RNAi in human cells: Basic structural and functional features of small interfering RNA
RNAi in human cells: Basic structural and functional features of small interfering RNA
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DOI：
10.1016/s1097-2765(02)00652-4
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发表时间：
2002-09-01
期刊：
影响因子：
16
通讯作者：
Rana, TM
中科院分区：
文献类型：
--
作者：
Chiu, YL;Rana, TM
我们研究了RNA干扰（RNAi）在人类细胞中的作用机制。在这里，我们证明了siRNA反义链的5‘羟基末端的状态决定了RNAi活性，而3’末端阻滞在体内是耐受的。从人细胞中分离的反义链的5′羟基端被磷酸化，3′端生物素组不能有效去除。我们发现siRNA不需要完美的a型螺旋来产生干扰效应，但反义靶RNA双链需要a型结构。引人注目的是，补骨脂素对siRNA双链的交联并没有完全阻断RNA干扰，这表明siRNA螺旋的完全解绕对于体内RNAi活性不是必需的。这些结果表明，通过RNA依赖的RNA聚合酶进行RNA扩增对人类细胞中的RNAi并不是必需的。
We investigated the mechanism of RNA interference (RNAi) in human cells. Here we demonstrate that the status of the 5' hydroxyl terminus of the antisense strand of a siRNA determines RNAi activity, while a 3' terminus block is tolerated in vivo. 5' hydroxyl termini of antisense strands isolated from human cells were phosphorylated, and 3' end biotin groups were not efficiently removed. We found no requirement for a perfect A-form helix in siRNA for interference effects, but an A-form structure was required for antisense-target RNA duplexes. Strikingly, crosslinking of the siRNA duplex by psoralen did not completely block RNA interference, indicating that complete unwinding of the siRNA helix is not necessary for RNAi activity in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.