SomaGenics, Inc., Santa Cruz, California 95060, USA.
RNA. 2010 Jan;16(1):106-17. doi: 10.1261/rna.1894510. Epub 2009 Dec 1.
Small hairpin RNAs (shRNAs) are widely used in RNAi studies and typically consist of a stem of 19-29 base pairs (bp), a loop of at least 4 nucleotides (nt), and a dinucleotide overhang at the 3' end. Compared with shRNAs with 21-29 bp stems, we have found that shRNAs with 19-bp or shorter stems (sshRNAs) possess some unique structure-activity features that depend on whether the antisense strand is positioned 5' or 3' to the loop (L- or R-type sshRNAs, respectively). L sshRNAs can have IC(50)s in the very low picomolar range, and sshRNAs with nominal loop sizes of 1 or 4 nt were at least as active as those with longer loops. L sshRNAs remained highly potent even when the 3' end of the antisense strand was directly linked with the 5' end of the sense strand. In this case, the sense strand can be shorter than the antisense strand, and the loop can be formed entirely by the 3' end of the antisense strand. Monomer sshRNAs are not processed by recombinant Dicers in vitro. Although they can form dimers that are sometimes Dicer substrates, their RNAi activity is not dependent on the formation of such structures. Our findings have implications for the mechanism of action of sshRNAs, and the ability to design highly potent shRNAs with minimal length is encouraging for the prospects of the therapeutic use of direct-delivered shRNAs.
小发夹 RNA（shRNA）被广泛应用于 RNAi 研究，通常由 19-29 个碱基对（bp）的茎部、至少 4 个核苷酸（nt）的环和 3' 端的双核苷酸突出组成。与具有 21-29 bp 茎部的 shRNA 相比，我们发现具有 19-bp 或更短茎部的 shRNA（sshRNA）具有一些独特的结构-活性特征，这些特征取决于反义链位于环的 5' 端还是 3' 端（分别为 L-或 R-型 sshRNA）。L sshRNA 的 IC（50）可低至皮摩尔级，并且名义上大小为 1 或 4 nt 的 loop 的 sshRNA 与具有更长 loop 的 sshRNA 一样具有活性。即使反义链的 3' 端与正义链的 5' 端直接连接，L sshRNA 仍然保持高度活性。在这种情况下，正义链可以短于反义链，并且 loop 可以完全由反义链的 3' 端形成。单体 sshRNA 不会在体外被重组 Dicer 加工。尽管它们可以形成二聚体，有时是 Dicer 的底物，但它们的 RNAi 活性并不依赖于这些结构的形成。我们的发现对 sshRNA 的作用机制有影响，并且能够设计具有最小长度的高效 shRNA 对直接递送 shRNA 的治疗应用前景令人鼓舞。
