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Effect of siRNA with an asymmetric RNA/dTdT overhang on RNA interference activity.

ABSTRACT: Small interfering RNAs (siRNAs) guide RNA-induced silencing complexes (RISC) to target mRNAs for sequence-specific silencing. A fundamental aspect of this highly coordinated process is a guide strand-specific loading of the siRNA duplex into the RISC for the accurate target recognition, which is currently dictated by certain duplex parameters such as thermodynamics. Here, we show that minor changes in the overhang structure have profound effects on the extent to which the individual strands of the siRNA duplex participate in RNAi activity. We demonstrate that the two strands of the siRNA are similarly eligible for assembly into RISC for the siRNAs with symmetric overhangs, whereas those with asymmetric RNA/deoxythymidine dinucleotide (dTdT) overhangs exhibit a distinct preference in favor of a strand with an RNA overhang that drives a mature RISC affinity to the desired target. We believe that this additional determinant provides a plausible and simple approach for improving the strand selection, thereby considerably increasing a specificity of target silencing.

SUBMITTER: Park JH

PROVIDER: S-EPMC4162433 | biostudies-other | 2014 Oct

REPOSITORIES: biostudies-other

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Publications

Effect of siRNA with an asymmetric RNA/dTdT overhang on RNA interference activity.

Park June Hyun JH Hong Sun Woo SW Yun Soyeong S Lee Dong-Ki DK Shin Chanseok C

Nucleic acid therapeutics 20141001 5

Small interfering RNAs (siRNAs) guide RNA-induced silencing complexes (RISC) to target mRNAs for sequence-specific silencing. A fundamental aspect of this highly coordinated process is a guide strand-specific loading of the siRNA duplex into the RISC for the accurate target recognition, which is currently dictated by certain duplex parameters such as thermodynamics. Here, we show that minor changes in the overhang structure have profound effects on the extent to which the individual strands of t ...[more]

PMID: 25211666

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Project description:Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype-phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.

Dataset Information

Effect of siRNA with an asymmetric RNA/dTdT overhang on RNA interference activity.

Publications

Effect of siRNA with an asymmetric RNA/dTdT overhang on RNA interference activity.

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