By Lindsey Peacock
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Yang, D. , Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells, Proc. Natl. Acad. Sci. , 99, 9942, 2002. 31. Calegari, F. , Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA, Proc. Natl. Acad. Sci. , 99, 14236, 2002. 32. , Gene synthesis machines: DNA chemistry and its uses, Science, 230, 281, 1985. pmd 34 7/26/2004, 10:57 AM 35 Basics of siRNA Design and Chemical Synthesis 33.
The consequence of this is the necessity to design three or more duplexes per target to ensure selection of a functional siRNA. For example, in a study to assess siRNA as a high throughput tool in a screen for regulators of apoptosis,12 62% (31/50) of a preliminary set of duplexes was found to exhibit silencing functionality of F70 or more. With this in mind, three siRNAs each were designed to target a panel of 510 genes. Genes selected for further analyses were then targeted by two additional duplexes to confirm sequence specific silencing (total of five).
Furthermore, by modulating the affinity of RISC for the sense strand, this strategy may reduce competition between the two strands thus enhancing the potency of the siRNAs and reducing the effective concentration needed to induce silencing. Preliminary work using modified nucleosides in key positions of the duplex supports this hypothesis of biased strand selection by RISC (data not shown). Certain positional modifications in the sense strand had no effect on duplex silencing capacity yet effectively eliminated sense strand off-target effects.
AH-1 Huey Cobra by Lindsey Peacock