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siRNA Design: It's All in the Algorithm

ictates what mRNA sequences are targeted for degradation. Schwarz et al. noted that the strand of the siRNA whose 5' end had a lower G/C content was preferentially loaded. In fact, siRNAs with high duplex stability at one end and low duplex stability at the other exhibited such significant strand bias that one strand could be incorporated into RISC to the exclusion of the other strand. The authors hypothesized that an RNA helicase responsible for unwinding siRNAs selects a strand for incorporation into the RISC based on the ease with which it can unwind the first 4-5 nucleotides of the duplex (Figure 5). These findings help explain the sequence bias between effective and ineffective siRNAs that was observed by Cenix. An siRNA target site with high G/C content at positions 1-4 and low G/C at positions 16-19 results in an siRNA whose antisense strand (the strand complementary to the desired mRNA target) has a low G/C at the 5' end and a high G/C at the 3' end. According to Schwarz et al., the antisense strand would be preferentially incorporated into RISC and target the proper mRNA for degradation.

Preferential uptake of the siRNA antisense strand by RISC has two important consequences: (1) The siRNA tends to have higher efficacy since the correct strand is efficiently taken up by the enzyme complex responsible for mRNA degradation and (2) the siRNA has higher specificity since the sense strand is not taken up by RISC and thus
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