siRNA
MYCOsiRNA: an effective and specific siRNA designer for fungi and oomycete RNAi based on the assessments of Target site accessibility and Genome-wide Off-target selection
User-submitted target sequence for siRNA design
Users need to submit sequences (no more than 50 sequeces) as target for siRNA design. A typical siRNA target sequence can be a cDNA, EST, Unigene, mRNA or transcript of gene from genomic sequencing project, etc. The server will design potential siRNAs on the basis of user submission. Prior to analysis, back-end pipeline will check users submission by the following standards:
  • A valid sequence can only be FASTA format or single sequence without FASTA header(Pure Sequence, see above figures);
  • Server only allow users to submit one sequence for analysis once and maximal submission size is 15K;
  • The submitted sequence should be between 30 and 15360 nt in length, and pipeline will report error for invalid submission;
  • Only 'ATCGU' are valid sequence letters, other characters will be deleted.
Parameters for siRNA design
  • siRNA length: Input an integer between 19 and 40 as the length of the designed siRNA, default is 21 nt.
  • siRNA GC% between: Set the range of GC% content. The default is from 40 to 60, which means that the GC content of the siRNA antisense strand is between 40% and 60%.
  • Target accessibility: Set a percentage of non-helical nucleotides in the last 10 nucleotides of the target site. The default is 70 which means 70% or more in last 10 nucleotides of the target site are non-helical structure, indicated by “.”.
  • Species name: Choose the species name in which design siRNA will use to silence users’ submitted sequence. Selecting the species can automatic load its cDNA/transcript libraries for the assessment of genome wide off-target of siRNAs.
  • Maximum # of off-target genes:In order to get specific siRNA, there should be no off-target. Specific siRNA can only silence users’ submitted transcripts without affecting to other genes. However, practically it is not possible to get every siRNA without off-target. Therefore, users can minimize the maximum number of off-target, default is 5, to find those siRNA which is specific and cause less off-target silencing.Off-target gene were detected for a candidate siRNA using Blastn program with the query of antisense sequences, wordsize of 13 and e-value of 1.
Motifs
siRNA should be removed which contains toxic motifs. Studies showed that sequence motifs UGUGU, GUCCUUCAA (1,2), or UGGC (3) in siRNA show immunostimulatory activity and resulting reduce cell viability in the transfected animal cells (1,4-6). Moreover, some sequence motifs are commonly present among several genes. Therefore, it must avoid targeting those common motif otherwise antisense leads to silencing non-specific targets (7,8). Thus, we avoid uninterrupted >2x(CUG) (7,8), >2x(CCG), 2x(CGG) (9,10) repeat or WUAAAUW (11) motifs in the antisense strand of animal siRNA. We also avoid contiguous motif of >2x(CAN), or repeat of more than 4 same nucleotides like, AAAA, CCCC, GGGG, UUUU in siRNA of both animals and plants.
References:
  1. Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K. and MacLachlan, I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature biotechnology, 23, 457-462.
  2. Hornung, V., Guenthner-Biller, M.,z Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., Noronha, A., Manoharan, M., Akira, S., de Fougerolles, A. et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nature medicine, 11, 263-270.
  3. Fedorov, Y., Anderson, E.M., Birmingham, A., Reynolds, A., Karpilow, J., Robinson, K., Leake, D., Marshall, W.S. and Khvorova, A. (2006) Off-target effects by siRNA can induce toxic phenotype. RNA, 12, 1188-1196.
  4. Armstrong, M.E., Gantier, M., Li, L., Chung, W.Y., McCann, A., Baugh, J.A. and Donnelly, S.C. (2008) Small interfering RNAs induce macrophage migration inhibitory factor production and proliferation in breast cancer cells via a double-stranded RNA-dependent protein kinase-dependent mechanism. Journal of immunology, 180, 7125-7133.
  5. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H. and Williams, B.R. (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol, 5, 834-839.
  6. McAllister, C.S. and Samuel, C.E. (2009) The RNA-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic RNA sensors. The Journal of biological chemistry, 284, 1644-1651.
  7. Yu, Z., Teng, X. and Bonini, N.M. (2011) Triplet repeat-derived siRNAs enhance RNA-mediated toxicity in a Drosophila model for myotonic dystrophy. PLoS genetics, 7, e1001340.
  8. Lawlor, K.T., O'Keefe, L.V., Samaraweera, S.E., van Eyk, C.L. and Richards, R.I. (2012) Ubiquitous expression of CUG or CAG trinucleotide repeat RNA causes common morphological defects in a Drosophila model of RNA-mediated pathology. PloS one, 7, e38516.
  9. Sofola, O.A., Jin, P., Botas, J. and Nelson, D.L. (2007) Argonaute-2-dependent rescue of a Drosophila model of FXTAS by FRAXE premutation repeat. Human molecular genetics, 16, 2326-2332.
  10. Krzyzosiak, W.J., Sobczak, K., Wojciechowska, M., Fiszer, A., Mykowska, A. and Kozlowski, P. (2012) Triplet repeat RNA structure and its role as pathogenic agent and therapeutic target. Nucleic acids research, 40, 11-26.
  11. Ahmed, F., Benedito, V.A. and Zhao, P.X. (2011) Mining functional elements in messenger RNAs: overview, challenges, and perspectives. Frontiers in Plant Science, 2.