siRNA design guidelines
Selected regionFor better efficacity, it is recommended to avoid 5' and 3'UTRs. These regions contain binding sequences for regulatory proteins that may affect the accessibility of the RNA target sequence to the RISC complex. However, we and others have successfully silenced the expression of several genes by targeting the 5' or 3'UTRs (1, 2, 3, 4). Therefore, 5' and 3'UTRs could also be considered when selecting a region on your target gene.
siRNA sizesiRNA-mediated RNAi is based on using dsRNA < 30 nt to avoid nonspecific silencing. According to Hannon et al. siRNA of 25-29 nt are generally more effective than shorter ones. However, we and others found that hairpin siRNAs with duplex lengths of 19-21 nt are as effective as longer hairpin siRNAs (5, 6, 7).
Thermodynamic properties of siRNAStatistical analysis of published siRNA sequences reveals that functional duplexes display lower internal stability at the 5' antisense end relative to the rest of the sequence than non-functional duplexes (8, 9).
Duplexes with a G at the 5'end and a A or T at the 3' end of the sense strand are favored (displayed in blue). Duplexes with a A at the 5' end and a A or T at the 3' end are less favored (displayed in black in "Advanced Search"). And duplexes with a G or C at the 3'end are discarded.
Starting nucleotideThe first nucleotide of the siRNA sequence can either be an A or a G. The pyrimidines C and T should be avoided because expression of RNAs from RNA polymerase III promoters is only efficient when the first transcribed nucleotide is a purine. In cases where your siRNA sequence starts with a C or T, we recommend to add an A as the first nucleotide. This addition will not affect the activity of your siRNA since it will generate a T at the end of the antisense siRNA strand that will be included in the termination signal maintaining its complementarity with the target sequence. This point is important since according to current knowledge recognition of the specific gene target is achieved by the antisense siRNA strand.
When using vector with the 7SK promoter, a G as the starting nucleotide should be preferred to a A. On the contrary, when using vector with the H1 promoter, a A as the starting nucleotide is more efficient than a G. That's the reason why we recommend to use 7SK vectors, since a G as starting nucleotide is preferred for thermodynamic stability (see above).
GC content of the siRNA sequenceIt is usually recommended to choose sequences with low GC content (between 30% and 55%). However, there are many examples of active siRNAs with high GC content (5, 11, 12).
BLAST against mRNA databasesIn order to increase specificity, siRNA Wizard filter candidate siRNA sequence to remove non-unique sequences by BLAST against an unique and comprehensive mRNA database. According to Jackson et al. (13) canditate siRNA presenting more than 13 contiguous nucleotides of sequence identity with another mRNA are discarded.
Due to the increase of the database that drastically reduce the number of siRNA candidate, only siRNA presenting homology with more than 5 Unigene sequence are discarded. The list of homolog genes to each putative siRNA with their expression profiles and link to Unigene cluster enables the user to choose siRNA corresponding to his/her needs.
BLAST against miRNA SEED databasesTo avoid off target effect, siRNA Wizard filter candidate siRNA sequence to remove sequence displaying a known miRNA SEED recognition region at 3' end.
Loop for short hairpin siRNAs (shRNA)We and others have tested a variety of sequences for the loop between the two complementary regions of a shRNA, ranging from 3 to 9 nt in length. Similar effectiveness have been obtained for loops of 5, 7 or 9 nt. We use a 7 nt loop sequence (TCAAGAG) for the psiRNA vectors.
References:1- Yokota T. et al., 2003.Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs. EMBO reports AOP.
2- Yu JY. et al., 2002. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells, PNAS 99(9):6047-6052
3- Rubinson DA. et al., 2003. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nature Genetics 33:401-406
4- Mcmanus MT. et al., 2002. Gene silencing using micro-RNA designed hairpins. RNA 8:842-850
5- Kim MH. et al., 2002. Successful inactivation of endogenous Oct-314 and c-mos genes in mouse preimplantation embryos and oocytes using short interfering RNAs. BBRC 296:1372-1377
6- Yu JY. et al., 2003. Simultaneous inhibition of GSK3a andGSKb using hairpin siRNA expression vectors. Molecular Therapy 7(2):228-236
7- Song E. et al., 2003. RNA interference targeting Fas protects mice from fulminant hepatitis. Nature Medicine 9(3):347-351
8- Khvorova, A. et al., 2003. Functional siRNAs and miRNAs Exhibit Strand Bias. Cell 115(2):209-216
9- Schwarz, D.S. at al., 2003. Asymmetry in the Assembly of the RNAi Enzyme Complex. Cell 115(2):199-208
10- Tiscornia G. et al., 2003. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. PNAS 100(4):1844-1848
11- Hasuwa H. et al., 2002. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Letters 532:227-230
12- Bertrand JR. et al., 2002. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. BBRC 296:1000-1004
13- Jackson A.L. et al., 2003. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 21(6): 635-637