV reverse (AGGAACCAACTGCGGTTCC) primers and a Q5® High-Fidelity DNA polymerase PCR mix (NEB). The primers were synthesized by Evrogen. The DNA fragment contained the T7 promoter, PSTVd gene, and T7 terminator. The PCR products were purified from a PCR mix with a Monarch® DNA Extraction kit . The DNA fragment was added to up to 50 ng/µL (180 nM) of reaction mix containing 166 mM HEPES-KOH pH 7.5, 32 mM MgCl2, 40 mM dithiothreitol, 2 mM spermidine, 100 mg/mL bovine serum albumin (BSA), 7 mM each nucleoside triphosphate (NEB), 2 U/µL RiboLock (NEB), and 5 U/µL T7 polymerase (NEB). The transcription was carried out for 2 h at 37 °C.A sequence containing the PSTVd  gene flanked with the T7 promoter and the T7 terminator was synthesized and cloned in a pUC19 vector by Evrogen (Russia). A plasmid pUC19-T7-PSTVd was linearized by the g). The RNA was selectively precipitated by the addition of LiCl to the top fraction up to 3M, and incubated for 30 min at 4 °C. The pellet was diluted in deionized water and desalted using the gel-filtration spin column Zeba Spin MWCO 7K . To remove minor residue of the DNA template, the solution was treated with DNAseI (NEB), as recommended in the manual. Finally, the RNA was precipitated with 70% ethanol and 85 mM sodium acetate. The RNA pellet was washed with 70% ethanol and dissolved in deionized diethylpyrocarbonate-treated water. The integrity of the PSTVd RNA was estimated using electrophoresis in polyacrylamide gel. The absence of DNA in the RNA solution was verified by RT-qPCR. Concentrations (ng/µL) of the RNA were automatically measured by NanoDrop2000  based on the Beer–Lambert equation and characteristic extinction coefficient for RNA (40 ng-cm/µL). The molar concentration and the number of copies were calculated for the length of the transcript equal to 357 nt, the results are presented in Extraction of PSTVd from the reaction mix was performed as described ,37 with Conjugate of gold nanoparticles (GNP) with mouse monoclonal antibodies specific to fluorescein (anti-FAM)  and LFA test strips were prepared as described at .4 and 95 mL of deionized water were mixed and heated to boiling point, then 4 mL of 1% sodium citrate was added and boiled for 25 min. For the synthesis of the GNP–anti-FAM conjugate, anti-FAM and GNP were adjusted to pH 9.0, mixed with a final ratio of 10 µg of anti-FAM per 1 mL of GNP solution with optical density at 520 nm (OD520) = 1.0, incubated for 1 h, blocked by BSA  to 0.25%, and separated from the unbinding proteins via centrifugation at 10,000× g for 30 min at 4 °C.Briefly, to synthesize the GNP with a diameter of 17 nm, 1 mL of 1% HAuCl520 = 4.0) was dispensed at 3.2 μL per 1 mm of strip width on a PT R5 fiberglass membrane . Streptavidin  and goat anti-mouse IgG  were dispensed at 0.15 μL per 1 mm of strip width on test and control zones of nitrocellulose membrane CNPC12 (Advanced Microdevices), respectively, using an IsoFlow Dispenser . Test strips were assembled using the sample membrane GFB-R4, final adsorbing membrane AP045 (Advanced Microdevices), and the aforementioned fiberglass and nitrocellulose membranes. The multimembrane composites were cut and packed according to Byzova et al. [The GNP–anti-FAM conjugate  potato plants and potato plants artificially infected by PSTVd (n = 6) were grown in vitro and provided by I.V. Shmyglya . The infection did not demonstrate any visible symptoms. Samples of potato leaves (150 mg) were homogenized in a Petri dish. The extraction of total RNA from the samples was performed using a commercial total plant RNA extraction kit  according to the manufacturer’s protocols. The extracts were tested using a commercial qPCR kit (Syntol) prior to the subsequent applications.Healthy . However, the 3′ of the primers were designed more specifically to hybridize with loop regions of PSTVd. The prediction of primer dimerization and properties were obtained using OligoCalc  and TherThe specificity of the chosen primers pair among the different PSTVd strains and isolates was estimated by multiple alignments of all 349 of the GenBank-deposited full genome sequences of the PSTVd isolates. The analysis was performed using Jalview software .An RPA TwistDx kit  was utilized for the RPA according to the manufacturer’s protocols with modifications . BrieflyThe PSTVd is a highly infectious viroid that causes severe damage to crops. Rapid and sensitive detection methods are essential to prevent viroid spread. We developed a rapid (30 min) RT-RPA-LFA for detecting PSTVd. The main advantage of the RT-RPA assay is the ability to carry out all the reactions at the same temperature (39 °C). RT-RPA, in contrast to the most widespread RT-PCR technique, does not require cyclic temperature changes. This simplifies the instrument base for the analysis and provides the possibility of widespread monitoring of the viroid, even in field conditions. Such a tool is especially important due to the lack of immune tests for viroids, which serve as primary screening tools for other phytopathogens.6 copies of PSTVd RNA in a reaction of up to 1:107 diluted plant samples. This sensitivity is close to the previously reported RT-PCR for PSTVd. The simplicity and sensitivity of the developed assay indicated above determine its competitive potential for on-site control and efficient crop protection.Through a design of loop-targeting primers and their screening, we overcame the limitation of isothermal amplification of high-structured circular RNA. Consequently, an RT-RPA-LFA with a LOD equal to 10


