This is a site with links to several siRNA services, including siRNA base sequence searches, specificity searches, known siRNA molecule searches, and target sequences. One of the sites, called siSVM, allows users to predict efficacy of siRNAs given their base sequence using features derived from the siRNA sequence. siSVM is designed to allow common methods of siRNA design to be included in the search. This includes motif rules,energy conditions and specificity searching. The second site it links to, siRNA specificity prediction, allows users to perform a specificity search for siRNAs to avoid off-target effects. SpecificityServer is designed to help you identify potential non-specific matches to your siRNA. It incorporates the latest information about non-specific matches (sequence-specific only). The third site it links to, siRNAdb, is a database of known siRNA molecules. It provides a list of sirnaID, target, geneID, geneAcc, TargetStart, and targetEnd.Category: RNA sequence databases

VIRsiRNAdb is a curated database of experimentally validated viral siRNA / shRNA targeting diverse genes of 42 important human viruses including influenza, SARS and Hepatitis viruses. Submissions are welcome. Currently, the database provides detailed experimental information of 1358 siRNA/shRNA which includes siRNA sequence, virus subtype, target gene, GenBank accession, design algorithm, cell type, test object, test method and efficacy (mostly quantitative efficacies). Further, wherever available, information regarding alternative efficacies of above 300 siRNAs derived from different assays has also been incorporated. The database has facilities like search, advance search (using Boolean operators AND, OR) browsing (with data sorting option), internal linking and external linking to other databases (Pubmed, Genbank, ICTV). Additionally useful siRNA analysis tools are also provided e.g. siTarAlign for aligning the siRNA sequence with reference viral genomes or user defined sequences. virsiRNAdb would prove useful for RNAi researchers especially in siRNA based antiviral therapeutics development.

The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

The canonical seed region, positions 2–7, is underlined in the guide strand. 3′ overhangs are in lowercase. The modified (M) versions are identical in sequence to the unmodified siRNA and contain a 2′-O-methyl ribosyl substitution at position 2 of the guide strand.

A database that serves as a repository for both, sequences of published functional siRNA molecules targeting human genes and important technical details of the corresponding gene silencing experiments. It aims at supporting the setup and actual procedure of specific RNAi experiments in human cells.

siRNA sequences used in this study.

*Stealth-990 corresponds to the siRNA against EV71 VP2.

The VIRsiRNA database contains details of siRNA/shRNA which target viral genome regions. It provides efficacy information where available, as well as the siRNA sequence, viral target and subtype, as well as the target genomic region.

DNA sequence and relationships for survivin siRNA 1 (dna)

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA-mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24 nt long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA-targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants.

Protein-Protein, Genetic, and Chemical Interactions for Hartig JV (2009):Endo-siRNAs depend on a new isoform of loquacious and target artificially introduced, high-copy sequences. curated by BioGRID (https://thebiogrid.org); ABSTRACT: Colonization of genomes by a new selfish genetic element is detrimental to the host species and must lead to an efficient, repressive response. In vertebrates as well as in Drosophila, piRNAs repress transposons in the germ line, whereas endogenous siRNAs take on this role in somatic cells. We show that their biogenesis depends on a new isoform of the Drosophila TRBP homologue loquacious, which arises by alternative polyadenylation and is distinct from the one that functions during the biogenesis of miRNAs. For endo-siRNAs and piRNAs, it is unclear how an efficient response can be initiated de novo. Our experiments establish that the endo-siRNA pathway will target artificially introduced sequences without the need for a pre-existing template in the genome. This response is also triggered in transiently transfected cells, thus genomic integration is not essential. Deep sequencing showed that corresponding endo-siRNAs are generated throughout the sequence, but preferentially from transcribed regions. One strand of the dsRNA precursor can come from spliced mRNA, whereas the opposite strand derives from independent transcripts in antisense orientation.

