Of Sirna Specificity And Off-target Effects

RNA interfering (RNAi) is an evolutionarily conserved process where small interfering RNA (siRNA) specifically represses the expression of target genes. The effectors of this process are siRNA duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytoplasmasiRNAs are widely expected to become next generation of biological therapeutics, and they are initially anticipated to play a major role in treatment of diseases involving single nucleotide polymorphisms (SNPs) where discrimination against single nucleotide variation between wild-type and mutant alleles is demanded.In RNA interference, siRNAs can silence target genes specifically by binding corresponding mRNA. But recent researches confirm that siRNAs are likely to act on non-target genes and degrade them. This is so-called "off target effects".siQuant is a reporter-based siRNA validation system that provides a rapid and precise approach to evaluate individual siRNA in high-throughput manner. The efficacy of a siRNA could be read out by measuring the activity of a reporter enzyme, of which the mRNA was tagged by a short sequence representing the target site of tested siRNA.Silencing specificity and off target effects are critical issues in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th,12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 260 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.