Article

Naito, Y. et al. dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Nucleic Acids Res. 33, W589-W591

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Nucleic Acids Research (Impact Factor: 9.11). 08/2005; 33(Web Server issue):W589-91. DOI: 10.1093/nar/gki419
Source: PubMed

ABSTRACT

Off-target effects are one of the most serious problems in RNA interference (RNAi). Here, we present dsCheck (http://dsCheck.RNAi.jp/), web-based online software for estimating off-target effects caused by the long double-stranded RNA (dsRNA) used in RNAi
studies. In the biochemical process of RNAi, the long dsRNA is cleaved by Dicer into short-interfering RNA (siRNA) cocktails.
The software simulates this process and investigates individual 19 nt substrings of the long dsRNA. Subsequently, the software
promptly enumerates a list of potential off-target gene candidates based on the order of off-target effects using its novel
algorithm, which significantly improves both the efficiency and the sensitivity of the homology search. The website not only
provides a rigorous off-target search to verify previously designed dsRNA sequences but also presents ‘off-target minimized’
dsRNA design, which is essential for reliable experiments in RNAi-based functional genomics.

Download full-text

Full-text

Available from: Kumiko Ui-Tei
    • "For non-target species, the likelihood of off-target effects due to gene homologs has been shown to be affected by both the number and position of mismatches between the siRNA strand(s) and the gene sequence it cross-hybridizes with (Du et al., 2005;Holen et al., 2005). Efforts at minimization of RNAi off-target effects has seen development of software such as siDirect (Naito et al., 2004) and dsCheck (Naito et al., 2005). In the case of transgenic plants, long undiced hairpin RNA (hpRNA) has been shown to promote more efficient RNAi in insects compared to siRNA readily processed by the host plant. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.
    No preview · Article · Nov 2015 · Journal of insect physiology
  • Source
    • "To ensure dsRNA specificity and to exclude potential non-specific targets, we used the E-RNAi [43], dsCheck [44] and DEQOR [45] programs to determine the dsRNA sequence. However, because the A. fluviatilis gene sequences are not available, we performed the analyses based on equivalent regions of the GSK-3 orthologous genes and genome sequences of other species (A. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W-) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism.
    Full-text · Article · Jun 2014 · PLoS ONE
  • Source
    • "However, to reinforce this inactivation, we introduced either another UAS- zfh-2RNAi insertion or the UAS-Dicer-2 (Dcr-2) construct (Dietzl et al., 2007) into some of our mutant combinations. The two RNA interference constructs are directed against different regions of the zfh-2 RNA (data not shown) and the RNAi construct previously described (Terriente et al., 2008) was checked with the DsCheck software (Naito et al., 2005; http://dscheck.rnai.jp) for possible offtargets (with 0 mismatches) and found to have none. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.
    Full-text · Article · Oct 2013 · Developmental Biology
Show more