Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

Small interfering RNAs (siRNAs) are applied for post-transcriptional gene silencing by binding target mRNA. A target coding region is usually chosen, although the 3′-untranslated region (3′-UTR) can also be a target. This study elucidates whether the coding region or 3′-UTR elicits higher repression. pFLuc and pRLuc are two reporter plasmids. A segment of FLuc gene was PCR-amplified and inserted behind the stop codon of the RLuc gene of the pRLuc. Similarly, a segment of RLuc gene was inserted behind the stop codon of FLuc. Two siFLuc and two siRLuc were siRNAs designed to target the central portions of these segments. Therefore, the siRNA encountered the same targets and flanking sequences. Results showed that the two siFLuc elicited higher repression when the FLuc segment resided in the coding region. Conversely, the two siRLuc showed higher repression when the RLuc segment was in the 3′-UTR. These results indicate that both the coding region and the 3′-UTR can be more effective targets. The thermodynamic stability of the secondary structures was analyzed. The siRNA elicited higher repression in the coding region when the target configuration was stable, and needed to be solved by translation. A siRNA may otherwise favor the target at 3′-UTR.