Greater expectations: meeting clinical needs through broad and rapid genomic testing. Adapted from Addgene and Biosettia. siRNAmod is the first comprehensive platform of 4894 cm-siRNAs with 128 unique chemical modifications. Chemical structures can be hand drawn and used to search for their presence or absence in the database. volume6, Articlenumber:20031 (2016) Sipa, K. et al. Display of search output with cm-siRNA details (a) Result output of search or browse (b) Details page of individual entry (c) Structure and other chemical information of modification. Enter your siRNA name for reference (optional) For-profit organizations are requested to contact the corresponding author. Nucleic acids research 33, D108111, 10.1093/nar/gki131 (2005). Google Scholar. Synthetic oligonucleotides were used in the late 1970s for gene inhibition known as antisense technology8. For structure drawing and search, MarvinSketch tool is provided. Nucleic acids research 37, D146149, 10.1093/nar/gkn817 (2009). Your comment will be reviewed and published at the journal's discretion. Further, compatibility of RNAi protein machinery with cm-siRNA format has been investigated50,51. Furthermore, structure search option is also available to draw the structure and search against the database. 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. doi:10.1038/35078107. 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. 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. The RNA Modification Database, RNAMDB: 2011 update. The database contains information about siRNA molecules from two sources: (i) siRNAs collected from the literature that have experimentally verified efficacy and (ii) siRNAs selected computationally to target the REFSEQ ( 9 ) curated human gene set (20410 NM sequences and 6767 XM sequences). Non-specific off-target effects caused by siRNAs matching genes other than their intended target gene render experimental results hard to interpret or useless. (, Yuan,B., Latek,R., Hossbach,M., Tuschl,T., Lewitter,F., Naito,Y., Yamada,T., Ui-Tei,K., Morishita,S., Saigo,K. and Saigo,K. Nature 464, 10671070, 10.1038/nature08956 (2010). restriction site. The database is a collection of siRNA experiments. The output includes siRNATarget.set.out : list of the siRNA target sequences 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. Exploration of chemical modifications and analysis in siRNAs and has been accelerated in last decade and data is continuously growing. & Tiemann, K. Current progress of siRNA/shRNA therapeutics in clinical trials. In the meantime, to ensure continued support, we are displaying the site without styles Filipowicz, W. RNAi: the nuts and bolts of the RISC machine. 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. Chemical modifications remain the critical aspect for development of siRNA therapeutics to reduce their inherent limitations6,44. PubMed Article 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. Small interfering RNA (siRNA) technology has vast potential for functional genomics and development of therapeutics. The querying of the underlying SQL database is implemented using Java servlets running on an Apache Tomcat server. (C) shRNA construct for insertion into expression vectors. (, Chalk,A.M., Wahlestedt,C. (, Naito,Y., Yamada,T., Ui-Tei,K., Morishita,S. 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. 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. If siRNAdb lacks predictions for a gene it is recommended that the user manually search for siRNA sequences using one, or a combination of siRNA prediction servers. Amarzguioui, M., Holen, T., Babaie, E. & Prydz, H. Tolerance for mutations and chemical modifications in a siRNA. (optional), Enable C quadruplet "The fascinating world of RNA interference". Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. As specialists in innate immunology, InvivoGen is a global leader of resources for the next breakthroughs in the field. S3. Besides, phosphate modifications were also employed in different studies to enhance the properties of siRNAs28,29. The black curve is that of the current siRNA. If such siRNAs exist, the researcher will be interested in the conditions under which the siRNA was tested, and the reference to the article where the siRNA was published for further investigation. Soutschek, J. et al. 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. Potenza, N. et al. (i) Name of chemical modifications for example 2-O-methyl. Naked siLNA-mediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration. [7] Whereas humans only express one isoform of the Dicer enzyme, Drosophila and Arabidopsis express two and four Dicer paralogues (known as Dicer-like proteins or DCLs) respectively. siRNA target sequence GC% between: and First nucleotide of the target sequence: Enable C quadruplet Enable A quadruplet Enable G quadruplet Database: miRNA SEED Database: Selective Antibiotics Our state-of-the-art facilities allow us to produce large quantities of high quality, endotoxin-free antibiotics with purity levels exceeding 95%. 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. 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. RNAcentral is a comprehensive database of non-coding RNA sequences that represents all types of ncRNA from a broad range of organisms. 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. siRNAmod browsing option helps user to surf via these five categories: i) modification in sense strand, ii) position of modification in sense strand, iii) modification in antisense strand, iv) position of modification in antisense strand and v) reference. Short interfering RNAs (siRNAs) enable the inhibition of single genes at the nucleotide level. Here, we have combined the similar types of modifications into single categories as synonyms to represent the same or similar modifications reported in different studies. Pie chart depicting their distribution is presented in supplementary Fig. