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6 - Design and synthesis of small interfering RNA (siRNA)
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- By Queta Boese, Dharmacon, Inc., William S. Marshall, Dharmacon, Inc., Anastasia Khvorova, Dharmacon, Inc.
- Edited by Krishnarao Appasani, GeneExpression Systems, Inc., Massachusetts
- Foreword by Andrew Fire, Stanford University, California, Marshall Nirenberg
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- Book:
- RNA Interference Technology
- Published online:
- 31 July 2009
- Print publication:
- 17 January 2005, pp 103-117
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- Chapter
- Export citation
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Summary
Efficient RNA interference (RNAi) depends on siRNA design and synthesis
RNAi is a powerful technology with tremendous utility for functional genomic analysis, drug discovery strategies and therapeutic applications (Appasani 2003). While this pathway for post-transcriptional gene regulation is ubiquitous among eukaryotes, species-specific variations in the mechanism impact the utility of this pathway. These species-specific distinctions have strong implications with regard to the design, production, and delivery of the functional silencing intermediates. For example, in Caenorhabditis elegans (C. elegans), simple exposure by soaking (Tabara et al. 1998; Timmons and Fire 1998), feeding (Fraser et al. 2000; Timmons et al. 2001), or injecting (Fire et al. 1998) the nematode with long dsRNA is sufficient to induce prolonged and potent gene knockdown. Silencing efficiency appears to be due to siRNA-primed amplification of additional dsRNA from the mRNA target resulting in a secondary pool of Dicer processed duplexes (Sijen et al. 2001; Tijsterman et al. 2002). This mechanism is characteristic of post-transcriptional gene silencing in nematodes and other lower eukaryotes and is mediated by an RNA-dependent RNA polymerase [(RdRP) (Sijen et al. 2001; Martens et al. 2002)]. Invariably, several of the newly generated siRNAs will be capable of proficient gene-specific knockdown thereby eliminating the need to carefully design and synthesize a single siRNA silencing intermediate.
In mammalian cell culture models, preliminary attempts to induce RNAi using long dsRNA met with limited success (Tuschl et al. 1999; Caplen et al. 2000; Zhao et al. 2001).