The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA.
The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA.
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DOI：
10.1371/journal.pone.0002602
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发表时间：
2008-07-02
期刊：
影响因子：
3.7
通讯作者：
Weinberg MS
中科院分区：
文献类型：
--
作者：
Saayman S;Barichievy S;Capovilla A;Morris KV;Arbuthnot P;Weinberg MS
RNA干扰（RNAi）效应器已用于抑制哺乳动物细胞中的流氓RNA。然而，快速进化的序列，如人类免疫缺陷病毒1型（HIV-1），需要多种靶向方法，以防止逃逸变体的出现。表达的长发夹RNA（lhRNA）最近已被用作产生靶向高度变异序列的多个短干扰RNA（siRNA）的策略。我们的目的是通过设计包含siRNA编码序列的不同组合的IhRNA来表征表达的IhRNA产生沉默三个非连续HIV-1位点的独立siRNA的能力。所有IhRNA都能够沉默个别靶序列。然而，沉默效率连同单个IhRNA衍生的siRNA的浓度从发夹的茎基部（第一位置）朝向环侧减小。针对HIV-1的沉默功效主要由位于茎基部的siRNA序列介导。可以通过调整第一和第二位置siRNA连接处的间隔排列来改善第一和第二位置siRNA，但第三位置siRNA的沉默在很大程度上仍然无效。尽管IhRNA为组合RNAi提供了优势，但我们表明，用编码两个以上相邻独立siRNA的序列难以实现跨IhRNA双链体跨度的良好沉默功效。
RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.
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