1] Department of Biology, University of York, York, UK [2] Current address: Institute of Cancer Therapeutics, University of Bradford, Bradford, UK.
Department of Biology, University of York, York, UK.
Mol Ther Nucleic Acids. 2014 Jan 7;2(1):e141. doi: 10.1038/mtna.2013.68.
Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3' end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3'-DNA/ds siRNA and includes a 3'-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3'-DNA/ds siRNA, (II) passenger-3'-DNA/ds siRNA, (III) guide-3'-DNA/ss siRNA, and (IV) passenger-3'-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I-III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3'-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3'-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3'-DNA/siRNA constructs are discussed.Molecular Therapy-Nucleic Acids (2014) 2, e141; doi:10.1038/mtna.2013.68; published online 7 January 2014.
通过 RNA 干扰（RNAi）进行选择性基因沉默涉及双链小干扰 RNA（ds siRNA），其由单链（ss）向导和过客 RNA 组成。siRNA 被 Ago2 和 C3PO（RNA 诱导沉默复合物（RISC）的内切核酸酶）识别和加工。RISC 切割过客 RNA，暴露向导 RNA，使其与同源 mRNA 靶标进行碱基配对。值得注意的是，过客 RNA 的 3'端可以容纳长达 19 个核苷酸的 DNA 延伸，而不会丧失 RNAi 功能。这种构建体称为过客-3'-DNA/ds siRNA，并包含 3'-核酸酶抗性迷你发夹结构。为了进一步测试这种新的修饰，我们现在比较了以下构建体：（I）向导-3'-DNA/ds siRNA、（II）过客-3'-DNA/ds siRNA、（III）向导-3'-DNA/ss siRNA 和（IV）过客-3'-DNA/ss siRNA。RNAi 靶标是 SIRT1，一种癌症特异性存活因子。构建体 I-III 均在非癌细胞和癌细胞中诱导 SIRT1 mRNA 和蛋白的选择性敲低，同时癌细胞发生凋亡性细胞死亡。缺乏 SIRT1 向导链的构建体 IV 没有作用。重要的是，3'-DNA 迷你发夹赋予构建体 I 和 II 核酸酶抗性。抗性需要双链 RNA 结构，因为单链向导-3'-DNA/ss siRNA（构建体 III）易受血清核酸酶的影响，导致 RNAi 活性丧失。3'-DNA/siRNA 构建体的潜在应用正在讨论中。分子治疗-核酸（2014 年）2，e141；doi:10.1038/mtna.2013.68；在线发表于 2014 年 1 月 7 日。