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. 1. doi:10.1126/science.286.5441.950. Commonly used cell lines were HeLa, HEK 293, Hepa 16, Vero, SH-SY5Y etc. In both cases it is important to identify potential sequence-specific off-target effects of the siRNA. Large-scale screens and high throughput platforms using different permutation and combinations of chemical modifications were explored to check their effect24,32. Further effect on enhanced efficacy due to chemical modifications was tested using different siRNAs concentrations49. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. In viruses, RNA and DNA can carry information. [4] This achievement led to the widespread use of synthetic siRNAs as a laboratory tool to selectively knock-down the activity of specific genes. 4. Bovine milk-derived exosomes have recently emerged as a promising nano-vehicle for the encapsulation and delivery of macromolecular biotherapeutics. Nucleic acids research 40, 57875793, 10.1093/nar/gks204 (2012). "Gene discovery by ribozyme and siRNA libraries". ADS Our24-ntsiRNAdataincluded3.6millionreadsforA.thaliana and5.1millionforA.lyrata.Wefocusedonreadsthathadperfect matches to a TE sequence, with 78% and 70% of reads in Nature Reviews Molecular Cell Biology 6: 413-422. 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. Ren, Y. et al. Butora, G. et al. Short interfering RNAs (siRNAs) are a popular method for gene-knockdown, acting by degrading the target mRNA. 128 unique chemical modifications incorporated on various components of nucleotides are archived. There are two search options provided (i) basic search (ii) structure based search. Reverse complement based chemical modification of siRNA position wise on sense and antisense siRNA sequences. CAS 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. This includes efficacy, cell type, efficacy assay and information about the target gene. 2. Prakash, T. P. et al. One of the most famous ta-siRNAs is the trans-acting short-interfering RNA-auxin response factor (tasiR-ARF). Similarly, other groups also studied terminal modifications like 5-phosphate53 or serinol modification54. and Turner,D.H. Besides, inhibition concentration-50 (IC50) values are sometimes provided in the studies instead of percentage inhibition. Summary statistics are self-evident, and energy profiles are described in ( 6 ). User can choose exact or containing operators. 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. mRNA knockdown by single strand RNA is improved by chemical modifications. 17, 27 The sequences of circRNA_45478 were obtained from the Arraystar database while miR-190a-5p while from miRDB . Get the most important science stories of the day, free in your inbox. It includes the information about the chemical moiety and all its related details for both sense and antisense strand distinctly. . It provides manually curated information about chemical modification, sequences, siRNA activity and experimental details. [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. The RNAi Consortium, or TRC, is a public-private effort based at the Broad whose mission is to create a shRNA library as well to validate tools and methods that will enable the scientific community to use RNAi to determine the function of human and mouse genes. Chemical modifications on siRNAs avoid Toll-like-receptor-mediated activation of the hepatic immune system in vivo and in vitro. Kenski, D. M., Willingham, A. T., Haringsma, H. J., Li, J. J. A link to PubMed with a pre-formulated siRNA query search is also made available to allow the user to easily check for new siRNA articles relating to the gene of interest literature. We are currently seeking additional sequences to add to our database. Elbashir,S.M., Lendeckel,W. Of the 500 siRNAs, 12.8% give knockdown efficacy >90% while 55.8% give efficacy >50%. Database Profile VIRsiRNAdb General information Classification and Labelling Contact information Publications VIRsiRNAdb: a curated database of experimentally validated viral siRNA/shRNA. The database architecture is shown in Fig. [13], Small interfering RNA (siRNA) is also known as short interfering RNA or silencing RNA. Nature 391 (6669): 80611. PMID: 12110901. MIT Researchers: Submit sequences directly through the submission page or contact us for assistance. The reagents are composed of short hairpin sequences carried in lentiviral vectors arrayed in 96-well plates. So, there is further scope to explore these combinations. Our state-of-the-art facilities allow us to produce large quantities of high quality, endotoxin-free antibiotics with purity levels exceeding 95%. "A species of small antisense RNA in posttranscriptional gene silencing in plants". PubMed Central 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. on what positions the modifications are reported along with their biological effects. p53-siRNA-1 and p53-siRNA-2). & Kjems, J. and Maglott,D.R. p53-siRNA), with constructs targeting the same gene distinguished by the addition of numbers after the construct name (e.g. Haringsma, H. J. et al. The results are clearly visualized as a band on an immunochromatic strip. 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. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. For data retrieval and usage we have designed user-friendly tools and web pages. In Arabidopsis thaliana, siRNA-mediated cytosine methylation has been shown to play an active role in chromatin remodeling and to be crucial for the maintenance of the heterochromatic state. This work is licensed under a Creative Commons Attribution 4.0 International License. These data were gathered from 55 articles. The user interface is gene-centric, allowing the user to search by nucleotide accession number, free text, sequence or by viewing the list of genes with verified siRNAs. FPT, a 2-Aminotetralin, Is a Potent Serotonin 5-HT. This analysis revealed a strong preference for G or C nucleotides at position 1. the sequence of each siRNA strand (This option is recommended if you wish your siRNA to have 3' termini other than dTdT or UU.) We accept user submissions of siRNA sequences or publications via email. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. The database enables . All authors read and approved the final manuscript. 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. We calculate and display these factors. (Spaces and numbers will be ignored) miRBase: the microRNA database. A Pie-chart depicting up-regulation of 27% of the genes in SiHa-R cells as revealed by the RNA-seq data.B Volcano plot of genes in SiHa-R, displaying up-regulation of a majority of the genes.C An increase in expression of genes involved in stemness in SiHa-R was observed by analysis of RNA-seq data . User-friendly web interface is provided to retrieve the desired curated information with ease. Intracellular stability of 2-OMe-4-thioribonucleoside modified siRNA leads to long-term RNAi effect. siRNAs are thought to have most likely evolved as a primitive immune response evoked by the presence of foreign nucleic acids. Sense sequence: CGTTGTGAGTGGTATTATTTT. Nucleic acids research 39, D195-201, 10.1093/nar/gkq1028 (2011). As part of our rigorous quality control procedures, each . Aboul-Fadl, T. Antisense oligonucleotides: the state of the art. Peel et al. Mini reviews in medicinal chemistry 10, 578595 (2010). and Jayasena,S.D. positions on the siRNA sequence where the modifications are incorporated. However, negative efficacy values were also reported with respect to control taken in a particular experiment. NS siRNA - Non-specific siRNA PRAD - Prostate Adenocarcinoma . 15. However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. - Gannon, G (2002). Hamilton A, Baulcombe D (1999). Article [6] siRNA or siRNA-like molecules (such as rasiRNA) are represented in nearly every kingdom (the exception being bacteria), including algae (Chlamydomonas reinhardtii), plants (A. thaliana), fungi (S. pombe) and both vertebrate and invertebrate members of the animal kingdom (D. melanogaster, C. elegans, M. musculus, H. sapiens). The database implementation and verified siRNAs are available at http://siRNA.cgb.ki.se for non-commercial use. siRNAs have been found to be effective in Arabidopsis thaliana , Drosophila melanogaster , Caenorhabditis elegans and mammals ( 2 ). hypoxanthine, 2,4-di-fluorotoluene, dihydrouridine, 2-thiouridine, pseudouridine12,25,26 etc. Combinations of different unique modifications i.e. 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. Studies related to chemical modification for their synthesis, lacking complete biological information and reviews were excluded. The database, which MIT's ICBP made available in July, is currently limited to sequences provided by the lab of Phillip Sharp. and Khvorova,A. Woessmann, W., Damm-Welk, C., Fuchs, U. Convert standard siRNA to Stealth RNAi siRNA : Find out more about Stealth RNAi siRNA, the next generation RNAi molecule or read about the benefits of BLOCK-iT siRNA. 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). 4b,c). Please check for further notifications by email. By submitting a comment you agree to abide by our Terms and Community Guidelines. 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. We have also integrated chemical information about modifications introduced in the siRNAs. Paste the sequence of your target gene. S.D. CAS Diagrammatic representation of siRNAmod architecture. Huang, Y. et al. This may be probably due to ease of end modifications and their role in nuclease resistance55. and JavaScript. As more siRNAs are verified this database will become increasingly useful for developing siRNA design tools. InvivoGen provides a set of cutting-edge tools to knockdown efficiently the expression of mammalian genes. (, Reynolds,A., Leake,D., Boese,Q., Scaringe,S., Marshall,W.S. [5], siRNAs hold nearly limitless potential for use as a tool, which has led to their rise as the most widely used gene-silencing approach. These dsRNA are recognized by the RNA binding domain (RBD) of the Dicer complex, which contains a pair of RNaseIII type endonuclease catalytic domains. Nature 431: 343-349. Butora et al. The derived x-mers are then used to probe an mRNA or cDNA sequence database for matching sequences. "siRNA-mediated gene silencing in vitro and in vivo". Annals of Neurology 57: 773-776. But the database administrator said she has identified researchers from other MIT labs who have published papers in which siRNA and shRNA reagents had "good" results knocking down mRNA or proteins. 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. Oligonucleotides 18, 187200, 10.1089/oli.2008.0123 (2008). 12. We use two methods for calculating potential sequence-specific off-target effects. 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. In addition to the local siRNA Database, submissions to the MIT/ICBP siRNA Database are added to the NCBI's Probes Database. We utilize a low-cost enrichment method combining casein . View of Human AKT1 siRNA data. "The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucelolar RNA processing center". Currently sIR database consists of only ~1200 siRNA sequences. Petersen, M. & Wengel, J. LNA: a versatile tool for therapeutics and genomics. It elucidates that maximum 5 different combinations of modifications are seen so far in cm-siRNAs. PMID: 15372041. Due to various mechanisms of antiviral response to long dsRNA, RNAi at Langkjaer, N., Pasternak, A. "Unlocking the potential of the human genome with RNA interference". & Burrows, C. J. PMCID: PMC1693862. 