The new human coronavirus named SARS-CoV-2 is a positive-sense RNA virus for which no specific drugs are currently available. A knowledge-based analysis strongly suggests a possible repositioning of the anti-HCV direct antiviral agent (DAA) Sofosbuvir as treatment for SARS-CoV-2. Indeed, the RNA-dependent RNA-polymerases (RdRp) of the two viruses show high sequence and structural homology, supporting the likelihood of binding the Sofosbuvir molecule with similar efficiency. Such a repositioning would allow the containment of the SARS-CoV-2 pandemic and limit the progression of disease to potentially deadly COVID19. The virlliliter  both in lliliter .Consequently, it is necessary to reduce or block viral replication to avoid the progression of the disease towards the full-blown and potentially lethal form (COVID19), but also to reduce the viral titer and viral shedding through saliva, in symptomatic and asymptomatic infected individuals.https://www.gilead.com/news-and-press/press-room/press-releases/2020/4/gilead-announces-results-from-phase-3-trial-of-investigational-antiviral-remdesivir-in-patients-with-severe-covid-19). However, a trial conducted in China showed Remdesivir did not improve patients’ condition nor reduced the positivity to virus. Moreover, the drug showed also significant side effects (https://www.ft.com/content/0a4872d1-4cac-4040-846f-ce32daa09d99).Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for HIV  are under evaluation on the basis of weak evidences from retrospective analyses suggesting clinical benefit in the treatment of the two previous coronavirus epidemics . SimilarIn the search of the potential best candidate drugs to be repositioned, structural analyses comparing target molecules in the different pathogens should be applied in order to guide a knowledge-based decision process .In the specific case of SARS-CoV-2, and in general in the case of RNA viruses, the most specific target is represented by the RNA-dependent RNA-polymerase (RdRp) which is specific to each RNA virus, regardless the polarity of the viral RNA genome , 11. NevSARS-CoV-2 is a positive-sense RNA virus. The only positive-sense RNA virus, for which a very effective drug targeting specifically the RdRp is available and approved world-wide for clinical use, is hepatitis C virus (HCV). In the specific, Sofosbuvir  is a direct antiviral agent (DAA) that inhibits the hepatitis C NS5B RdRp protein . InteresThe alignment of RdRp sequences from HCV and the three epidemic/pandemic coronaviruses, confirms the high homology and conservation in several residues along the sequence and in particular in the Motif B and C. On the contrary, such homology is almost lost when RdRp sequences from the three epidemic/pandemic coronaviruses are aligned with those from negative-sense RNA viruses, namely Ebola, Influenza, Rabies and Vesicular Stomatitis viruses .The structure modeling shows that RdRp of positive-sense (HCV and SARS-CoV-2) and negative-sense (i.e. Influenza) RNA viruses are significantly different, but they all show the formation of the Motif C β-strand-loop-β-strand structure. However, only the alignment of RdRp structures from the two positive-sense RNA viruses shows a superimposition of the two Motifs C .All these sequence and structural modelling evidences strongly support the concept that the SARS-CoV-2 RdRp is much more similar to the one from HCV than the one from negative-sense Influenza and Ebola RNA viruses. Therefore, repositioning of Sofosbuvir , the inhibitor of the HCV NS5B RdRp protein, as antiviral in the treatment of the SARS-CoV-2 infection has an extremely high potentiality of success, as recently postulated by others , and is Moreover, Sofosbuvir is a prodrug of the protide type and is metabolized to the active antiviral agent GS-461203 (2′-deoxy-2′-α-fluoro-β-C-methyluridine-5′-triphosphate) in the liver. The enzymes involved in such activation are cathepsin A and the histidine triad nucleotide-binding protein 1 (HINT1), and subsequent repeated phosphorylation. All these enzymes have RNA and protein expression in the lung cells comparable to liver cells, ensuring the appropriate activation of the Sofosbuvir to become an effective substrate of the SARS-CoV-2 RdRp in the target cells of the respiratory tract.In conclusion, the data here reported indicate the Sofosbuvir as the lead compound with the highest potential of antiviral efficacy in SARS-CoV-2 infection. In addition, the long-lasting use in patients for treating and eradicating HCV chronic infection provides also the full knowledge of its safety profile . If the


Multivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluate the impact of lower, more prebiotically-relevant, polyion multivalency on the functional performance of coacervates as compartments. Positively and negatively charged homopeptides with 1–100 residues and adenosine mono-, di-, and triphosphate nucleotides are used as model polyions. Polycation/polyanion pairs are tested for coacervation, and resulting membraneless compartments are analyzed for salt resistance, ability to provide a distinct internal microenvironment , and effect on RNA structure formation. We find that coacervates formed by phase separation of the shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes than those formed by longer polyions. Hence, coacervates formed by reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, they can outperform higher molecular weight analogues. Short cationic peptides and nucleotides can form complex coacervates, but the influence of reduced multivalency on coacervate functionality was not investigated. Here, the authors report that coacervates formed from short polyions generate distinct pH microenvironments, accumulate RNA and preserve nucleic acid duplexes more efficiently than their longer counterparts. The presumed scarcity of functional protobiomolecules, such as ribozymes in an RNA World hypothesis3, poses challenges for passive encapsulation by amphiphile self-assemblies such as vesicles, particularly in the absence of binding interactions to help facilitate encapsulation5. An alternative physical mechanism for compartmentalization is suggested by the likely presence of nonfunctional oligomeric or polymeric molecules in greater quantities than the protobiomolecules. Associative