Lee Lim | PubMed Health

PubMed2006

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity.

Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended ("off-target") consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3' UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with dependence on siRNA concentration similar to that of silencing of the target transcript. Thus, short stretches of sequence complementarity to the siRNA or shRNA seed region are key to the silencing of unintended transcripts.

3' Untranslated Regions↗

PubMed2006

Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

Base Sequence↗