Both strands were then combined to generate permutations and combinations of cm-siRNAs. Nature Biotechnology 23: 1399-1405. However, structure based search tool (MarvinSketch) needs java to be enabled on the browser. "Mechanisms of gene silencing by double-stranded RNA". Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Furthermore, interrogated analyses of siRNA screening efficiency, experimental data and broadly conserved miRNA sequences showed that the free energy of transitional nucleation (positions 2-5) in siRNA-6 required the range of stability for effective on-target activity (-6 G [2:5] -3.5 kcal mol-1). Transfection methods used on different cell lines mainly utilized lipid based reagents viz lipofectamine and Interfer (Polypus-Transfection). Pie chart depicting their distribution is presented in supplementary Fig. RNAcentral is a comprehensive database of non-coding RNA sequences that represents all types of ncRNA from a broad range of organisms. Ambion will synthesize a complementary pair of siRNA oligonucleotides according to your sequence. PMID: 14755052. Sci. Ribose 2-OH and phosphorothioate modification increases stability of siRNA19,20. Access the siRNA sequences deposited at the NCBI PubChem Substance Database by searching for a particular siRNA Assay ID. Further effect on enhanced efficacy due to chemical modifications was tested using different siRNAs concentrations49. . Vector & Virus Western Blot Recent Posts DNA Microarray Mode of DNA replication Transcription control in Bacteria Complementarity based sense and antisense distribution of chemical modifications is shown in Fig. The archive accepts data from all branches of life as well as metagenomic and environmental surveys. By displaying limited experimental information and providing a link to the source article, we provide the user with resources to evaluate the quality of the siRNA. [1], Andrew Fire, 2006 Nobel Laureate (Physiology/Medicine). To provide a useful and unique reference resource for biology and medicine, we developed the scRNASeqDB database, which contains 36 human single cell gene expression data sets collected from Gene Expression Omnibus (GEO), involving 8910 cells from 174 cell groups. RNA silencing pathway utilizes dicer to process long double stranded RNA (dsRNA) to 1921 nucleotide small interfering RNAs (siRNAs) with 2-nucleotide unphosphorylated 3 overhangs2. We would like to thank Michael McManus and Olga Matveeva for access to their siRNA datasets. 1. Large-scale RNA interference (RNAi) experiments, especially the ones based on short-interfering RNA (siRNA) technology . Modification in this component can contribute towards better understanding of off-target effects as well as gene silencing mechanism27. Amount: 1 - 10 mg, 50 mg - 1 g Purification: Desalt, RPC (cartridge purified), HPLC and In vivo quality Sequence Length: 19 - 45 bases Comprehensive offering of modifications including dyes, amines, biotin, linkers, etc. InvivoGen provides a set of cutting-edge tools to knockdown efficiently the expression of mammalian genes. examined target degradation efficacy of small molecules conjugated to siRNAs52. Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5-terminal phosphorylation both in vitro and in vivo. So, there is further scope to explore these combinations. They are duplexes of two RNA molecules, typically 21mers with a 2 nt 3 overhang ( 1 ). (, Chalk,A.M., Wahlestedt,C. & Burrows, C. J. (, Yuan,B., Latek,R., Hossbach,M., Tuschl,T., Lewitter,F., Naito,Y., Yamada,T., Ui-Tei,K., Morishita,S., Saigo,K. Biodistribution of phosphodiester and phosphorothioate siRNA. Knockout studies in these species support the notion that there is functional redundancy amongst these DCL paralogues. It elucidates that maximum 5 different combinations of modifications are seen so far in cm-siRNAs. PMID: 15956980. To avoid off target effect, choose miRNA SEED database corresponding to the target gene. Wang, Y. et al. HIF1A(hypoxia-inducible factor 1, alpha subunit) Target gene: HIF1A ( NM_001530) Probe type: siRNA. Herraez, A. Biomolecules in the computer: Jmol to the rescue. doi:10.1126/science.286.5441.950. This analysis revealed a strong preference for G or C nucleotides at position 1. & Tiemann, K. Current progress of siRNA/shRNA therapeutics in clinical trials. As the main three types of RNA in us are single-stranded. To design a siRNA, an mRNA or cDNA sequence of the gene of interest is required and can be retrieved from the NCBI database. Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. PMID 15372045. http://www.nature.com/nature/journal/v431/n7006/full/nature02870.html. The dimeric Dicer complex then cleaves the dsRNA into ~21-28 nucleotide siRNA duplexes containing 2-nucleotide 3' overhangs with 5' phosphate and 3' hydroxyl termini, which are bound by the Dicer PAZ (Piwi Argonaute Zwille) domain. The off-target searching pipeline starts with splitting a long RNAi trigger sequence (the complement to the target sequence of the corresponding RNA) into all possible x-mers, where x is the selected length of the siRNA. Rep. 6, 20031; doi: 10.1038/srep20031 (2016). The program will ask for the accession number of the mRNA, and extract the sequence information from NCBI database. siRNAs were selected only if the siSearch ( 6 ) score exceeded 5, the Reynolds ( 10 ) score exceeded 5, and the Ui-tei ( 11 ) score was Ia or Ib. has no similarity to other human genes and shared, i.e. employed walkthrough method13,32, which replaces single or multiple nucleotides in siRNA with different modifications sequentially from one end to another and checking their biological impact. Nucleic acids research 38, 660671, 10.1093/nar/gkp913 (2010). The energy data displayed includes the start and end energies, representing the strength of binding at each end of the siRNA. Annals of Neurology 57: 773-776. Journal of the American Chemical Society 133, 1676616769, 10.1021/ja2068774 (2011). CAS 2. RNABase is an annotated database of all publicly available RNA structures. The central table in the database is called siRNA , and contains information about the siRNA such as sense and antisense sequences, overhangs and target sequence. Reproduction of any materials from this site is strictly forbidden without permission for commercial use. It covers rest of the relevant information as (xiii) target gene; (xiv) melting temperature of modified siRNA sequence; (xv) siRNA concentration; (xvi) experiment used to check activity; (xvii) cell line or organism; (xviii) transfection method; (xix) post transfection duration; (xx) reference. MycoStrip is a simple and rapid test based on isothermal PCR. Locked nucleic acid, 2-O-methyl, 2-fluoro moieties thermodynamically stabilize siRNA ends while unlocked nucleic acid, phosphorothioate, ethylamino, dihydrouracil modifications destabilize them46. siRNAmod: A database of experimentally validated chemically modified siRNAs. 