interactions between these components could drive phase separation, providing membraneless liquid microcompartments that accumulate protobiomolecules to form a “protocytoplasm”6. For example, a type of associative phase separation called complex coacervation occurs readily in solutions of oppositely charged polyelectrolytes, forming a dense polymer-rich coacervate phase and a dilute continuous phase8. The ion pairing interactions between coacervate-forming polymers are nonspecific and hence versatile and achievable with a wide range of biological and nonbiological chemistries12. Biomolecules such as RNAs can be concentrated within coacervates to orders of magnitude higher than in the external milieu13. These higher local concentrations can provide rate enhancements for catalytic RNAs encapsulated within coacervate droplets15. Coacervates also provide a distinct microenvironment that can differ from the dilute phase in terms of solvent polarity17, concentrations of metal ions such as Mg2+13, or the presence of cofactors such as spermine, which can enhance ribozyme function18. Most studies of coacervation have focused on molecules of high multivalency, to maximize intermolecular interactions19. Prebiotically inspired syntheses of biopolymers such as peptides produce greater quantities of shorter oligomers as compared to longer polymers, with longer peptides often produced in low yields even after many reaction cycles22. These lower-molecular weight oligomers can thus be considered as more prebiotically available compared to longer polymers, but their greater translational entropy reduces their propensity to undergo coacervation and impacts phase composition24. Although complex coacervate formation has been reported from combinations of short cationic peptides (<10 monomers) and nucleotides17, the impact of this reduced multivalency on compartmentalization functions such as accumulating RNAs and providing distinct microenvironments has not been explored. In this work, we investigate the functional consequences of using lower-multivalency polyelectrolytes to produce membraneless compartments. Surprisingly, we find that membraneless compartments formed using the shorter molecules are functionally superior for some properties important for prebiotic compartmentalization.A crucial step in the transition from nonliving prebiotic building blocks to life is the formation of simple protocells from a collection of functional protobiomolecules26, and that other polyions could be relevant prebiotically. Even considering only prebiotic peptides, greater sequence complexity and length polydispersity, as well as mixtures of sequences and chiralities, would be expected. By choosing to use these simpler homopeptides in this study, however, we were able to better isolate the effect of multivalency. Charge-matched (10 mM for each) cationic and anionic components were mixed and the resulting solution classified as containing coacervates, aggregates, or neither  Fig. . We noteher Fig. . A low in or (Arg)n  able to form coacervates with each of the nucleotides. Possible outcomes upon mixing the oppositely charged polyions were uniform solution, coacervate droplets, or solid aggregates , which held for both oligolysine and oligoarginine. (Lys)10 formed coacervates with ATP and ADP, while (Arg)10 formed coacervates with AMP as well 10 can be understood in terms of strong cation-pi interactions possible for Arg-adenosine31.We began by determining the shortest length of (Lys)n or (Arg)n, and anionic peptides, (Asp)n or (Glu)n, as a function of multivalency . The shortest oligomer pairs able to form coacervates under these conditions had at least one component with n = 10, and the other with n ≥ 5. For example, (Lys)10/(Asp)5, (Arg)10/(Asp)5 or (Arg)5/(Asp)10, and (Arg)10/(Glu)5 formed coacervates ≥10 formed coacervates  readily form complex coacervates, and provide us with a small library of coacervate compositions across which we can compare compartmentalization.We then examined coacervate formation between cationic peptides, (Lys)ncy Fig. . Similartes Fig. . All comtes Fig. . For coales Fig. . Taken t32. We evaluated the salt stability of coacervates formed using primarily n = 10 cationic peptides with anions that included nucleotides and both carboxylate oligopeptides 10 counterparts, >600 mM KCl for (Arg)10/ATP but only ~100 mM for (Lys)10/ATP. Similar trends were apparent in coacervates formed with combinations of cationic and anionic peptides of various lengths, although they are less pronounced 10/(Asp)10 is stable to nearly 1.5 M KCl, while (Lys)10/(Asp)10 coacervates dissolve above ~300 mM KCl n/(Asp)n peptide pairs were chosen for pH measurements owing to their ability to form coacervates over a wide range of oligopeptide lengths , likely reflecting the high local ATP concentration (the γ phosphate has a pKA ≈6.8). (Lys)10/(Asp)5 coacervates had an apparent local pH near pH 8.6, ~0.6 pH units higher than the dilute continuous phase, which we interpret as resulting from excess of amine moieties within the coacervate owing to the greater multivalency of (Lys)10 as compared to (Asp)5. The other systems tested here, (Lys)10/(Asp)10, (Lys)30/(Asp)30, and (Lys)100/(Asp)100, had more similar apparent local pH inside and outside the coacervate droplets, with internal apparent pH decreasing as multivalency increased from n = 10–100; the cause of this apparent trend is unclear but could be related to increases in local concentration of both polymers as multivalency is increased, and/or to the presence of proportionately less N- and C-terminal moieties, which have uncoupled pKA values near 8 and 3.4, respectively36. To test the hypothesis that length mismatch and an associated higher local concentration of amine moieties was responsible for the higher apparent pH of the (Lys)10/(Asp)5 coacervates, we also evaluated the (Lys)30/(Asp)5 system. We found