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Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.

ABSTRACT: Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region-a total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in targeting binding-free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also use this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNA•RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous.

SUBMITTER: Joseph TT

PROVIDER: S-EPMC3935976 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.

Joseph Thomas T TT Osman Roman R

Proteins 20120210 5

Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region-a total of 21 substitutions. We report results from s ...[more]

PMID: 22275138

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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

Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.

Publications

Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.

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