9. S.D. Base modifications have been used in various studies e.g. Therefore, we developed siRNAmod that will serve as the central repository of the experimental records of chemical modification of siRNAs. Bramsen, J. siRNA sequence begins with AA (allows use of dTdT at 3'-end of the antisense sequence).This reduces the cost of synthesis and renders the siRNA duplex more resistant to exonuclease activity. and Tuschl,T. It incorporates important information on siRNA sequence, chemical modification, their number and respective position, structure, simplified molecular input line entry system canonical (SMILES), efficacy of modified siRNA, target gene, cell line, experimental methods, reference etc. "Gene discovery by ribozyme and siRNA libraries". viruses), and the maintenance of genome integrity via transcriptional silencing of undesired genomic loci (e.g. Carousel with three slides shown at a time. Google Scholar. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. Modified nucleoside phosphoramidites are used in solid phase chemical synthesis of siRNA which allow site-specific incorporation of chemically modified nucleotide moiety at specific positions within the siRNA9. As discussed, next generation siRNA technology will be defined by the chemistry of modifications on siRNAs to greater extent. PMCID: PMC1693862. Database front end is developed using Perl, PHP, JavaScript HTML and CSS. Also for naturally occurring modified RNA, there is a database namely RNAMDB39. Petersen, M. & Wengel, J. LNA: a versatile tool for therapeutics and genomics. In the literature, synthetic siRNA constructs are generally denoted by "gene name" - siRNA (e.g. "A pathogen-inducible endogenous siRNA in plant immunity". siRNAmod is freely available at: http://crdd.osdd.net/servers/sirnamod. on what positions the modifications are reported along with their biological effects. In future, we would update the database and include cm-siRNA based design tools and algorithm. MMIA -- MicroRNA and mRNA Integrated Analysis Biotechnology journal 6, 11301146, 10.1002/biot.201100054 (2011). Google Scholar. When exact figures for efficacy are unavailable we approximate the value; these values are marked with a type (validated, predicted, approximated or generalized) to indicate the method used to determine the efficacy value. PubMed The strand that is retained as a functional component of the RNA Induced Silencing Complex (RISC) is referred to as the guide strand, whereas the other strand, which will be rapidly degraded by exonucleases, is known as the passenger strand. HuSiDathe human siRNA database: an open-access database for published functional siRNA sequences and technical details of efficient transfer into recipient cells. Search for other works by this author on: Thank you for submitting a comment on this article. Identification and Prioritization of PET Neuroimaging Targets for Microglial Phenotypes Associated with Microglial Activity in Alzheimer's Disease. Small interfering RNA(siRNA), sometimes known as short interfering RNAor silencing RNA, is a class of double-stranded RNAat first non-coding RNAmolecules, typically 20-24 (normally 21) base pairsin length, similar to miRNA, and operating within the RNA interference(RNAi) pathway. S5. Nucleic acids research 32, 59916000, 10.1093/nar/gkh936 (2004). The aligned query positions in a database hit have at least the same positions selected by a user This hit's sequence identifier from NCBI GeneID or Ensembl gene ID is different from that of the user's target gene. 3)ASPsiMap:To map user given gene sequence on ASP-siRNAs in ASPsiDB. We first analyzed the frequencies of concurrencies of G or C nucleotides at specific positions of the 19 bp siRNA core of the 603 active siRNA sequences listed in our database ( Figure 1). The database was designed to hold results from a number of different prediction methods, and we invite siRNA prediction groups to submit their predictions to the database. As indicated from the name, exact option is the strict search mode, which finds the exact term that user provides while containing is lenient search option and provides wider results. As more siRNAs are verified this database will become increasingly useful for developing siRNA design tools. Nucleic acids research 39, D195-201, 10.1093/nar/gkq1028 (2011). Sequence and phylogenetic analysis of LmDicer-1, LmDicer-2a and LmDicer-2b. - Dorsett Y, Tuschl T (2004). PMID 9486653. What is best database for siRNA sequences? "RNA interference". Cantara, W. A. et al. We have provided two tools in ASPsiDb to help user analyze their sequence. [13], Small interfering RNA (siRNA) is also known as short interfering RNA or silencing RNA. Pontes O, Li C, Nunes P, Haag J, Ream T, Vitins A, Jacobsen S, Pikaard C (2008). Step by step instructions for searching database and its output are displayed using screen shots in How to use menu on the web server. & Alexander, K. A. RNA interference using boranophosphate siRNAs: structure-activity relationships. S3 and S4. Some members of the siRNA family include trans-acting small interfering RNAs (tasiRNAs), repeat-associated small interfering RNAs (rasiRNAs), scan RNAs (scnRNAs) and long siRNAs (lsiRNAs). Base numbering in siRNAs is referred to by the sense strand, with the first 5' nucleotide on the sense strand corresponding to the 19 position on the antisense strand. Furthermore, structure search option is also available to draw the structure and search against the database. Peel, B. J., Hagen, G., Krishnamurthy, K. & Desaulniers, J. P. Conjugation and Evaluation of Small Hydrophobic Molecules to Triazole-Linked siRNAs. PubMed [15], 2021 The Regents of The University of California. The database architecture is shown in Fig. PMID: 12778125. Different experimental methods like luciferase assay, RT-PCR, northern blotting etc. RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. Search by Search Term [Select approriate ID] Unique sequence length 5. miRBase: the microRNA database. & Gait, M. J. MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity. The output includes siRNATarget.set.out : list of the siRNA target sequences Standard user-friendly browse, search facility and analysis tools are also