RISC Preference And Off-target Effect Of Asymmetric Sirna

Since its discovery, siRNA has been widely used in research work, especially in reverse genetics. Recently, more and more siRNAs have been tested for their potential in therapeutic use including prevention of infectious disease like HIV-1infection. Although some siRNAs have made their way to clinical trials, there are inherent disadvantages for the use of siRNA. One of the biggest caveats is side effects, most of which are caused by off-target effect. siRNA is a species of double stranded RNA of19-22bp. The most widely used structure of siRNA consists of19base pairs and2-nucleotide overhangs at the3'-end of both strand. This structure inevitably caused significant off-target effects. It was previously reported that asymmetric siRNA with one strand shortened caused less off-target effects. Based on an siRNA targeting CCR5, a coreceptor for HIV-1, we designed a series of asymmetric siRNAs and compared their potency, specificity, off-target effects with their19+2 counterpart. The asymmetric siRNA showed comparable silencing ability with the symemetric19+2siRNA, despite their extreme unstability. Microarry analysis revealed that asymmetric siRNA caused significantly less off-target effects, which could not have been a result stemed from classical RNAi pathway, implicating an unidentified silencing mechanism that was inhibited by strand shortening.19+2siRNA is symmetric in topology, so it is necessay to choose siRNA sequence bearing significant thermodynamic asymmetry between its two ends to guide proper RISC loading. To investigate the correlation between siRNA structure and RISC preference, we compared the ability of different siRNA to inhibit the EGFP fluorescence using a pair of EGFP reporter plasmids. It was showed that3'-overhang was necessary for strand selection. Having both of the two3'-overhang, RISC loading was guided by thermodynamic asymmetry, so loading of both19+2siRNA and5'-shortened siRNA was dominated by this rule. But when siRNA was shortened from3'-end in one strand and only one3'-overhang was left, it seemed that the RISC loading was biased by structural asymmetry. The strand without3'-overhang was more likely to be abandoned. In summary, asymmetric design of siRNA is a promising approach to improve its performance.