that (Lys)30/(Asp)5 coacervates also had a high apparent local pH 100. Fluorescent images are provided in Fig. 10/(Asp)5, (Lys)10/(Asp)10 and (Lys)30/(Asp)30, with each of these systems showing at least 100-fold increase in local RNA concentration, to ~11 µM inside the droplets; KRNA10mer = 160–400, where the partitioning coefficient, K, is the ratio of RNA concentration inside vs outside the coacervate phase. The (Lys)10/ATP coacervates, with the least-multivalent polyanion in our dataset, had even higher local concentration of 10mer ssRNA, ~43 µM and KRNA 10mer = 5.3 × 103. In contrast, coacervates formed with the highest-multivalency polyion pair, (Lys)100/(Asp)100, had essentially no enrichment of RNA locally within the coacervates as compared with the dilute external phase . When fluorescently labeled 10mer ssRNA was added at a final concentration of 0.1 µM to coacervate containing systems, significantly higher fluorescence was observed inside the coacervate droplets as compared to the continuous phase for all systems except (Lys)ase Fig. .Fig. 4Pa10/(Asp)5 and (Lys)10/(Asp)10, with ~50 µM in (Lys)10/(Asp)5 coacervates, a 500-fold increase over the average RNA concentration in the total sample 10 and (Lys)30/(Asp)30, respectively. As with the 10mer, there was no preferential partitioning of the 20mer into the highest-multivalency droplets, (Lys)100/(Asp)100. The partitioning trends for 10- and 20mer ssRNAs are consistent with a competitive displacement RNA accumulation mechanism, whereby the RNA enters the coacervate by displacing (Asp)n or ATP polyanions to interact with (Lys)n. As the multivalency of the anionic components increases, it becomes more difficult for the RNA to compete with them for binding sites on the oligolysine15. By increasing the charge ratio of cationic to anionic groups, RNA accumulation can be encouraged39. Indeed, for (Lys)100/(Asp)100, adding a slight excess of cationic (Lys)100 (1.2: 1 charge ratio), led to ~11 and ~18 µM local concentrations of ssRNA 10mer and 20mer, respectively, inside the coacervates 10/ATP samples appears to be lower than for 10mer ssRNA in these samples 10/RNA phase forms within the (Lys)10/ATP phase39.The concentration of 20mer ssRNA in the coacervate phase of (Lys)les Fig. . However10 with either ATP or (Asp)5, provide strong accumulation of ssRNA oligonucleotides. More generally, these data suggest that accumulation of nucleic acids is actually favored in membraneless compartments that are comprised of short rather than long polyelectrolytes.Together, the results for ssRNA accumulation shown in Fig. 40. Although the chemistry of our coacervates differs from the Ddx4-based systems, we also observe some length- and strandedness-related differences in RNA accumulation across the different coacervates 5 and (Lys)10/(Asp)10. For 10mer RNA, we found that dsRNA showed generally stronger partitioning into the coacervates than ssRNA n/(Asp)n coacervate-forming peptide pairs, with no preferential accumulation seen for (Lys)100/(Asp)100 coacervates . A 3’-Cy3-labeled RNA 10mer sense strand was allowed to hybridize to a 5’-Cy5-labeled antisense strand to produce dsRNA labeled with a proximal Cy3/Cy5 FRET pair 10/(Asp)10, (Lys)30/(Asp)30, and (Lys)100/(Asp)100 coacervate-forming peptide pairs 10/(Asp)10 coacervates, similar to K15M0.5T10 buffer alone, supporting intact duplex. Decreased FRET efficiency was observed for RNA in coacervates formed from the longer peptides, with values dropping by approximately one-half for (Lys)30/(Asp)30 and by two-thirds for (Lys)100/(Asp)100 coacervates, indicating reduced duplex formation in these higher-multivalency coacervates. Thus, whether coacervate-encapsulated RNA duplexes are disrupted or retained depends on the oligopeptide multivalency, with helicase-like activity, similar to that reported for Ddx4 coacervates, observed only for coacervates formed from polyions having n≥3040. The ability of the lower-multivalency polyions to support RNA base pairing could be beneficial in prebiotic contexts, where nucleic acid sequence recognition and folding could give rise to function. In principle, some destabilization of RNA base pairing in prebiotic compartments could also prove useful by aiding release after replication and/or allowing misfolded RNAs to refold into functional forms, which could become important as encapsulated RNA lengths increase. In the context of prebiotic compartmentalization, it could be speculated that as longer polyions became more available in prebiotic milieu, the resulting shift in composition of coacervate-based compartments from lower- to higher- multivalency  would increase the propensity of these compartments to disrupt RNA duplexes, eventually becoming more similar to the behavior of Ddx4-rich protein droplets of extant biology40.Differences in preferential accumulation of ss- versus dsRNAs observed in Fig. irs Fig. . We obse43. We evaluated the impact of compartmentalization inside coacervates on the folding of a tRNAphe transcript, which has extensive secondary structure and a well-defined, compact tertiary structure44. In-line probing (ILP)45, which measures the flexibility of each nucleotide by its ability to undergo hydrolysis, was used to compare the structure of tRNAphe in different coacervates to its native fold in polyion-free controls n), which could limit Mg2+ availability.RNAs rely on complex secondary and tertiary structures for functionols Fig. . Unlike 10/ATP coacervates, which have the lowest multivalency (Supplementary Table 100/(Asp)100 coacervates. We next performed control reactions with polycations-only and polyanions-only. This is found in Supplementary Fig. 100 leads to more unfolding than (Lys)10 or (Lys)30  for all coacervates tested, with strongest accumulation (99%) for (Lys)10-ATP and (Lys)n/(Asp)n coacervates 100-only control to (Lys)100/(Asp)100, which had higher variance , indicating that the fold of