Abstract
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.
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