1.Introduction. Both 2-O-methyl and 2-deoxy are natural RNA modifications. Kamiya, Y. et al. Complementary positions from 1 to 19 of cm-siRNAs show comparatively similar frequency distribution of cumulative modifications as depicted in Fig. 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). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. But we currently lack the platform to explore the complete spectrum of chemical modifications synthesized and plotted with relation to siRNAs so far. Rna 14, 12011213, 10.1261/rna.1007408 (2008). Of those, 42155 siRNAs from 12888 genes were also found to be specific using BLAST criteria; 14189 genes have no siRNAs matching these criteria, owing to the strict requirements for the automated predictions. Journal of controlled release : official journal of the Controlled Release Society, 10.1016/j.jconrel.2014.05.030 (2014). S5. Collingwood, M. A. et al. "RNA interference". They are duplexes of two RNA molecules, typically 21mers with a 2 nt 3 overhang ( 1 ). This information is derived form public chemical databases namely PubChem58, ChemSpider (http://www.chemspider.com), ChEMBL (https://www.ebi.ac.uk/chembl). [15], 2021 The Regents of The University of California. "Improved and automated prediction of effective siRNA". Therefore, for the sake of simplicity, we have reported negative efficacies as zero. For example, 2-fluoro, 2-O-methyl, 2-deoxy, unlocked nucleic acid, 2-hydroxy and phosphorothioate modification increase siRNA serum stability19,20. Some programs can take a RefSeq ID as a sequence input which usually looks like 'NM_xxxxxx'. 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. 5. RNAcentral is the world's largest RNA secondary structure database. Vector & Virus Western Blot Recent Posts DNA Microarray Mode of DNA replication Transcription control in Bacteria Journal of medicinal chemistry 48, 42474253, 10.1021/jm050044o (2005). GC content: Ideally the GC content is < 50% (most software defaults range between 40 to 50%) Stretches of nucleotide repeats. International Journal of Biological Sciences 5(2): 97-117. http://www.biolsci.org/v05p0097.htm PMID: 19173032. The resource is hosted on IBM SAS 3800 machine. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. It encompasses information relevant to cm-siRNA sequences and includes (ix) sequence of unmodified siRNA i.e. In contrast, synthetically produced injected/transfected siRNAs yield a more transient knockdown of gene activity that is largely dependent on the rate of cell division. [16], - Dykxhoorn D, Novina C, Sharp P (2003). Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo. The database enables . & Sczakiel, G. Phosphorothioate-stimulated uptake of siRNA by mammalian cells: a novel route for delivery. MMIA -- MicroRNA and mRNA Integrated Analysis However, fluorine modification at 2-OH on different nucleotide positions of siRNA maintains silencing potency as well as increases thermal stability22,23. ( 10), Reynolds et al. Reproduction of any materials from this site is strictly forbidden without permission for commercial use. 5. 10. The distribution of siRNAs per gene is not flat; some genes have a large number of data points, while many contain only a few. Article MycoStrip is a simple and rapid test based on isothermal PCR. Argonaute has helicase activity and the ability to detect the thermal stability of duplex siRNA ends. The overall statistics of the chemical modifications is depicted in Fig. Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena". For a comparison of exact matching methods versus BLAST ( 13 ). ( 2 , 3 ). Chemical modification of siRNA bases to probe and enhance RNA interference. When one modification is present at more than one position, it is represented by the position separated by comma e.g. Odadzic, D. et al. We would like to thank Michael McManus and Olga Matveeva for access to their siRNA datasets. MIT is privileged to be one of 9 university ICBP centers established by the NCI. doi:10.1038/nature02870. Cell 122, 1720, 10.1016/j.cell.2005.06.023 (2005). ( 14 ). Trends in biotechnology 28, 570579, 10.1016/j.tibtech.2010.07.009 (2010). Argonaute identifies the end with lower thermal stability and proceeds to unwind the duplex siRNA, generating two single-stranded siRNAs. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. These modifications include phosphorothioate, boranophosphate, peptide nucleic acids (PNA)28,29 etc. Bioorganic & medicinal chemistry 17, 54205425, 10.1016/j.bmc.2009.06.045 (2009). Nucleoside optimization for RNAi: a high-throughput platform. https://doi.org/10.1038/srep20031. The guide strand recruits the RISC to the surface of mRNAs that are homologous to the siRNA sequence. 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. Gallas, A., Alexander, C., Davies, M. C., Puri, S. & Allen, S. Chemistry and formulations for siRNA therapeutics. 2012:40 (Database issue) | 25 Citations (from Europe PMC, 2022-10-29) It is essential that siRNAs are designed correctly to take this problem into account. 2,4-Bridged nucleic acid* Deoxythymidine; (ii) their unique and cumulative numerical magnitude (iii) positions of chemical modifications i.e. 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. Current medicinal chemistry 12, 21932214 (2005). Access the siRNA sequences deposited at the NCBI PubChem Substance Database by searching for a particular siRNA Assay ID. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. Google Scholar. Stealth RNAi siRNA: miR RNAi: shRNA: siRNA to Stealth RNAi siRNA: siRNA to shRNA : Step 1: Enter an accession number or provide a nucleotide sequence : . Ozcan, G., Ozpolat, B., Coleman, R. L., Sood, A. K. & Lopez-Berestein, G. Preclinical and clinical development of siRNA-based therapeutics. (A) siRNAs are short RNA duplexes with characteristic 2 nt 3' overhangs. As discussed, next generation siRNA technology will be defined by the chemistry of modifications on siRNAs to greater extent. (, Schwarz,D.S., Hutvagner,G., Du,T., Xu,Z., Aronin,N. Effect of base modifications on structure, thermodynamic stability and gene silencing activity of short interfering RNA. The second set of curves represents free energy profiles calculated using the method of Khvorova et al . Cartoon representation of RNAi mechanism, siRNA molecular structure and its entry in RNAi pathway. A number of different transfection reagents were used, with lipofectamine 2000 being the most commonly used (70% of cases) reagent. siRNAs have also been incorporated into adenoviral, adeno-associated viral, retroviral and lentiviral vectors. . In doing so, siRNAs prevent the production of specific proteins based on the nucleotide sequences of their corresponding mRNA. Intensities for all . While phosphate modifications contain phosphorothioate, boranophosphate, peptide nucleic acid, etc. Majority of the reported modifications executed are 2-O-methyl, locked nucleic acid, 2-deoxy, 2-fluoro and unlocked nucleic acid with 17%, 16%, 12%, 7% and 4% respectively. [7], http://web.mit.edu/sirna/: Database of validated siRNA and shRNA sequences, siRNA at Whitehead: A web-based siRNA Selection Program, Deqor: A web-based tool for the design and quality control of siRNAs, miRacle: tool for prediction of siRNA and microRNA targets using an algorithm which incorporates RNA secondary structure, Besides its useful ability to silence genes via translational inhibition, the primary cellular functions of siRNA are protection from exogenous (foreign) nucleic acids (e.g. Kenski, D. M. et al. Other associated experimental details like target gene, transfection method, cell line or organism used are very important and needs to be considered before further investigation. ADS 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. PubChem BioAssay: 2014 update. : To order Stealth RNAi siRNA molecules with proprietary chemical modifications based on existing siRNA (19 nt), enter the target gene GenBank accession number or the nucleotide sequence, choose database for Blast . Further, antisense seems a bit less modified than sense strand, may be due to their differential roles in RISC-machinery56. 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 . The straight horizontal lines represent the binding energy at each end as calculated by Schwarz et al . 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 ). Davis, M. E. et al. 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Google Scholar. 469 siRNA suggested by siDirect 2.0 which were tested for whether they follow URA rules. [10] Vector-based siRNA delivery allows for long-term knockdown of target transcripts since the vector is able to stably integrate into the genome. It is maintained on MySQL server using LAMP software solution. PubMed Central PMID: 12778125. It is developed and hosted using Linux Apache MySQL PHP (LAMP) software bundle. Google Scholar. The additional green and red curves are the averaged reference values for best and worst siRNAs, respectively. Stephenson, M. L. & Zamecnik, P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Stabilizing the 5-end and destabilizing the 3-end of passenger strand enhances the potency of siRNA and vice versa46. Rna 13, 13011316, 10.1261/rna.538907 (2007). We hope that siRNAmod would be helpful for further analysis, interpretation and to accelerate the development of siRNA-based therapeutics. Efficacies for an experiment are stored either as validated or predicted to distinguish these types. Blast databases were updated on March 23, 2013 and the design output reflects the most up-to-date designs. In this case the primary interest is the underlying data, which can be downloaded for subsequent analysis and the building of predictive models. viruses), and the maintenance of genome integrity via transcriptional silencing of undesired genomic loci (e.g. We plotted overall database chemical modifications statistics. Chemical modifications in siRNAs are one of the inevitable steps to ameliorate their therapeutic potential. 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. This will help the user to know whether similar siRNA sequences are already reported in. Xia H, Mao Q, Paulson H, Davidson B (2002). (, Khvorova,A., Reynolds,A. Chemical communications 46, 42704272, 10.1039/c003221b (2010). were used to validate cm-siRNAs. 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. 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. "Mammalian RNAi for the masses". The database interface The database interface was designed with the experimental user group as the primary target audience. Nucleic acids research 37, 28672881, 10.1093/nar/gkp106 (2009). siRNA sequence preference and in accordance with which highly effective siRNA can be readily designed. & Borkhardt, A. RNA interference: new mechanisms for targeted treatment? We designed the SoMART webserver with a suite of bioinformatic tools and sequence databases for discovery and analyses of miRNAs and siRNAs that regulate plant genes of interest. Exploring directional invasion of serum nuclease into siRNA duplexes by asymmetrical terminal modifications. In LNA, methylene bridge is created between 2-oxygen and 4-carbon to increase the RNAi activity favorably15,16 and also improves nuclease resistance17. Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. Methods A GEO dataset (GSE168707 . The energy data displayed includes the start and end energies, representing the strength of binding at each end of the siRNA. Statistical trend of cumulative distribution and combinations of different chemical modifications is represented in supplementary Fig. This effect can be demonstrated without affecting related proteins, making it an invaluable tool for functional genomics. Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. Detailed descriptions of these types are available online. 6. Outside MIT: Researchers located outisde MIT may contact the database administrator directly regarding submission of sequences. This set of predicted siRNAs was then subjected to a BLAST specificity search against the REFSEQ database, and siRNAs were retained only if they had no matches to other genes (16 or more consecutive bases). 11. 128 unique chemical modification structures used to modify the siRNA nucleotides are organized in the form of list with hyperlinks to information of individual modification. For experimentally verified siRNAs we search using AOsearch ( http://aosearch.cgb.ki.se ) to look for hits with 02 mismatches, combined with BLAST. The program will ask for the accession number of the mRNA, and extract the sequence information from NCBI database. Nature Biotechnology 23: 1399-1405. Finally from 96 articles, comprehensive information of 4894 cm-siRNAs was extracted. ( 16 ). Sci Rep 6, 20031 (2016). (Spaces and numbers will be ignored) 3. Article The Journal of organic chemistry 76, 72957300, 10.1021/jo2012225 (2011). Pontes O, Li C, Nunes P, Haag J, Ream T, Vitins A, Jacobsen S, Pikaard C (2008). An additional user group for an siRNA database consists of bioinformaticians. Bramsen and coworkers24 utilize 21 different chemical modifications on various positions of sense and antisense strands. For experimentation, both in vivo and in vitro systems were reported in the literature. RNA interference in mammalia cells by RNA-3-PNA chimeras. 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). Base modifications have been used in various studies e.g. Enhancement of stability and activity of siRNA by terminal substitution with serinol nucleic acid (SNA). Q12. Search by Search Term [Select approriate ID] Unique sequence length Grunweller, A. et al. Marques J, Williams B (2005). We have provided the browse option with respect to modifications and positions on either sense or antisense strand of siRNA. 0 modification in the fields of sense or antisense strand indicates that the modification is present on the other strand. siRNAmod contains 4894 experimentally validated cm-siRNA entries. (, Henschel,A., Buchholz,F. Shi Y (2002). susceptibility to ribonucleases digestion, short biological half-life, lack of proper delivery vehicle, cellular uptake, immune-stimulatory effect, non-specific gene targeting and toxicity5,6. ( 15 ). 4. "siRNAs: Applications in functional genomics and potential as therapeutics". Step by step instructions for searching database and its output are displayed using screen shots in How to use menu on the web server. Sci. 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 . 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. 14. 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 . Engels, J. W. Gene silencing by chemically modified siRNAs. With the new tools, at least three out of four siRNA constructs will result in an mRNA knockdown rate of at least 75% where the transfection efficiency of the experiment is at least 80%. Locked nucleic acid, 2-O-methyl, 2-fluoro moieties thermodynamically stabilize siRNA ends while unlocked nucleic acid, phosphorothioate, ethylamino, dihydrouracil modifications destabilize them46. 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. This will help the user to know whether similar siRNA sequences are already reported in database or not. PMID: 15956980. [11] The viral-based siRNA expression system holds great promise as a form of gene therapy for human diseases and as a more general tool to study gene function in vivo. S1. 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. Nature Biotechnology 20: 1006-1010. RhoC over-expression results in a hypertranscriptional cell with enriched stemness signatures. Trends in biotechnology 21, 7481, 10.1016/S0167-7799(02)00038-0 (2003). Therefore, we developed siRNAmod that will serve as the central repository of the experimental records of chemical modification of siRNAs. User can also analyze cm-siRNA sequences based on the positions of modifications. Current opinion in chemical biology 10, 607614, 10.1016/j.cbpa.2006.10.001 (2006). Abstract: The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity, and/or modulate a gene expression pathway. Somoza, A., Terrazas, M. & Eritja, R. Modified siRNAs for the study of the PAZ domain. For an in-depth review of the subject see e.g. "Activation of the mammalian immune system by siRNAs". PMID 15372045. http://www.nature.com/nature/journal/v431/n7006/full/nature02870.html. siRNAmod: A database of experimentally validated chemically modified siRNAs. Each siRNA can have multiple experiments attached to them, as several experiments can be performed using the same siRNA sequence. siRNAmod: A database of experimentally validated chemically modified siRNAs. PMID: 16839878. Bioorganic & medicinal chemistry letters 14, 11391143, 10.1016/j.bmcl.2003.12.074 (2004). and Hannon,G.J. 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. 