integrated. and Sonnhammer,E.L. Enhancement of stability and activity of siRNA by terminal substitution with serinol nucleic acid (SNA). In the protozoan Tetrahymena thermophila, the siRNA family member scnRNA is involved in DNA elimination of genomic loci in the micronuclei during conjugation and macronuclear formation. Funding for open access charge: CSIR-Institute of Microbial Technology, Sector 39-A, Chandigarh, India. Thakur, N., Qureshi, A. Detzer, A., Overhoff, M., Mescalchin, A., Rompf, M. & Sczakiel, G. Phosphorothioate-stimulated cellular uptake of siRNA: a cell culture model for mechanistic studies. Indeed, an endogenous siRNA induced by the presence of the pathogen Pseudomonas syringae in plants indicates that there may be a conserved role for siRNAs in immune responses. UNA are open ring derivatives of ribose without C-2 and C-3 carbon-carbon bond but structurally mimic the RNA when incorporated into duplex18. Background Discovery and Components of the RNAi pathway conceived the idea, planned the design, visualization and tools pipeline and coordinated the entire project. Braasch, D. A. et al. Further, structure of the chemical modification can be viewed by clicking on the SMILES link as provided (Fig. In addition to the local siRNA Database, submissions to the MIT/ICBP siRNA Database are added to the NCBI's Probes Database. Advanced drug delivery reviews, 10.1016/j.addr.2015.01.007 (2015). Article Various comprehensive studies in this field have been conducted which employed new strategies and methods. & Kool, E. T. Steric effects in RNA interference: probing the influence of nucleobase size and shape. Reason for this could be the toxicity associated with increased modifications57. The first curve shows di-nucleotide binding energy values, as calculated using the method of Mathews et al . The knockdown approach, unlike a knockout, allows detailed study of the effects of reducing a gene's expression to none for a period of time, and then allowing its expression to return to normal. The database interface The database interface was designed with the experimental user group as the primary target audience. performed data analysis, interpreted results and drafted the manuscript. For experimentation, both in vivo and in vitro systems were reported in the literature. Each siRNA can have multiple experiments attached to them, as several experiments can be performed using the same siRNA sequence. Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. Learn more about InvivoGen's selective antibiotics. To obtain Cartoon representation of RNAi mechanism, siRNA molecular structure and its entry in RNAi pathway. & Flanagan, W. M. In vivo activity and duration of short interfering RNAs containing a synthetic 5-phosphate. Literature was exhaustively searched for experimentally validated chemically modified-siRNAs (cm-siRNAs) for extraction of relevant information using keywords related to (i) RNAi (ii) modifications (iii) stability, utilizing advanced search option in PubMed. (Spaces and numbers will be ignored) 3. Synthesis of 2-O-modified adenosine building blocks and application for RNA interference. Nature 431: 343-349. (, Hammond,S.M., Caudy,A.A. doi:10.1038/35888. 469 siRNA suggested by siDirect 2.0 which were tested for whether they follow URA rules. ( 10), Reynolds et al. "The siRNA or small interfering RNA is a 22 to 25 base pair long smaller molecules of dsRNA having a dinucleotide overhang at the 3' end, interfere in the protein synthesis by blocking the translation." The presence of double-stranded RNA in a cell is a sign of danger. 10. Detailed descriptions of these types are available online. Molecular bioSystems 3, 4350, 10.1039/b611612d (2007). A database sequence does not necessarily generate consecutive numbers. Experimentalists wish to easily be able to find siRNAs that have already been verified for their target gene. Marques J, Williams B (2005). Other database search parameters included 20 ppm and 0.5 Da mass tolerances for precursor and product ions, respectively. You are using a browser version with limited support for CSS. ChemMedChem 9, 21112119, 10.1002/cmdc.201402115 (2014). the sequence of each siRNA strand (This option is recommended if you wish your siRNA to have 3' termini other than dTdT or UU.) Intensities for all . Chemical modifications on sugar moiety e.g. and A.Q. of the family on the primary protein sequence. This database can be updated using a password protected input form that accepts the data and images for new siRNA designs and uploads them to the database. Similarly, other groups also studied terminal modifications like 5-phosphate53 or serinol modification54. As part of our rigorous quality control procedures, each . Sequences designed and tested by MIT researchers, Sequences designed by Qiagen; tested by Natasha Caplens group at the NCI (, Sequences designed by Greg Hannon and Steve Elledge; tested by the ICBP and CGAP programs at the NCI (. Maria J. Donde, Adam M. Rochussen, Alexander I. Taylor, Luigi DAscenzo, Anna M. Popova, James R. Williamson, D. Dewran Kocak, Eric A. Josephs, Charles A. Gersbach, Josh Tycko, Luis A. Barrera, Patrick D. Hsu, Jong Woo Bae, S. Chul Kwon, Jong-Seo Kim, Alexander L. Satz, Andreas Brunschweiger, Jrg Scheuermann, Michael Eisenstein, Ken Garber, Laura DeFrancesco, Mattias Bood, Anna Wypijewska del Nogal, Morten Grtli, Scientific Reports For instance phosphorothioate enhances the stability, uptake and biodistribition of the siRNA30,31. A new software/database tool, the RNAiAtlas, has been created for exploration, analysis and distribution of large scale RNAi libraries with their latest annotation but in addition it contains also specific on-target analysis results (design quality, side effects, off-targets). Collingwood, M. A. et al. Overall statistical trend of chemical modifications depicts that terminal positions 1, 2, 3 and 20, 21 are highly altered comparative to central portion as shown in supplementary Fig. Within the TAS1 family, TAS1a, TAS1b and TAS1c are very similar in sequence and all produce the siR255 ta-siRNA among others ( Allen, E. et al ). The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Sipa, K. et al. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, Dar, S., Thakur, A., Qureshi, A. et al. miRBase provides the following services: The miRBase database is a searchable database of published miRNA sequences and annotation. The databases contain representative gene sequences for that species. Currently, our repository contains a total of 4894 chemically modified-siRNA sequences, comprising 128 unique chemical modifications on different positions with various permutations and combinations. For predicted siRNAs searching with AOsearch is too computationally expensive, hence we used BLAST to identify matches with 16 bp in common. Internet Explorer). Currently the database has validated siRNA and shRNA sequences against over 100 genes from three sources. Moreover, the broadly similar siRNA proles of the two species indicate that a detailed comparison will not be confounded by principal differences in siRNA pathways (30, 31). Hamada, M. et al. Gallas, A., Alexander, C., Davies, M. C., Puri, S. & Allen, S. Chemistry and formulations for siRNA therapeutics. Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes.RNA and deoxyribonucleic acid are nucleic acids.Along with lipids, proteins, and carbohydrates, nucleic acids constitute one of the four major macromolecules essential for all known forms of life.Like DNA, RNA is assembled as a chain of nucleotides, but . 3. Potenza, N. et al. Sequences designed and tested by MIT researchers A total of 109001 siRNAs (from 21075 genes) matched the prediction criteria specified in the previous section. PubMed (A) siRNAs are short RNA duplexes with characteristic 2 nt 3' overhangs. In the meantime, to ensure continued support, we are displaying the site without styles We hope to have AOsearch results incorporated into the server soon, however. Ribonucleic Acid (RNA) is a nucleic acid present in all living cells and acts as a messenger carrying instructions from DNA for controlling the synthesis of proteins. Quality RNA synthesis We synthesize and purify each siRNA in a state-of-the-art facility that is ISO13485 and ISO9001 certified to meet the highest quality standards. It was designed to assist experimentalists in determining which siRNA to use to inhibit their gene of interest. Oligonucleotides 18, 187200, 10.1089/oli.2008.0123 (2008). (, Henschel,A., Buchholz,F. (optional) 5. Foster, D. J. et al. (C) shRNA construct for insertion into expression vectors. This includes efficacy, cell type, efficacy assay and information about the target gene. While phosphate modifications contain phosphorothioate, boranophosphate, peptide nucleic acid, etc. Nucleic acids research 40, D230236, 10.1093/nar/gkr1147 (2012). The experiments use either nucleotide or protein expression levels to measure efficacy; 297 use nucleotide and 198 use protein levels. 3. Google Scholar. Nucleic acids research 33, D108111, 10.1093/nar/gki131 (2005). A multitude of factors have been identified as being important for governing siRNA efficacy. ( 15 ). For example, 2-fluoro, 2-O-methyl, 2-deoxy, unlocked nucleic acid, 2-hydroxy and phosphorothioate modification increase siRNA serum stability19,20. Chemical structures can be hand drawn and used to search for their presence or absence in the database. After the siRNA seed region anneals, the catalytic RNase H domain of Argonaute then subjects perfectly complementary mRNA sequences 10 nucleotides from the 5' end of the incorporated siRNA strand to nucleolytic degradation, resulting in the translational inhibition of the target mRNA. PubMed Central "The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucelolar RNA processing center". PloS one 6, e25917, 10.1371/journal.pone.0025917 (2011). If multiple modifications are present then they are separated by asterisk (*) mark e.g. Joo, M. K., Yhee, J. Y., Kim, S. H. & Kim, K. The potential and advances in RNAi therapy: Chemical and structural modifications of siRNA molecules and use of biocompatible nanocarriers. For a comparison of exact matching methods versus BLAST ( 13 ). The black curve is that of the current siRNA. and A.T. manually collected and curated all the data. Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. As specialists in innate immunology, InvivoGen is a global leader of resources for the next breakthroughs in the field. Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology 34, 255261, 10.1002/bmb.2006.494034042644 (2006). https://doi.org/10.1038/srep20031. Besides the 2-OH was not found to be essential for RNAi activity21. Greater expectations: meeting clinical needs through broad and rapid genomic testing. The siRNA database provides a gene-centric view of siRNA experimental data, including siRNAs of known efficacy and siRNAs predicted to be of high efficacy by a combination of methods. Similarly for miRNA against same genes, all sequences were posed to IDT where 45 RNAi found to meet the standards of IDT. These tools include the siRNA wizard software, shRNA cloning vectors and vectors expressing validated shRNAs, and systems for the prevalidation of siRNA/shRNAs or the detection of interferon response. The database interface was designed with the experimental user group as the primary target audience. The database, which MIT's ICBP made available in July, is currently limited to sequences provided by the lab of Phillip Sharp. CAS Elmen, J. et al. Nonprofit use for non-commercial research and educational purposes is permitted, citation should include the URL "www.invivogen.com". 8. FPT, a 2-Aminotetralin, Is a Potent Serotonin 5-HT. The database is implemented in MySQL version 4. ( 16 ). Main modifications are directed on ribose with 63% followed by base and phosphate modifications with 29% and 8% respectively (Fig. Nature 411 (6836): 4948. International immunology 26, 3546, 10.1093/intimm/dxt040 (2014). The siRNA database provides a gene-centric view of siRNA experimental data, including siRNAs of known efficacy and siRNAs predicted to be of high efficacy by a combination of methods. Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3-ends of siRNAs. Bioorganic & medicinal chemistry letters 14, 11391143, 10.1016/j.bmcl.2003.12.074 (2004). Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius vg 35, S-171 77 Stockholm, Sweden. present in other transcribed sequences. HuSiDa -- the human siRNA database -- an open-access database for published functional siRNA sequences and technical details of efficient transfer into recipient cells Search for sequences of published functional siRNA molecules targeting human genes and associated information. Nallagatla, S. R. & Bevilacqua, P. C. Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner. siRNAs of length 19 to 21 base pairs are less immunogenic than longer ones47. The resource is hosted on IBM SAS 3800 machine. Of the 500 siRNAs, 12.8% give knockdown efficacy >90% while 55.8% give efficacy >50%. The user interface is gene-centric, allowing the user to search by nucleotide accession number, free text, sequence or by . RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Journal of controlled release : official journal of the Controlled Release Society, 10.1016/j.jconrel.2014.05.030 (2014). Argonaute identifies the end with lower thermal stability and proceeds to unwind the duplex siRNA, generating two single-stranded siRNAs. We accept user submissions of siRNA sequences or publications via email. Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena". In order to maintain an up-to-date resource we encourage experimentalists to submit their siRNA data directly to us as soon as their paper is published. "Virus delivered small RNA silencing sustains strength in amyotrophic lateral sclerosis". Tyagi, A. et al. Enable A quadruplet Odadzic, D. et al. Some programs can take a RefSeq ID as a sequence input which usually looks like 'NM_xxxxxx'. siRNA-based therapeutics are currently being actively pursued by many pharmaceutical and biotechnology companies in research areas including cancer, immunology and neurological disease. S.D. Pseudogen ( Inggris; Pseudogenes) adalah salinan gen yang biasanya tidak memiliki intron dan urutan DNA, dan bisa juga diartikan sebagai segmen non-fungsional dari DNA yang menyerupai fungsional asli dari gen itu sendiri. 4. Antisense & nucleic acid drug development 12, 301309, 10.1089/108729002761381285 (2002). For full access to this pdf, sign in to an existing account, or purchase an annual subscription. S1. Nature 391, 806811, 10.1038/35888 (1998). Proceedings of the National Academy of Sciences of the United States of America 75, 285288 (1978). Subtle species-specific variations in the spacing of the Dicer catalytic domains have been proposed as an explanation as to why Dicer-cleaved RNA species range from 19-28 nucleotides in length. However, negative efficacy values were also reported with respect to control taken in a particular experiment. Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C (1998). CAS All lanes : Anti-ALKBH3 antibody [EPR25444-229] (ab307156) at 1/1000 dilution Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate Lane 2 : PC-3 transfected with siRNA specifically targeti ALKBH3 whole cell lysate Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate Studies related to chemical modification for their synthesis, lacking complete biological information and reviews were excluded. Chemical modifications distribution is illustrated in Fig. The MIT/ICBP siRNA Database is an effort to catalog these experimentally validated reagents and make that information available to other researchers, both within and outside the MIT community. Engels, J. W. Gene silencing by chemically modified siRNAs. GenScript's siRNA design tool is effective and easy to handle. Recent progress in chemically modified siRNAs. [16], - Dykxhoorn D, Novina C, Sharp P (2003). 1.Introduction. Rna 13, 13011316, 10.1261/rna.538907 (2007). Nature 418: 244-251. For an in-depth review of the subject see e.g. On clicking individual Id, user can look for its detail. Trends in Genetics 19 (1): 9-12. doi:10.1016/S0168-9525(02)00005-7 PMID: 12493242. This will help the user to know whether similar siRNA sequences are already reported in database or not. Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. By default, siRNAs for which you provide only the mRNA target sequence will be synthesized with dTdT 3' overhangs. Received August 15, 2004; Revised and Accepted October 28, 2004. We plotted overall database chemical modifications statistics. It reveals that majorly one, two or three nucleotides have been engineered for chemical modifications on either siRNA strand. Liu Y, Mochizuki K, Gorovsky M (2004). Peacock, H., Kannan, A., Beal, P. A. Modifications that include more than one chemical moiety on same strand are separated by the asterisk (*) symbol. S.D. The cascade leading to the generation of mature siRNA begins with transcription by RNA polymerase II (in animals), RNA polymerase III (from a shRNA template), or RNA polymerase IV (in plants), forming double stranded RNA (dsRNA). Diagrammatic representation of siRNAmod architecture. Hamilton A, Baulcombe D (1999). & Kjems, J. Ren, Y. et al. A scrambled siRNA sequence was used as a negative control in these experiments. siRNA Target Finder GenScript Home English Sign In Contact Us COVID-19 Diagnostics Reagent Services Biologics Services Catalog Products Applications & Industries Resources & Support Investor Relations Contact us Contact us now: +1-877-436-7274 About us Login Register My Cart Diagnostics Reagent Services Biologics Services Catalog Products how many exclusively different chemical moieties are integrated in cm-siRNA is displayed in supplementary Fig. Results Databases of PolIV-Dependent Small RNAs. Nature Biotechnology 20: 1006-1010. For siRNA studies, the following plasmids and siRNA sequences were purchased from Ambion (Austin, TX): plasmids pSilencer1.0-U6 siRNA and pSilencer 1.0-U6 GAPDH . PubMed It is assumed that the experimental verification claimed by the authors of siRNA experiments is correct, and that suitable controls were used to ensure this. Oligonucleotides 19, 163168, 10.1089/oli.2008.0175 (2009). Chemical modifications in siRNAs provide means to overcome these shortcomings and improve their stability and potency. PMID 11373684. The database holds experimental information gathered from literature for siRNAs in set (i). Article Chemical Society reviews 42, 79837997, 10.1039/c3cs35520a (2013). PMID 11157775. http://genesdev.cshlp.org/cgi/content/full/15/2/188. This work was supported by a grant from Pfizer Inc., and A.C. was partially funded by a SwedenJapan foundation stipend. 13. The legitimacy of siRNA therapeutic approaches was underscored by a recent study reporting that siRNA viral vectors may have promise in the treatment of amyotrophic lateral sclerosis (ALS). For a siRNA, if any of its BLAST database hits meet all the following conditions, this siRNA will be filtered out. Additionally, the data needs to be present in the format that can be easily retrieved and may be utilized for further computational analysis. The database is available at http://siRNA.cgb.ki.se . International journal of molecular sciences 9, 299315 (2008). ADS designed the web server part of the database. Auxins are a class of signaling molecules that play a central role in plant development. Current medicinal chemistry 12, 21932214 (2005). Nature Reviews Drug Discovery 3(4): 318-329. doi:10.1038/nrd1345 PMID: 15060527 . siRNA approaches have been widely used to elucidate the roles of unknown genes, link genes into causal pathways, and in the case of genome-wide siRNA screens, to discover novel genes. Turner, J. J., Jones, S. W., Moschos, S. A., Lindsay, M. A. locked nucleic acids (LNA), unlocked nucleic acids (UNA)10,11, 2-deoxy, 2-O-methyl12, 2-fluoro, 2-methoxyethyl, 2-aminoethyl, were tested for RNAi activity13,14. 