RNA Interference by Single-and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure
RNA Interference by Single-and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure
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DOI：
10.1038/mtna.2013.68
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发表时间：
2014-01-01
影响因子：
8.8
通讯作者：
Milner, Jo
中科院分区：
文献类型：
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作者：
Allison, Simon J.;Milner, Jo
RNA 干扰 (RNAi) 选择性基因沉默涉及由单链 (ss) 引导 RNA 和过客 RNA 组成的双链小干扰 RNA (ds siRNA)。 siRNA 由 Ago2 和 C3PO（RNA 诱导沉默复合物 (RISC) 的核酸内切酶）识别和加工。 RISC 切割乘客 RNA，暴露引导 RNA，以便与其同源 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 19 个核苷酸的 DNA 延伸，而不会丧失 RNAi 功能。该构建体被称为passenger-3'-DNA/ds siRNA，并包含3'-核酸酶抗性微型发夹结构。为了进一步测试这种新颖的修饰，我们现在比较了以下构建体：（I）guide-3'-DNA/ds siRNA，（II）passenger-3'-DNA/ds siRNA，（III）guide-3'-DNA/ss siRNA，和（IV）passenger-3'-DNA/ss siRNA。 RNAi 的目标是 SIRT1，一种癌症特异性生存因子。构建体 I-III 均诱导非癌细胞和癌细胞中 SIRT1 mRNA 和蛋白质的选择性敲低，并伴随癌细胞中的细胞凋亡。构建体 IV 缺乏 SIRT1 引导链，没有效果。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guide-3'-DNA/ss siRNA（构建体III）对血清核酸酶敏感，并伴有RNAi活性的丧失。讨论了 3'-DNA/siRNA 构建体的潜在应用。
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.