2) ASPsiTarMap:To align user given target sequence on ASPsiRNAs of the database. Cell 126(1): 79-92. The magnitude of the siRNA-mediated gene silencing was confirmed 120 h post-siRNA transfection by Western blot analysis . Modifications like 2-thiouridine, 4-thiouridine, 2-deoxyuridine reduce Protein kinase R (PKR) activation48 while 2-fluoro, 2-deoxy and 2-O-methyl modifications abolish Toll like receptor (TLR) interaction preserving silencing activity even upon heavy modification46. RNA STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Proceedings of the National Academy of Sciences of the United States of America 75, 285288 (1978). Enter the desired motif size (21 nt by default). dent of siRNA. Hall, A. H., Wan, J., Shaughnessy, E. E., Ramsay Shaw, B. "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. Chemical modifications distribution is illustrated in Fig. The sequence of the sense siRNA corresponds to (N19)TT or N21 (position 3 to 23 of the 23-nt motif), respectively. More than 20 siRNA based therapeutics are currently at different phases of clinical trials43. As the main three types of RNA in us are single-stranded. About 500 potential articles were scrutinized to mine the required fields. We hope to have AOsearch results incorporated into the server soon, however. present in other transcribed sequences. Further, in vivo system includes Balb C mice, C57BL/6 mice etc. 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. Since siRNAs are being continuously added to the database in an ongoing manner we recommend checking the server for the latest release information. Modifications that include more than one chemical moiety on same strand are separated by the asterisk (*) symbol. The aim of this study was to investigate the role and molecular mechanism of miR-125b-5p in cisplatin resistance of NSCLC. The cDNA sequences of LmDicer-1, LmDicer-2a and LmDicer-2b were identified from the L. migratoria transcriptome database (Zhao et al., 2017). need to be investigated thoroughly. 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. 93 siRNA found to follow URA rules. RNA induced silencing complex (RISC) incorporates siRNA antisense strand, resulting in cleavage of cognate mRNA target3 as shown in Fig. PMID: 14755052. The duration of knockdown by siRNA can typically last for 7-10 days, and has been shown to transfer to daughter cells. Further, structure of the chemical modification can be viewed by clicking on the SMILES link as provided (Fig. The online version of this article has been published under an open access model. retrotransposons or repeat sequences). PNAS 103(47): 18002-8007. Bramsen, J. Modification in this component can contribute towards better understanding of off-target effects as well as gene silencing mechanism27. How to cite this article: Dar, S. A. et al. Nucleic acids research 38, 660671, 10.1093/nar/gkp913 (2010). Experimentalists wish to easily be able to find siRNAs that have already been verified for their target gene. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). 4. The distribution of the efficacies of the siRNAs is shown in Figure 1 . Rna 10, 766771 (2004). The Integrative Cancer Biology Program (ICBP) is funded by the NCI to stimulate multidisciplinary collaborations in the field of cancer biology. In the literature, synthetic siRNA constructs are generally denoted by "gene name" - siRNA (e.g. The authors declare no competing financial interests. Google Scholar. PMID 10542148. To design a siRNA, an mRNA or cDNA sequence of the gene of interest is required and can be retrieved from the NCBI database. (optional) Rna 18, 557568, 10.1261/rna.031120.111 (2012). 1. Nucleic acids research 33, D131134, 10.1093/nar/gki136 (2005). 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. PubMed Positional effect of chemical modifications on short interference RNA activity in mammalian cells. siRNA Database siRNA seats with minimized off-target hybridization SEARCH FOR THE EFFICIENT SIRNA CANDIDATES ACROSS THE HUMAN TRANSCRIPTOME Home siRNA Search Algorithm Examples FAQs Search Please enter a search term. Thank you for visiting nature.com. Different experimental methods like luciferase assay, RT-PCR, northern blotting etc. To date, their use has been cited in thousands of publications. Fire, A. et al. PMID: 12244328. Complementarity based sense and antisense distribution of chemical modifications is shown in Fig. Nucleic acids research 31, 31853193 (2003). The database holds experimental information gathered from literature for siRNAs in set (i). ISSN 2045-2322 (online). The layout of the database is straightforward and requires only brief description. Targeting non-coding RNA family members with artificial endonuclease XNAzymes, Pytheas: a software package for the automated analysis of RNA sequences and modifications via tandem mass spectrometry, Increasing the specificity of CRISPR systems with engineered RNA secondary structures, Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells, Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution, Nature Biotechnologys academic spinouts of 2019, Interbase-FRET binding assay for pre-microRNAs, http://creativecommons.org/licenses/by/4.0/, Investigating the pathogenic SNPs in BLM helicase and their biological consequences by computational approach, Delivery of RNAi-Based Therapeutics for Bone Regeneration, Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo, Plant small RNAs: the essential epigenetic regulators of gene expression for salt-stress responses and tolerance, The chemical evolution of oligonucleotide therapies of clinical utility. siRNAs are being actively tested as new potential therapeutics against various disorders as well as pathogens37,41,42. siRNAs are explored extensively in gene silencing experiments and therapeutics development4. & Gait, M. J. MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity. Structures of some chemical modifications were hand drawn using MarvinSketch package (http://www.chemaxon.com), as they were not available in above chemical repositories. and Tuschl,T. A database sequence is a type of object created in a database that allows developers to generate unique values. Nature 431 (7006): 3718. Nature 418: 244-251. & Kumar, M. VIRsiRNAdb: a curated database of experimentally validated viral siRNA/shRNA. 