Functional RNAi based screening is affected by large numbers of false positive and negative hits due to prevalent sequence based off-target effects. We performed a druggable genome targeting siRNA screen intended to identify novel regulators of E-cadherin (CDH1) expression, a known key player in epithelial mesenchymal transition (EMT). Analysis of primary screening results indicated a large number of false-positive hits. To address these crucial difficulties we developed an analysis method, SENSORS, which, similar to published methods, is a seed enrichment strategy for analyzing siRNA off-targets in RNAi screens. Using our approach, we were able to demonstrate that accounting for seed based off-target effects stratifies primary screening results and enables the discovery of additional screening hits. While traditional hit detection methods are prone to false positive results which are undetected, we were able to identify false positive hits robustly. Transcription factor MYBL1 was identified as a putative novel target required for CDH1 expression and verified experimentally. No siRNA pool targeting MYBL1 was present in the used siRNA library. Instead, MYBL1 was identified as a putative CDH1 regulating target solely based on the SENSORS off-target score, i.e. as a gene that is a cause for off-target effects down regulating E-cadherin expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5′-untranslated region (5′-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options.

Mechanistic Investigation of Resistance to SHR-A1811 and T-DXd, the Third Generation of HER2-ADC, in Breast Cancer

Yellow lupine (Lupinus luteus L.) belongs to a legume family that benefits from symbiosis with nitrogen-fixing bacteria. Its seeds are rich in protein, which makes it a valuable food source for animals and humans. Yellow lupine is also the model plant for basic research on nodulation or abscission of organs. Nevertheless, the knowledge about the molecular regulatory mechanisms of its generative development is still incomplete. The RNA-Seq technique is becoming more prominent in high-throughput identification and expression profiling of both coding and non-coding RNA sequences. However, the huge amount of data generated with this method may discourage other scientific groups from making full use of them. To overcome this inconvenience, we have created a database containing analysis-ready information about non-coding and coding L. luteus RNA sequences (LuluDB). LuluDB was created on the basis of RNA-Seq analysis of small RNA, transcriptome, and degradome libraries obtained from yellow lupine cv. Taper flowers, pod walls, and seeds in various stages of development, flower pedicels, and pods undergoing abscission or maintained on the plant. It contains sequences of miRNAs and phased siRNAs identified in L. luteus, information about their expression in individual samples, and their target sequences. LuluDB also contains identified lncRNAs and protein-coding RNA sequences with their organ expression and annotations to widely used databases like GO, KEGG, NCBI, Rfam, Pfam, etc. The database also provides sequence homology search by BLAST using, e.g., an unknown sequence as a query. To present the full capabilities offered by our database, we performed a case study concerning transcripts annotated as DCL 1–4 (DICER LIKE 1–4) homologs involved in small non-coding RNA biogenesis and identified miRNAs that most likely regulate DCL1 and DCL2 expression in yellow lupine. LuluDB is available at http://luluseqdb.umk.pl/basic/web/index.php.