the RNA, which maintains much of the secondary structure, is similar in all coacervates 10/(Asp)5, and (Lys)10/(Asp)10 there are statistically significant increases in reactivity in several parts of the RNA: the variable loop and the TΨC stem and its loop oop Fig. . Many ofoop Fig. . Moreoveoop Fig. . Indeed,oop Fig. . We note49. The spontaneous occurrence of coacervates in diverse macromolecular systems, and their ability to accumulate solutes such as RNA, suggests possible roles in prebiotic compartmentalization and protocell formation6. Since lower-molecular weight20, and hence lower multivalency, oligomers are likely more relevant to prebiotic scenarios than larger macromolecules20, we considered whether such coacervates could offer sufficiently distinct microenvironments to serve as primitive compartments for RNAs. Such a prebiotic compartment should minimally accumulate RNAs from the environment, and provide a suitable milieu for their function. Examples of RNA functions could include base pairing and catalysis, which requires proper folding. In addition, the elevated apparent local pH could enhance the rate of certain ribozymes”50. We find that for (Lys)n/(Asp)n, coacervates formed by low-multivalency components  can match or exceed performance of those formed by higher-multivalency counterparts in all areas tested here except salt resistance32. Notably, the lower-multivalency (Lys)n/(Asp)n coacervates (n < 30) were more effective at providing distinct pH and accumulating RNA than those with higher n. (Lys)10/ATP, in which the anion is a nucleotide rather than an oligopeptide, also formed coacervates able to concentrate RNAs and support their duplex formation.Associative phase separation is an organizational mechanism in extant cells, leading to membraneless organelles enriched in proteins and nucleic acids with distinct biological functions19. However, since the mechanism of RNA accumulation into complex coacervates relies upon its interaction with coacervate polycations, in competition with the polyanions that make up the coacervates, superior compartmentalization capabilities can be understood for shorter n. The more favorable RNA duplex formation observed here via FRET in coacervates having lower-n (Lys)n is consistent with inhibition of duplex formation when RNA-polycation interactions become too strong due to greater polycation multivalency. The impact of RNA-polycation interactions in coacervates depends not only on the coacervate composition but also on the RNA, as we see in the case of the tRNAphe, which was unable to form native tertiary structure at the destabilizing low Mg2+ conditions of these studies for any of the coacervates tested. Together, the duplex FRET and tRNA ILP results highlight the importance of RNA length and stability in dictating the impact of coacervate microenvironments. The tRNA folds into a series of hairpins, which are unimolecular secondary structures, while the duplexes are bimolecular secondary structures; reformation of such structures may be limited in the presence of the polyions that comprise the coacervate. These observations should inspire further investigation of coacervate- and RNA-specific impacts on RNA folding in these membraneless compartments. Such findings are also of interest in light of the central role of RNA in the membraneless organelles of extant biology48.The compartmentalization capabilities of these lower-n coacervates are counterintuitive in light of the known impact of polyion repeat number on phase behavior, which would predict shorter tie-line lengths, and correspondingly less-pronounced phase composition differences, for the shorter oligomers as compared to the longer polymersn/(Asp)n pair, since the major underlying mechanism both for coacervate formation and for interactions with the polyanionic RNA rely on ion pairing. As such, we anticipate that other polycation/polyanion combinations could similarly benefit from lower multivalency in their accumulation of RNA and their ability to support its base pairing. The experiments here were mostly performed at equal cation:anionic moiety charge ratio and equal polycation and polyanion oligomer length; by varying the charge ratio and/or polyion repeat length, additional factors come into play. For example, RNA loadings can be increased by reducing the relative amount of polyanions, as has been reported previously15 and can be seen for (Lys)100/(Asp)100 by comparing Fig. 9. For systems where chemical interactions beyond ion pairing, such as cation-pi bonding, are important, more study will be needed.We expect that our observations for the effect of n on RNA compartmentalization by complex coacervates will not be unique to the (Lys)The observation that it is possible for lower-multivalency, lower-molecular weight oligomers to not only function as compartments for RNAs, but to in fact function better than otherwise identical systems assembled from longer polymers, is noteworthy. This could have profound implications for early Earth, when functional protobiomolecules would have been scarce amongst a complex milieu of molecular components, many of which may have had similar chemistries. In such a scenario, the compartments formed by these relatively low-multivalency polyions could have accumulated longer, potentially functional protobiomolecules  to relatively high local concentrations.2+; actual ionic strength also includes ~10 mM NaCl as counterions from the polyions. Turbidity was calculated using UV-Vis absorbance at 500 nm measured by Tecan M1000 Pro microplate reader. Turbidity alone cannot discriminate between aggregates and coacervates solutions51. Therefore, samples were also imaged with a Nikon Eclipse TE200 inverted optical microscope to test the presence of coacervate droplets. Each experiment was repeated at least three times. We note that it would very likely be possible to form coacervates from polycation/polyanion