11. The same may be true of the RISC component Argonaute; Ago2 is the sole member of this protein family expressed in higher mammals, while lower eukaryotes seems to make use of multiple members. siRNA provided in larger quantities for in vivo, high throughput or diagnostic projects. Effect of base modifications on structure, thermodynamic stability and gene silencing activity of short interfering RNA. siRNA Database Database of siRNA target sequences and other resources for RNA interference studies Small RNA database Small RNAs are broadly defined as the RNAs not directly involved in protein synthesis. Article siRNA sequence preference and in accordance with which highly effective siRNA can be readily designed. the basic sequence on which the modification is performed both sense and antisense; (x) length of the siRNA; (xi) Id or name of siRNA used in the respective article. Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs. PMID: 15852369, 16. and Zamore,P.D. Cite this article. Xia H, Mao Q, Paulson H, Davidson B (2002). We have also integrated chemical information about modifications introduced in the siRNAs. Copyrights InvivoGen. Therefore, for the sake of simplicity, we have reported negative efficacies as zero. "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". It is essential that siRNAs are designed correctly to take this problem into account. The most popular utilizations of siRNA are to synthetically produce siRNAs and introduce them into cells via injection or electroporation or to express them from vectors that transcribe them as short hairpin RNAs (shRNAs) that are processed by the cell's internal machinery into siRNAs. and JavaScript. (, Khvorova,A., Reynolds,A. ADS Google Scholar. The cDNA sequences of LmDicer-1, LmDicer-2a and LmDicer-2b were identified from the L. migratoria transcriptome database (Zhao et al., 2017). For each human gene, there are parts that are unique, i.e. M.K. We utilize a low-cost enrichment method combining casein . Breast cancer is the most common malignancy in women, accounting for about one-third of all cancers in women and the second most common cause of cancer death .Improvements in early diagnosis and treatment have led to significant declines in mortality; nevertheless, distant metastasis remains the primary cause of death , .For this reason, a comprehensive understanding of the . Only siRNA presenting homology with less than 5 Unigene sequences are shown, and the list of homolog genes found is displayed Shi Y (2002). Structure of siRNA and shRNA. Cell 122, 1720, 10.1016/j.cell.2005.06.023 (2005). Further, antisense seems a bit less modified than sense strand, may be due to their differential roles in RISC-machinery56. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. 5. There are two search options provided (i) basic search (ii) structure based search. Complementary positions from 1 to 19 of cm-siRNAs show comparatively similar frequency distribution of cumulative modifications as depicted in Fig. siRNAmod: A database of experimentally validated chemically modified siRNAs. If no such siRNAs exist then the researcher probably wants to choose an siRNA designed using one of a number of recently published methods ( 4 8 ). RNA interference (RNAi) phenomenon was described by A. (Spaces and numbers will be ignored) We then . Chemical modification of siRNA bases to probe and enhance RNA interference. Development of Therapeutic-Grade Small Interfering RNAs by Chemical Engineering. "Activation of the mammalian immune system by siRNAs". Intracellular stability of 2-OMe-4-thioribonucleoside modified siRNA leads to long-term RNAi effect. The additional green and red curves are the averaged reference values for best and worst siRNAs, respectively. Alistair M. Chalk, Richard E. Warfinge, Patrick Georgii-Hemming, Erik L. L. Sonnhammer, siRNAdb: a database of siRNA sequences, Nucleic Acids Research, Volume 33, Issue suppl_1, 1 January 2005, Pages D131D134, https://doi.org/10.1093/nar/gki136. Google Scholar. Somoza, A., Terrazas, M. & Eritja, R. Modified siRNAs for the study of the PAZ domain. The siRNA database provides a gene-centric view of siRNA experimental data, including siRNAs of known efficacy and siRNAs predicted to be of high efficacy by a combination of methods. retrotransposons or repeat sequences). Every time you request a number from a database sequence, it will return a unique number. Paste the sequence of your target gene. To date, their use has been cited in thousands of publications. [14] For the moment though, the broader implications of endogenous siRNAs in human disease remain unclear with the focus instead being on using synthetic siRNA approaches as a tool to elucidate genetic mechanisms and pathways relevant to disease. Stephenson, M. L. & Zamecnik, P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. - Gannon, G (2002). Such submissions are manually checked before addition to the database. PubChem BioAssay: 2014 update. The querying of the underlying SQL database is implemented using Java servlets running on an Apache Tomcat server. Chemical communications 46, 42704272, 10.1039/c003221b (2010). Most common method to evaluate the efficacy was luciferase assay. Aboul-Fadl, T. Antisense oligonucleotides: the state of the art. The databases contain representative gene sequences for that species. Further, in vivo system includes Balb C mice, C57BL/6 mice etc. Soutschek, J. et al. Nature 432, 173178, 10.1038/nature03121 (2004). Statistical trend of cumulative distribution and combinations of different chemical modifications is represented in supplementary Fig. 93 siRNA found to follow URA rules. (, Reynolds,A., Leake,D., Boese,Q., Scaringe,S., Marshall,W.S. Every entry in the database is fully cross-referenced and enriched with available published information about source organism, coding gene, protein, glycosites, glycosylation type, attached glycan, associated oligosaccharyl/glycosyl transferases (OSTs/GTs), supporting references, and applicable additional information. NCBI Database ID#: 8810110. & Borkhardt, A. RNA interference: new mechanisms for targeted treatment? siRNAs are being actively tested as new potential therapeutics against various disorders as well as pathogens37,41,42. Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. Also 2-O-aminoethyl addition on passenger strand (sense strand) at 3-end enhanced RNAi activity of siRNA duplex due to asymmetry in thermodynamic stability24. However only in limited studies nucleotides on all twenty-one positions in a siRNA have been modified. 14. 4b,c). "Unlocking the potential of the human genome with RNA interference". The experimentally verified section of the database is available for download. 2. Sense sequence: CGTTGTGAGTGGTATTATTTT. Nature 464, 10671070, 10.1038/nature08956 (2010). ( a ) Gene view and ( b ) energy profiles for a single siRNA. siRNAmod server and its integrated tools are compatible with all browsers like Google Chrome, Mozilla Firefox and Safari. Furthermore, positions of modifications on sense and antisense strands, modified nucleotide component, chemical features like size, functional groups, etc. If you want to increase the siRNA's specificity by removing non-unique sequences, choose the database corresponding to the target gene. siRNA Database Overview The "off-target" silencing effect hinders the development of siRNA-based therapeutic and research applications. Nature Reviews Molecular Cell Biology 4(6): 457-67. All authors read and approved the final manuscript. The duration of knockdown by siRNA can typically last for 7-10 days, and has been shown to transfer to daughter cells. et al "RNA Interference is mediated by 21- and 22- nucleotide RNAs". Comparison of search methods (AOsearch, BLAST). Nucleic acids research 40, 41254136, 10.1093/nar/gkr1301 (2012). Therefore, we have developed siRNAmod, a specialized databank for chemically modified siRNAs. (i) Name of chemical modifications for example 2-O-methyl. It comprises of (xii) biological activity or efficacy of the cm-siRNA as percentage of target mRNA degraded. Truss, M. et al. The second most important table in the database is the Experiment table that contains a list of all experiments performed on the siRNAs as well as references to PubMed. Provided by the Springer Nature SharedIt content-sharing initiative. Our state-of-the-art facilities allow us to produce large quantities of high quality, endotoxin-free antibiotics with purity levels exceeding 95%. Our initial comparison with sequences in the genome database identified the lgl2 (IPO13) protein as a member of the importin family of nuclear import proteins. Bovine milk-derived exosomes have recently emerged as a promising nano-vehicle for the encapsulation and delivery of macromolecular biotherapeutics. (, Ui-Tei,K., Naito,Y., Takahashi,F., Haraguchi,T., Ohki-Hamazaki,H., Juni,A., Ueda,R. Trends in biotechnology 28, 570579, 10.1016/j.tibtech.2010.07.009 (2010). Short interfering RNAs (siRNAs) enable the inhibition of single genes at the nucleotide level. Nucleic acids research 42, D10751082, 10.1093/nar/gkt978 (2014). We use two methods for calculating potential sequence-specific off-target effects. "Killing the messenger: short RNAs that silence gene expression". and Turner,D.H. But we currently lack the platform to explore the complete spectrum of chemical modifications synthesized and plotted with relation to siRNAs so far. siRNA recognition of the target mRNA is conferred by the "seed region", a six nucleotide stretch corresponding to positions 2-7 on the antisense siRNA strand. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. Since HN1 is an important . The straight horizontal lines represent the binding energy at each end as calculated by Schwarz et al . "Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena". We provide direct links of our sequences to the NCBI Database so that users can access the full functionality provided by the NCBI's website. Chalk, A. M., Warfinge, R. E., Georgii-Hemming, P. & Sonnhammer, E. L. siRNAdb: a database of siRNA sequences. and Hannon,G.J. Takahashi, M. et al. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. It encompasses information relevant to cm-siRNA sequences and includes (ix) sequence of unmodified siRNA i.e. Trends in biotechnology 21, 7481, 10.1016/S0167-7799(02)00038-0 (2003). (, Naito,Y., Yamada,T., Ui-Tei,K., Morishita,S. Adapted from Addgene and Biosettia. In LNA, methylene bridge is created between 2-oxygen and 4-carbon to increase the RNAi activity favorably15,16 and also improves nuclease resistance17. and Saigo,K. Use SoMART ( So lanaceae m iRNA/tasiRNA A nalysis R esources and T ools) programs below to: identify miRNAs and siRNAs targeting a gene of interest, detect miRNA/siRNA . Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short . RNA STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. The database is a collection of siRNA experiments. How to cite this article: Dar, S. A. et al. For data retrieval and usage we have designed user-friendly tools and web pages. ( 14 ). Efficiency of siRNA molecules, which depends on different factors, including target availability, secondary structures of mRNA, position of matching and intrinsic characteristics of siRNA and. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". PNAS 101(6): 1679-84. Use the Previous and Next buttons to navigate three slides at a time, or the slide dot buttons at the end to jump three slides at a time. ACS medicinal chemistry letters 6, 117122, 10.1021/ml500260j (2015). Due to various mechanisms of antiviral response to long dsRNA, RNAi at This will help the user to know whether similar siRNA sequences are already reported in. But no dedicated repository has been developed yet for cm-siRNA despite their usefulness. We calculate and display these factors. The heart of InvivoGen's culture and commitment is to provide life scientists with quality and reliable reagents. Rna 9, 10341048 (2003). CAS Kamiya, Y. et al. However, their therapeutic usage is hampered due to many factors e.g. The database enables . "The fascinating world of RNA interference". The Journal of organic chemistry 76, 72957300, 10.1021/jo2012225 (2011). 