Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity

Abstract

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.

Probing the shRNA characteristics that hinder Dicer recognition and consequently allow Ago-mediated processing and AgoshRNA activity

“…More potent target knockdown was observed in the luciferase reporter assays with the 17-to 19-bp minimized AgoshRNA compared with the 21-bp regular shRNAs, but a direct comparison remains difficult as different active RNA strands are generated that are probed on different reporter constructs. Such optimized AgoshRNA therapeutics may allow one to reduce the RNA dosage, thus reducing the chance of adverse effects, e.g., due to saturation of the components of the RNAi pathway or due to off-target effects on unrelated mRNAs (Jackson et al 2006). We also reasoned that AgoshRNAs can be improved further by increasing the base-pairing complementarity with the mRNA target by adaptation of the loop sequences.…”

Combinatorial RNAi for quantitative protein network analysis

“…Untarget effects, i.e., the repression of non-targeted proteins (7), increase with elevated concentrations of siRNA (9). Our results for actin and ErbB2 protein levels let us suppose that this increase of untarget effects is also true for the combinatorial RNAi strategy.…”

“…Furthermore, downregulation of mRNA is not always correlated with the amount of residual protein (7,9). Our data suggest that high fluctuations on mRNA level occur especially for genes which are related to the pathways under consideration, but these fluctuations do not necessarily reproduce on protein level.…”

Selective targeting of point-mutated KRAS through artificial microRNAs

“…Evidence shows that endogenous miRNAs possess multiple targets (33). It has also been proven that the presence of one or more perfect matches in 3′ UTR sequences of unintended targets can lead to considerable off-target effects, as with siRNAs when acting as miRNAs (34,35). Because our artificial RNA molecules were designed to act as endogenous miRNAs, possessing a central bulge, they also display extensive complementarity to the corresponding target sequence, like siRNAs, thus potentially reducing off-targeting.…”