SmartBase^TM siRNA Modifications

Effective siRNA Design Guidelines

Several guidelines have been proposed to design effective siRNA (3-5). The strategy for siRNA design is based on our present understanding of the biochemical mechanisms involved in RNA interference and, in particular, structural features that allow the antisense-strand of the siRNA duplex to be more efficiently incorporated into the RNA-induced silencing complex (RISC). The main characteristics are listed below (3).

Default parameters: (N19)TT

1. Low to medium GC content (30-50%).
2. Absence of internal repeats or palindromes.
3. Presence of an A at position 3 of the sense strand.
4. Presence of A at position 19 of the sense strand.
5. Absence of G or C at position 19 of the sense strand.
6. Presence of U at position 10 of the sense strand.
7. Absence of a G at position 13 of the sense strand.
8. At least 3 A/Us at positions 15-19 of the sense strand.

There are several other factors that influence effective functional performance of the designed siRNA; half-life of the siRNA is crucial for sustained activity and in many cases to increase effect for a longer time the dosage (concentration) is increased that leads to toxicity and off target effects. Similarly serum or cellular delivery methods can be improved. A common list of features for improvement is given below and possible sites in the nucleobase that can be modified to impart specific customized properties.

1. Increased nuclease resistance.
2. Increased duplex stability and manipulation of duplex stability.
3. Cellular delivery.
4. Surface attachment.

Common Modification Sites

1. Phosphodiester linkages.
2. Nucleic acid bases.
3. Sugar moieties.
4. Functional group addition.

References
1. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811. 2. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498. 3. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. & Khvorova, A. 2004. Rational siRNA design for RNA interference. Nat. Biotechnol. 22:326–330. 4. Amarzguioui, M., Lundberg, P., Cantin, E., Hagstrom, J., Behlke, M. & Rossi, J. 2006. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nature Protocols. 1:508-517. 5. Naito. Y., Yoshimura, J., Morishita, S. and Ui-Tei, K. 2009. siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect. BMC Bioinformatics 10:392. 6. Wang, H., Ghosh, A., Baigude, H., Yang, C-S., Qiu, L., Xia, X., Zhou, H., Rana, T.M. and Xu, Z. 2008. Therapeutic Gene Silencing Delivered by a Chemically Modified Small Interfering RNA against Mutant SOD1 Slows Amyotrophic Lateral Sclerosis Progression. J. Biol. Chem. 283: 15845-5852. 7. Jackson, A. L., Burchard, J., Schelter, J., Chau, B.N., Cleary, M., Lim, L. and Linsey, P.S. (2006) Widespread siRNA ‘‘off-target’’ transcript silencing mediated by seed region sequence complementarity.RNA 12:1179–1187. 8. Bramsen, J.B., Laursen, M.B., Nielsen, A.F., et al. 2009. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res. 37:2867–2881. 9. Vaish, N., Chen, F., Seth, S. et al. 2011. Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs. Nucleic Acids Res. 39:1823–1832.

Appendix References:
1. B.S. Sproat, A.I. Lamond, B. Beijer, P. Neuner, and U. Ryder, Nucleic Acids Res., 1989, 17, 3373. 2. T. Imanishi, and S. Obika, Chem Commun (Camb), 2002, 1653-1659. 3. S. Obika, Y. Hari, M. Sekiguchi, and T. Imanishi, Angew Chem Int Ed, 2001, 40, 2079-2081. 4. A.A. Koshkin, et al., Tetrahedron, 1998, 54, 3607-3630. 5. M. Petersen, and J. Wengel, Trends Biotechnol, 2003, 21, 74-81. 6. A. Sabahi, J. Guidry, G.B. Inamati, M. Manoharan, and P. Wittung-Stafshede, Nucleic Acids Res., 2001, 29, 2163-2170. 7. T. Ono, M. Scalf, and L.M. Smith, Nucleic Acids Res., 1997, 25, 4581-4588. *RNAi and siRNA
RNA interference (RNAi) is a specific and sequence dependent targeted gene silencing activity. RNAi acts by post transcriptional degradation of mRNA by small interfering RNAs (siRNA's) of the same sequence. The silencing approaches 100% and has to be empirically determined and optimized. Not every siRNA can effectively down regulate a gene. The process of RNA interference varies by individual siRNA while some do not exhibit any interference at all.