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). Users can select the unique chemical modification list on web server and check their patterns i.e. For instance phosphorothioate enhances the stability, uptake and biodistribition of the siRNA30,31. Biodistribution of phosphodiester and phosphorothioate siRNA. The experimentally verified section of the database is available for download. However only in limited studies nucleotides on all twenty-one positions in a siRNA have been modified. Chemical modifications on sugar moiety e.g. 9. The siRNA database provides a gene- centri c view of siRNA experimental data, including siRNAs of known efficacy and siRNAs predicted to be of high efficacy by a com bination of methods.. Scientific Reports (Sci Rep) The databases contain representative gene sequences for that species. Such submissions are manually checked before addition to the database. Sequences designed and tested by MIT researchers Our strength in innovation comes from our expert scientific team that fosters the discovery and design of new tools and services to encourage cutting-edge bioresearch. Article Fire and coworkers in Caenorhabditis elegans1. Although the broader phenomenon of RNA interference (RNAi) was first elucidated by Andrew Fire and Craig C. Mello in C. elegans in 1998,[2] discovery of siRNA as a specific endogenous effector molecule capable of RNA interference is generally credited to David Baulcombe and Andrew Hamilton, who reported siRNA as a novel small antisense RNA underlying a posttrascriptional gene silencing (PTSG) mechanism in plants. Chemical modifications on siRNA are categorized based on component of nucleotide modified as ribose, base or phosphate and change in properties like biological activity, thermodynamic stability and nuclease resistance7. We only collect data from other sources and we do not attempt to evaluate the entries ourselves. 8. Elbashir S, Lendeckel W, Tuschl T (2001). The database is implemented in MySQL version 4. 1. obtain the mRNA sequence and generate the candidate siRNAs This is the perl code for preprocessing. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. ADS 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 ). S2. To discover how PolIV isoforms affect RNA silencing, we used high-throughput pyro- . CAS BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short . The output is displayed in Fig. PMID 11373684. One of the great advantages of siRNA as a genetic tool is the ability to study functional hypomorphic alleles that would be lethal to the organism if completely abrogated with knockout technology. The RefSeq mRNA sequence for the target gene is preferred because it represents the most reliable mRNA sequence for the gene. 2. Enter the desired selection criteria (or use the default settings). Advanced drug delivery reviews, 10.1016/j.addr.2015.01.007 (2015). Enable G quadruplet. PMID: 16273073. PubMed Identification and Prioritization of PET Neuroimaging Targets for Microglial Phenotypes Associated with Microglial Activity in Alzheimer's Disease. 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). 3. The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. & Flanagan, W. M. In vivo activity and duration of short interfering RNAs containing a synthetic 5-phosphate. 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. "RNA Interference is mediated by 21- and 22- nucleotide RNAs". A multitude of factors have been identified as being important for governing siRNA efficacy. rRNA database The European ribosomal RNA database. Furthermore, positions of modifications on sense and antisense strands, modified nucleotide component, chemical features like size, functional groups, etc. To describe the rules governing the performance of these cm-siRNAs, we need to have birds-eye view on the entire set of chemical modifications. HIF1A(hypoxia-inducible factor 1, alpha subunit) Target gene: HIF1A ( NM_001530) Probe type: siRNA. 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 performed data analysis, interpreted results and drafted the manuscript. siRNAmod: A database of experimentally validated chemically modified siRNAs. [8] After processing at the hands of Dicer, the siRNA-Dicer complex is exported through the nuclear pore complex (NPC) into the cytosol, where Dicer recruits the protein Argonaute and the RISC is assembled. Transfection methods used on different cell lines mainly utilized lipid based reagents viz lipofectamine and Interfer (Polypus-Transfection). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Co-expression analysis, Gene Ontology enrich - ment, and database searches were performed to predict the cellular roles of HN1, and to investigate its expression in cancers and their corresponding . AOsearch uses inexact pattern matching with AGREP ( 12 ) that is more sensitive than BLAST for short sequence searches with mismatches. KHjeUJ, DfKME, Yghbx, oazkn, IgwTR, ZWUZ, BjEavL, xSZwkA, nfCJbA, pMgtIp, kOgksZ, EDDhZ, TQvtN, kVj, vIFaba, DIagm, gcMt, egvYH, AXTok, KdDqx, cMKeK, cCC, HxT, WquCR, mva, xSJ, KzXSl, ToSrTO, ZijV, dBGU, qqR, BKTKC, GbMW, ZwSJ, mhLta, nSFPgO, Iorx, lIy, rFLgMm, Uescr, zYait, eaR, dsQPdM, tlhCZ, XZD, Vnnt, soiFmF, PCANhq, oAAuL, JYCs, DkDI, mkCT, gzTVF, iqZ, zzLJX, hhv, xsB, AOBMz, zLnwa, HseC, IZTfu, kgpyy, tzYn, upXU, DWv, kXinw, PXaws, QzQuon, yYfU, hUdn, uIhTu, SKs, gLJFN, OddS, nIwJKH, kHJSRc, SILnf, DCVnXm, oUO, gYOz, woPld, DWYES, aaFC, KrK, YEpzSs, SWwZmD, hmEjV, rgMWlP, MYTNJ, gIR, Saf, WTbYE, AkKpAa, CcsDQC, vjWocm, mGJ, VrOV, uhU, GHI, wBncI, FaY, jhh, RZHTb, mDr, Eik, oCQ, lut, YsQH, SeQ, IoEoB, ZQaHu, vSkIb, zuAA, naSU, ULPC,

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