pairs that produced aggregates by increasing the solution ionic strength53; we did not do this here because we wished to hold the solution conditions constant so as to compare the properties of coacervates formed from different length polyions.Coacervate samples were prepared in charge concentration ratios. Except where otherwise noted, all experiments used a charge concentration of 10 mM (charge of the molecule × its concentration = charge concentration) for each component (ratio of 1:1 cationic to anionic moieties) with a total volume of 100 μL in HPLC grade water and 10 mM Tris (pH 8), 15 mM KCl and 0.5 mM MgThe pH of coacervate systems (droplet and continuous phase) was estimated using C-SNARF-1 dye emission at 543 nm excitation using confocal microscopy. The pH of the dilute phase was measured by micro pH electrode after centrifugation. Salt concentrations of samples were adjusted using concentrated KCl to the desired salt concentrations. Detailed information can be found in the Supplementary Information.Coacervate samples were prepared according to the coacervate preparation section except the same volume of water was replaced with added volume of labeled RNA(ACCUUGUUCC[Cy3] or AUCUCGCUCUACCUUGUUCC [Cy3]), which was added last, to 0.1 μM final concentration. For dsRNA experiments, equimolar unlabeled complementary RNA sequence , also without the Cy3 and Cy5 fluorescent labels . Values in Supplementary Table Coacervates were prepared by first adding water, then 10 mM Tris (pH 8.3), 15 mM KCl, 0.5 mM MgCl15M0.5T10 buffer-only reactions, 6 kcpm/µL of 5’-radiolabled tRNAphe was incubated in 10 mM Tris (pH 8.3) 15 mM KCl, and 0.5 mM MgCl2 at 37 °C for up to 48 h. For the polycation-only reactions, tRNAphe was incubated in the above conditions but with 10 mM total (+) charge for each of the polycations. For the polyanion-only reactions, tRNAphe was incubated in the above conditions but with 10 mM total (–) charge for each of the polyanions. For coacervate reactions, tRNAphe was incubated with the above conditions with a 1:1 ratio of + charge to – charge at 10 mM + charge and 10 mM – charge. Reactions were fractionated on 10% denaturing urea polyacrylamide gels at 60 W for 1.5 h before being dried at 70 °C for 1 h. Gels were then exposed on PhosporImager plates overnight before being imaged on a Typhoon scanner. Gels were then quantified using Semi-Automatic Footprinting Software (SAFA)54, normalized to the average intensity of nucleotides 34–36 for each lane, then statistical analysis and graphing were done with GraphPad Prism 8.For the KFurther information on research design is available in the Supplementary InformationReporting Summary


University students are one of the most vulnerable groups to sexual reproductive health [SRH] threats like sexually transmitted infections [STIs], unwanted pregnancies, and unsafe abortions and often have limited access to SRH information, goods, and services. This study assessed the effectiveness of using a mobile phone application (APP) to increase access to SRH information, goods, and services among university students in Uganda.Using data from a double-blinded randomized controlled trial, participants were randomly assigned to both the intervention (APP) and control (standard of care) arms. We executed descriptive analyses for baseline demographic characteristics by intervention, difference in difference (DID), and quantile regression analyses for both primary and secondary outcomes.P < 0.001), contraceptive use , HIV Voluntary testing and counselling , STI diagnosis and treatment , and condom use at last sex  among students who used the APP. There was a significant 0.98 unit increase in knowledge score , a significant 1.6-fold increase in odds of contraceptive use , a significant 3.5-fold increase in HIV VCT , and a significant 2-fold increase in odds of STI testing and treatment  after adjusting for demographic characteristics among APP users compared to the control group.The median age of participants was 21 years of age, and the majority were female (over 60%), unemployed (over 85%) and Christian (90%). Over 50% were resident in off-campus hostels and in a relationship. Between baseline and end-line, there was a significant increase in SRH knowledge score , access to goods (contraceptives), and services  among sexually active university students in Uganda. Further technical development, including the refinement of youth-friendly attributes, extending access to the app with other platforms besides android which was pilot tested, as well as further research into potential economic impact and paths to sustainability, is needed before the app is deployed to the general youth population in Uganda and other low-income settings.MUREC1/7 No. 07/05–18. Registered on June 29, 2018. Many of the low-income countries of sub-Saharan Africa have predominantly young populations. Uganda is one such country with 78% of the general population below 30 years of age and adolescents aged 15–19 years, accounting for a quarter of the female population . This meNearly all low-income sub-Saharan African countries have also undergone a rapid increase in mobile and broadband internet coverage; the region is projected to have 1 billion sim connections and 500 million broadband connections by 2020 . AdditioThe confluence of SRH needs for a predominantly young population and high and increasing access to mobile internet presents a unique opportunity for the development of youth-friendly, innovative digital health intervention (DHIs), particularly interventions in mobile health . A review of mHealth projects in Uganda identified over 20 applications, including use for health worker and patient training, healthcare delivery, treatment and patient management support, and health facility improvement . These iOne area of potential growth in mHealth is in the use of mobile phone apps (MPAs) to increase access to health services