Figures S1-S9. Figure S1. Schematic representation of repetitive DNA sequences present in the HPRT vectors used in this study. The location and length (bp) of each SINE/LINE fragment is based on the UCSC Genome Browser Database: Update 2006 (Nucleic Acids Res. 34:D590–D598, 2006). Figure S2. Impact of siRNA-mediated knockdown of DNA ligase I or IIIα on integration frequency. (A) The nucleotide sequence of LIG1 and LIG3 siRNA. These siRNAs were designed as reported previously (Nucleic Acids Res. 36: 3297–3310, 2008). (B) Western blot analysis for DNA ligase I and IIIα in siRNA-transfected Nalm-6 wild-type and LIG4-null cells. M, mock-transfected. (C, D) Integration frequency in wild-type and LIG4-null cells treated with LIG1 siRNA (C) or LIG3 siRNA (D). A non-targeting vector (pβactin-His; Nucleic Acids Res. 36: 6333–6342, 2008) was used for transfection. The integration frequency in mock-transfected wild-type cells was taken as 1, and the relative integration frequency was calculated. Figure S3. Structural features of gene-targeting vectors used for the analysis of integration frequency. (A) Fundamental structure of targeting vectors. In all the vectors, 5' and 3' arms flank the drug-resistance gene cassette (Puror), which is placed in the forward direction. (B) Structural features of the fourteen gene-targeting vectors used. Shown are the lengths of 5' and 3' arms and SINE/LINE sequences within each arm and the integration frequency. The length of SINE/LINE is based on the UCSC Genome Browser Database: Update 2006 (Nucleic Acids Res. 34:D590–D598, 2006). Figure S4. Integration frequency of targeting vector as a function of the length of repetitive DNA sequences. Integration frequencies of pHPRT8.9-Puro(+), pHPRT2.2-Puro(+), and twelve other gene-targeting vectors are shown as a function of the total length of SINE sequence (A), 5’-arm SINE length (B), 3’-arm SINE length (C), the total length of LINE sequence (D), 5’-arm LINE length (E), and 3’-arm LINE length (F). See also Figure 5. Figure S5. Correlation between the integration frequency and repetitive DNA sequences. (A) Integration frequencies of pHPRT8.9-Puro(+), pHPRT2.2-Puro(+), and twelve other gene-targeting vectors as a function of the total length of SINE/LINE sequences. Note that this graph is the same as Figure 5D. (B) Same as (A), except that the three vectors have been omitted from the data set. Note that the redrawn fitted curve reveals a stronger correlation between the total SINE/LINE length and the integration frequency. See text and Figure 5 for details. Figure S6. Gene-targeting efficiency is little affected by the length of homology arms. Targeting efficiencies are shown as a function of the total length of homology arms of the targeting vector. The twelve non-HPRT gene-targeting vectors were used for the analysis (see Figure S3B in File S1). Figure S7. PCR primers used to amplify the homology arms of pHPRT2.2-Puro vectors. The restriction sites used to construct the arm-deleted vectors are shown in red (SacI) or blue (SalI). Figure S8. PCR primers used to amplify the homology arms of non-HPRT targeting vectors. Red denotes attB sequences. Figure S9. Schematic representation of construction of imperfect pHPRT8.9-Puro vectors lacking the 3' arm (A) or 5' arm (B). (PDF)

Small interfering RNA (siRNA) has revolutionized biomedical research and drug development through precise post-transcriptional gene silencing technology. Despite its immense potential, siRNA therapy still faces technical challenges, such as delivery efficiency, targeting specificity, and molecular stability. To address these challenges and facilitate siRNA drug development, we have developed siRNAEfficacyDB, a comprehensive database that integrates experimentally validated siRNA efficacy data. This database contains 3544 siRNA records, covering 42 target genes and seven cell lines. It provides detailed information on siRNA sequences, target genes, inhibition efficiencies, experimental techniques, cell lines, siRNA concentrations, and incubation times. siRNAEfficacyDB offers a user-friendly web interface that makes it easy to query, browse and analyze data, enabling efficient access to siRNA-related information. In summary, siRNAEfficacyDB provides a useful data foundation for siRNA drug design and optimization, serving as a valuable resource for advancing computer-aided siRNA design research and nucleic acid drug development. siRNAEfficacyDB is freely available at https://cellknowledge.com.cn/siRNAEfficacy.

This is the supplementary data of the research. The supplementary data 1 is the multiple sequence alignment file. While the other one (no 2) is mainly about the structural annotation.

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5′ UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5′ untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5′ UTR RNA folding, and mutant 5′ UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5′ UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5′ end annealing or 3′ overhanging nucleotides. Replication promotion by per...

Protein-Protein, Genetic, and Chemical Interactions for He XJ (2009):NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. curated by BioGRID (https://thebiogrid.org); ABSTRACT: RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.