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Specific residues at every third position of siRNA shape its efficient RNAi activity.

ABSTRACT: Small interfering RNA (siRNA) induces sequence-specific post-transcriptional gene silencing in mammalian cells. Different efficacy of each siRNA is considered to result from sequence preference by protein components in RNAi. To obtain mechanistic insight into siRNA functionality, here we describe a complete data set of siRNA activities targeting all possible position of a single mRNA in human cells. Seven hundred and two siRNAs covering open reading frame of enhanced green fluorescent protein mRNA ( 720 bases) were examined with minimized error factors. The most important finding is that specific residues at every third position of siRNAs greatly influence its RNAi activity; the optimized base composition at positions 3n + 1 (4,7,10,13,16,19) in siRNAs have positive effects on the activity, which can explain the waving siRNA activity with 3 nucleotides (nt) periodicity in the sequential positions of mRNAs. Since there was an obvious correlation between siRNA activity and its binding affinity to TRBP, a partner protein of human Dicer, the 3-nt periodicity might correlate with the affinity to TRBP. As an algorithm ('siExplorer') developed by this observation successfully calculated the activities of siRNAs targeting endogenous human genes, the 3-nt periodicity provides a new aspect unveiling siRNA functionality.

SUBMITTER: Katoh T

PROVIDER: S-EPMC1851635 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Specific residues at every third position of siRNA shape its efficient RNAi activity.

Katoh Takayuki T Suzuki Tsutomu T

Nucleic acids research 20070126 4

Small interfering RNA (siRNA) induces sequence-specific post-transcriptional gene silencing in mammalian cells. Different efficacy of each siRNA is considered to result from sequence preference by protein components in RNAi. To obtain mechanistic insight into siRNA functionality, here we describe a complete data set of siRNA activities targeting all possible position of a single mRNA in human cells. Seven hundred and two siRNAs covering open reading frame of enhanced green fluorescent protein mR ...[more]

PMID: 17259216

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Project description:Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75-80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease.

Project description:Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi.

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Specific residues at every third position of siRNA shape its efficient RNAi activity.

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Specific residues at every third position of siRNA shape its efficient RNAi activity.

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