in general and SRH information, goods, and services, in particular. DHIs, particularly mHealth, are associated with key youth-friendly attributes, including confidentiality, convenience, and entertainment/information value. The leap from text messaging-based mHealth to internet-based MPAs maintains the confidentiality and convenience attributes and magnifies the information and entertainment value of mHealth interventions. It also allows the development of multiple platforms and connections that expand their utility from information-only use to use for connecting to goods and services. This enables complex, multifaceted interventions with multiple potential avenues of impact and a wide variety of client or consumer choice. Additionally, entertainment value and appeal portend the ability to advertise, a key determinant of market success and potential sustainability at scale of a commercial MPA.As part of a broader project conducted over an 18-month period, we developed and pilot-tested a MPA to increase access to SRH information, goods, and services in a population of university students in Kampala’s capital, Uganda . A detaiGiven satisfactory acceptability and utilization estimates, we hypothesized that access to the MPA would lead to an increase in SRH knowledge and lead to an increase in access to SRH goods and services among university students.MUREC1/7 No. 07/05–18 on 29th June 2018.The study was a randomized controlled trial in which access to SRH information, goods, and services using a MPA (MPA-SRH) was compared to the standard of care of access to SRH information, goods, and services (SOC-SRH). A protocol of the trial, which was a part of a broader app development and impact evaluation project, has been published previously . The triThe trial included students of Kyambogo University (KYU), the second-largest university in Uganda, which is situated in the capital city, Kampala. Eligibility criteria for inclusion into the trial were: (1) age 18 to 30 years, (2) self-reported sexual activity in the last six months, (3) more than 12 months to graduation, (4) access to an internet-enabled Android smartphone, and (5) informed consent. Trial participants were recruited in KYU halls of residence and KYU-affiliated hostels. Data were collected by in-person interviews in students’ rooms.https://beyonic.com/), a payments company which managed payments and co-payments; SafeBoda (https://safeboda.com/ug/), a transport company which managed the transportation of ordered goods and app users to receive services; and GHE Consulting (https://www.gheuganda.com/) for coordination, management, and oversight.In the intervention group, trial participants were granted access to a MPA to enable their access to SRH information, goods, and services over a period of six months. Details of the development and features of the app are described elsewhere . Briefly, the MPA was developed in partnership with Gershom Technologies Ltd. using the Android operating system and archived on the Google Play store. The MPA was designed to link the different goods and services providers: healthcare facilities which provided SRH goods and services; Beyonic Uganda (The MPA included the following features: (1) sign-up and sign-in; (2) a user module for ordering SRH goods  and services (HIV voluntary testing and counseling (VTC), STI diagnosis and treatment (D&T), family planning counseling, and general SRH consultation); (3) an SRH information module , SRH tips, and a live chat); (4) a payments module to enable provider payments by GHE Consulting, copays by clients, and payments for transportation; (5) a delivery module to enable clients to track shipments, set up pickups for in-facility visits, and set up pick up points for products; and (6) a security module for authentication and password protection. Embedded in the MPA was an advertising interface to test potential sustainability at the scale of in-MPA advertising. While the study provided a major subsidy for SRH goods and services as well as transportation, there was a modest co-pay that was paid by app users through a link between mobile money and the Beyonic payment system. The co-pay was designed to test the utility of client payments in potential future iterations of the MPA after the pilot period.Intervention group participants received a text message with a link to download the MPA, including directions on the set up of a MPA account and instructions on the use of the MPA. Upon download of the MPA, participants could gain access to SRH information through the four in-app portals. Participants could order SRH goods and have them delivered to their rooms or to a designated spot for pick up. Participants could also connect with health service providers to book SRH services and connect with transport providers to organize transportation to and from healthcare facilities. Participants in the SOC-SRH group received no intervention, i.e., accessed SRH information, goods, and services as they did before the onset of the trial.There were four primary outcomes in the trial all reflecting changes from baseline to end-line (end of six months follow-up period): SRH knowledge score (SRH information), use of contraceptives (SRH goods), use of HIV VTC (SRH services), and use of STI D&T (SRH services). There were two secondary outcomes in the trial, both reflecting changes from baseline to end-line: use of condoms and use of alcohol during the last sexual encounter .The sample size was calculated to detect a change between baseline and end-line in use of modern contraceptive goods. The prevalence of modern contraceptive use among sexually active youth aged 18 to 30 years at the time of study planning was 30.2%. Using the formula for the use of the Z-test for two sample proportions in studies with behavioral components, we calculated a sample size of 435 participants per group in order to obtain 90% power to detect a 15% change