Institute of Biotechnology in Medicine, Department of Biotechnology and Laboratory Science in Medicine, Yang-Ming University, Taipei 11221, Taiwan.
Biomed Res Int. 2013;2013:637850. doi: 10.1155/2013/637850. Epub 2013 Jun 12.
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.
小干扰 RNA（siRNAs）通过与靶 mRNA 结合来实现转录后基因沉默。通常选择靶编码区，但 3'非翻译区（3'-UTR）也可以作为靶区。本研究阐明了编码区或 3'-UTR 引起的抑制作用更高。pFLuc 和 pRLuc 是两种报告质粒。通过 PCR 扩增 FLuc 基因的一个片段，并将其插入 pRLuc 的 RLuc 基因的终止密码子之后。同样，RLuc 基因的一个片段被插入 FLuc 基因的终止密码子之后。设计了两个 siFLuc 和两个 siRLuc 来靶向这些片段的中心部分。因此，siRNA 遇到了相同的靶标和侧翼序列。结果表明，当 FLuc 片段位于编码区时，两个 siFLuc 引起的抑制作用更高。相反，当 RLuc 片段位于 3'-UTR 时，两个 siRLuc 显示出更高的抑制作用。这些结果表明，编码区和 3'-UTR 都可以作为更有效的靶区。还分析了二级结构的热力学稳定性。当靶结构稳定且需要通过翻译解决时，siRNA 在编码区引起更高的抑制作用。否则，siRNA 可能更倾向于 3'-UTR 上的靶标。
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