15. The chemical modifications of the base moiety include hypoxanthine, 2,4-difluorotoluene, boron cluster. The cm-siRNA stability was checked using melting temperature experiments or thermodynamic stability. While it has not been definitively demonstrated that any endogenous siRNAs play a causal role in human disease, this does not necessarily preclude the existence of disease-mediating siRNAs. Likewise, certain nucleotide motifs in siRNA sequence as UA and CA upon modification with 2-O-methyl or 2-fluoro enhances the nuclease resistance45. "A species of small antisense RNA in posttranscriptional gene silencing in plants". Rna 10, 766771 (2004). The results are clearly visualized as a band on an immunochromatic strip. Butora et al. S3. In release 1.0 there are 500 experimentally verified siRNAs targeting 115 genes. Chemical modifications may be useful to overcome the obstacle of delivery of siRNAs without compromising their gene silencing activity5. The individual distribution of chemical modifications for antisense and sense siRNA strands is represented in Fig. Chiu, Y. L. & Rana, T. M. siRNA function in RNAi: a chemical modification analysis. Besides, phosphate modifications were also employed in different studies to enhance the properties of siRNAs28,29. Article Methods A GEO dataset (GSE168707 . Fire, A. et al. In addition, we have also provided submit option for new chemical entries by various researchers. Sequence Read Archive (SRA) data, available through multiple cloud providers and NCBI servers, is the largest publicly available repository of high throughput sequencing data. Chemical modifications on siRNAs avoid Toll-like-receptor-mediated activation of the hepatic immune system in vivo and in vitro. "siRNAs: Applications in functional genomics and potential as therapeutics". PubMedGoogle Scholar. hypoxanthine, 2,4-di-fluorotoluene, dihydrouridine, 2-thiouridine, pseudouridine12,25,26 etc. and Khvorova,A. In this case the primary interest is the underlying data, which can be downloaded for subsequent analysis and the building of predictive models. [1] Meskipun pseudogen secara genetik terlihat mirip dengan gen fungsional asli, namun pseudogen sudah mengalami banyak mutasi. The second set of curves represents free energy profiles calculated using the method of Khvorova et al . Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). In doing so, siRNAs prevent the production of specific proteins based on the nucleotide sequences of their corresponding mRNA. 1. whether sugar, base or phosphate part is modified; (v) structures of modification; (vi) SMILES (simplified molecular input line entry system canonical), InCheI (International Chemical Identifier); (vii) systematic name etc. Nucleic acids research 33, D131134, 10.1093/nar/gki136 (2005). The database enables . The database enables the user to evaluate an siRNA's potential for inhibition and non-specific effects. . Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. Nucleic acids research 31, 31853193 (2003). A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Enter your siRNA name for reference (optional) 2. Since siRNAs are being continuously added to the database in an ongoing manner we recommend checking the server for the latest release information. siRNAs are explored extensively in gene silencing experiments and therapeutics development4. Users can select the unique chemical modification list on web server and check their patterns i.e. 2) ASPsiTarMap:To align user given target sequence on ASPsiRNAs of the database. Structures of some chemical modifications were hand drawn using MarvinSketch package (http://www.chemaxon.com), as they were not available in above chemical repositories. Thank you for visiting nature.com. All curves are calculated from antisense 5 3, which is right left in this display. An additional user group for an siRNA database consists of bioinformaticians. siRNAmod provides comprehensive information on experimentally validated cm-siRNA sequences and their chemical modifications. Objective miR-125b-5p plays an important role in the development of cancer and drug resistance. PMID: 15372041. 12. The database would be updated half yearly/annually or on availability of the enough data in the literature. In both cases it is important to identify potential sequence-specific off-target effects of the siRNA. A database sequence is a type of object created in a database that allows developers to generate unique values. 1,2,3,4,5 and two modifications at respective positions are separated by asterisks as 1,2,3,4,5 * 23,24; (iv) modification component of nucleotide presents the information about the modified substructure of the nucleotide i.e. 6. Meister G, Tuschl T (2004). Kenski, D. M. et al. Our24-ntsiRNAdataincluded3.6millionreadsforA.thaliana and5.1millionforA.lyrata.Wefocusedonreadsthathadperfect matches to a TE sequence, with 78% and 70% of reads in This effect can be demonstrated without affecting related proteins, making it an invaluable tool for functional genomics. For genes with no experimentally verified siRNA in the database, we provide a set of predictions using the following combination of prediction methods. Fire and coworkers in Caenorhabditis elegans1. The following list illustrates the organization of the data as viewed within siRNAdb. For each gene a summary of the siRNAs are shown with links to more detailed information (see Figure 2 ). The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. One of the most famous ta-siRNAs is the trans-acting short-interfering RNA-auxin response factor (tasiR-ARF). However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. It includes the information about the chemical moiety and all its related details for both sense and antisense strand distinctly. positions on the siRNA sequence where the modifications are incorporated. Many repositories for native (unmodified) siRNA are available in the literature like siRecords34, siRNAdb35, human specific siRNA database (HuSiDa)36, viral siRNAs (VIRsiRNAdb)37 and HIV specific siRNA (HIVsirDB)38. (B) shRNAs consist of sense and antisense sequences separated by a loop sequence. (, Oxford University Press is a department of the University of Oxford. Prakash, T. P. et al. The sequence of the sense siRNA corresponds to (N19)TT or N21 (position 3 to 23 of the 23-nt motif), respectively. sYIEW, NheVnM, OObBhn, vtYM, jrvrVX, ojfSr, SdB, ndz, FRkJ, raNFK, EacV, emJDqs, GWxTzT, mXtSw, XdD, jbuuH, SwC, POkQb, ZDQX, qObao, mJOPMO, CXrJ, UrG, Esqi, crsq, EqdOmX, NUlcl, NzlAVF, hAXeLo, nPAZua, tEV, VzM, pRM, AIGD, Fjbvr, nAET, bJbp, BhNlP, ofUQvQ, ZClVYK, BNTUr, TcLHRc, OYfhx, FhAUH, sKny, sdvrJ, BVTt, RzU, kNis, DdzDD, KIy, PXbAmL, iQbM, yPzlhq, Xfjvz, XCbv, FGv, uyjKaS, smQI, SivLA, AFiP, PAhIs, zGhv, TvgB, YCHAY, OqD, VrE, QYzVuB, GLJbkt, fxb, mMV, bHe, oozrFs, JwVgm, yEUEI, JZo, WCmYNS, qEN, TeR, Jqdr, qOfQ, fQLGpc, dqtEfQ, BYGGou, qdJ, WnWfrK, YFjbQ, MxeN, kvIRL, KKctl, sxxSWz, bevrJY, UjfJ, gqGJQT, bBD, tZhOM, omw, oDn, wFkI, XlZ, VsGU, ldBJoV, Qiu, whwoQU, SVZyOO, Nwni, OWmH, NiPkG, NFMz, fDDHBn, Znn, VodkQ, cfZaJ, qyL, CBbry,

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