Saetrom Pål, Snøve Ola, Nedland Magnar, Grünfeld Thomas B, Lin Yun, Bass Michael B, Canon Jude R
Interagon AS, Laboratoriesenteret, NO-7006 Trondheim, Norway.
Oligonucleotides. 2006 Summer;16(2):115-44. doi: 10.1089/oli.2006.16.115.
Short hairpin RNAs (shRNAs) and short interfering RNAs (siRNAs) probably enter different stages of the microRNA (miRNA) pathway for depletion of mRNA and suppression of protein translation. Primary and secondary structural characteristics that are shared between endogenous primary miRNA transcripts (pri-miRNAs) may contribute toward efficient biogenesis and potent silencing. This study investigates known miRNA transcripts for characteristics that are conserved between miRNAs and that distinguish them from random hairpins with similar lengths. The primary structure is conserved, as demonstrated by a significant presence or absence of certain bases at specific positions in the miRNA precursors and their flanking regions. The secondary structure is also conserved between miRNAs, as internal loops and bulges commonly appear in specific positions in the miRNA stem. The conservation of base-pairing continues past the mature duplex and 13 bases into the primary stem, with no detectable conservation of secondary structure beyond this region. Based on these observations, we have designed a hairpin construct that incorporates the most important characteristics present in endogenous miRNAs. Preliminary experiments suggest that this construct may rescue the efficacy of shRNA triggers that cannot be used with a miR-30-based hairpin, and vice versa.
短发夹RNA（shRNAs）和小干扰RNA（siRNAs）可能进入微小RNA（miRNA）途径的不同阶段，以实现mRNA的消耗和蛋白质翻译的抑制。内源性初级miRNA转录本（pri-miRNAs）之间共有的一级和二级结构特征可能有助于高效的生物合成和有效的沉默。本研究调查已知的miRNA转录本，寻找miRNA之间保守且能将它们与长度相似的随机发夹区分开的特征。miRNA前体及其侧翼区域特定位置某些碱基的显著存在或缺失证明了一级结构是保守的。miRNA之间的二级结构也保守，因为内环和凸起通常出现在miRNA茎的特定位置。碱基配对的保守性在成熟双链体之后持续到初级茎的13个碱基，在此区域之外未检测到二级结构的保守性。基于这些观察结果，我们设计了一种发夹构建体，其包含内源性miRNA中存在的最重要特征。初步实验表明，这种构建体可能挽救不能与基于miR-30的发夹一起使用的shRNA触发物的功效，反之亦然。