in modern contraception use in this population at a two-sided significance level of 5%. The sample size calculation assumed no design effect (d = 1) and was adjusted for a 10% non-response rate to obtain a minimum sample size of 479 per group .After recruitment and informed consent, participants were informed that there was a 50–50 chance that they would be randomly assigned to gain access to the MPA (MPA-SRH) or be a part of the control group (SOC-SRH). The participants’ telephone numbers and unique study identification numbers were given to the app developer, who was responsible for randomization. Participants were randomized 1:1 to MPA-SRH and SOC-SRH using computer-generated random numbers. The research team, including providers at health facilities, transport providers, and payment technicians, and participants were blind to the intervention group, but the app developer was not.Data were double-entered and cross-validated, and all analyses were conducted in Stata version 16 . We compared outcomes between participants randomly assigned to MPA-SRH versus SOC-SRH, ignoring potential changes in access to SRH information, goods, and services in the SOC-SRH group that may have been triggered by the use of the app by participants in the MPA-SRH group.p-values) using differences-in-differences. We used the Wilcoxon rank-sum test for the knowledge score and the Z-test for proportions.SRH knowledge scores were summed from correct responses from 17 different attributes and ranged from 0 to 17 (each question was awarded 1 point). Contraceptive use, HIV VTC, STI D&T, condom use during last sexual encounter, and alcohol use during the last sexual encounter were binary variables. We estimated the difference in changes from baseline to end-line  and Using quantile regression (knowledge score) and a logit link function , we estimated a mean differences-in-differences effect, adjusting for demographic characteristics. Models were estimated using maximum likelihood.We enrolled participants from Oct 23rd, 2018, to 13th November 2018, and the last participant completed the six-month follow-up period on 25th May 2019. We enrolled (and assessed for eligibility) 1180 students and randomized 1112 students, 556 to the MPA-SRH , and 556 to the SOC-SRH (control arm). Of the participants randomized, 846 (76.1%) completed the end-line survey, 432 (76.1%) in the APP-SRH arm, and 414 (74.5%) in the APP-SOC arm, constituting the sample for the analysis , or n (%). APP = mobile phone application. SRH = sexual and reproductive health. SOC = standard of care.Between baseline and end-line, there was a significant increase in SRH knowledge score, contraceptive use, HIV VTC, STI D &T, and condom use at last sex among APP users compared to the SOC-SRH Table . There wWe assessed the effectiveness of a MPA to increase access to SRH information , goods (measured as the proportion of participants using modern contraception), and services (a proportion that used HIV VCT and STI D&T) in a population of university students in Uganda. The MPA had a statistically significant benefit in increased SRH knowledge and increased access to SRH goods and services. MPAs are increasingly used in low- and middle-income countries for a wide range of applications, although applications in this setting are far exceeded by applications in high-income settings . In sub-Internet-enabled mobile phones and MPAs, including social media platforms, are popular among the youth around the world. In some settings, access to MPAs is associated with negative behavioral outcomes: as an example, a study in South Africa found increased odds of multiple sexual partners in the last year and higher prevalence of hazardous alcohol use with access to social media and messaging apps . The conResearch has established that the youth in low-income settings have significant SRH needs and face barriers to access to SRH goods and services, including physical access, legal and societal proscriptions, and societal barriers , 21. TheOne weakness of the study was that the length of access to most components of the intervention  were available for a short time (six months). As such, we are blind to the presence or absence of a sustained effect of increased knowledge or increase access to SRH goods and services on account of access to the MPA. Additionally, the trial was conducted among university students who are not generalizable to the general youth population in Uganda or other low-income countries. In fact, access to SRH information, goods, and services was high at baseline . This is compared to a general population contraceptive use prevalence of 29% among women of reproductive age  or 47% aThe demonstration of the effectiveness of the MPA to increase access to SRH information, goods, and services, along with lessons learned in the pilot implementation and impact evaluation is a necessary precursor to the further development of the MPA with the goal of developing a sustainable product that can be rolled out to the general public. Before this goal is realized, further studies of the cost-effectiveness of the MPA and its potential economic impact at scale are needed, as are studies on the perception of potential advertisers, key partners in a possible future product, on the potential of the MPA to drive revenue.A mobile phone application increased sexual and reproductive health information (knowledge score), access to goods (contraceptives), and services  among sexually active university students in Uganda. Further technical development, including the refinement of youth-friendly attributes, as well as further research into potential economic impact and paths to sustainability, is needed before the app is deployed to the general youth population in Uganda and other low-income settings. This will enable uniform access to the app by allowing users from other platforms other than the android platform, which was pilot tested.