SomaGenics, Inc., Santa Cruz, California 95060, USA.
RNA. 2010 Jan;16(1):118-30. doi: 10.1261/rna.1901810. Epub 2009 Nov 30.
Small hairpin RNAs (shRNAs) with 19-base-pair, or shorter, stems (short shRNAs [sshRNAs]) have been found to constitute a class whose mechanism of action appears to be distinct from that of small interfering RNAs (siRNAs) or longer shRNAs. These sshRNAs can be as active as canonical siRNAs or longer shRNAs. Their activity is affected by whether the antisense strand is positioned 5' or 3' to the loop (L or R sshRNAs, respectively). Dicer seems not to be involved in the processing of sshRNAs, although the mechanism of target gene suppression by these hairpins is through Ago2-mediated mRNA cleavage. In this study, the effects of chemical modifications on the potency, serum stability, and innate immune response of sshRNAs were investigated. Deoxynucleotide substitution and 2'-O-methyl (2'-OMe) modification in the sense strand and loop did not affect silencing activity, but, unlike with siRNAs, when placed in the antisense strand these modifications were detrimental. Conjugation with bulky groups at the 5'-end of L sshRNAs or 3'-end of R sshRNAs had a negative impact on the potency. Unmodified sshRNAs in dimer form or with blunt ends were immunostimulatory. Some modifications such as 3'-end conjugation and phosphorothioate linkages on the backbone of the sshRNAs could also induce inflammatory cytokine production. However, 2'-OMe substitution of sshRNAs abrogated the innate immune response and improved the serum stability of the hairpins.
小发夹 RNA（shRNA）具有 19 个碱基对或更短的茎（短 shRNA [sshRNA]），已被发现构成一类，其作用机制似乎与小干扰 RNA（siRNA）或更长的 shRNA 不同。这些 sshRNA 可以像经典 siRNA 或更长的 shRNA 一样活跃。它们的活性受反义链位于环的 5'端还是 3'端（分别为 L 或 R sshRNA）的影响。尽管这些发夹通过 Ago2 介导的 mRNA 切割抑制靶基因，但 Dicer 似乎不参与 sshRNA 的加工。在这项研究中，研究了化学修饰对 sshRNA 的效力、血清稳定性和固有免疫反应的影响。在有义链和环中脱氧核苷酸取代和 2'-O-甲基（2'-OMe）修饰不影响沉默活性，但与 siRNA 不同，当放置在反义链中时，这些修饰是有害的。L sshRNA 的 5'端或 R sshRNA 的 3'端与大体积基团的缀合对效力有负面影响。未修饰的 sshRNA 以二聚体形式或以平头末端存在具有免疫刺激性。一些修饰，如 3'端缀合和 sshRNA 骨架上的硫代磷酸酯键，也可以诱导炎症细胞因子的产生。然而，sshRNA 的 2'-OMe 取代消除了固有免疫反应并提高了发夹的血清稳定性。
