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

Katoh T, Suzuki T - Nucleic Acids Res. (2007)

Bottom Line: Different efficacy of each siRNA is considered to result from sequence preference by protein components in RNAi.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

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.

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FCS measurement of binding affinity of TRBP against various siRNAs. (A) FCS analysis measured translational diffusion velocity of TRBP–siRNA complex passing through a confocal laser spot in a mixture. Human TRBP was titrated in a range of 0–200 nM. Concentration of TAMRA-labeled siRNA, ssRNA or free TAMRA is fixed to 1 nM. (B) Competition analysis using non-labeled siRNAs. Competitor siRNA activities versus diffusion times are plotted with error bars. Competitor siRNA sequences targeting EGFP mRNA start from nt positions 8, 25, 37, 50, 64, 99, 123, 166, 176, 242, 264, 299, 359, 371, 383, 416, 417, 418, 450, 476, 485, 532, 591, 617, 626, 662 and 701. Concentration of competitor siRNA and TAMRA-labeled EGFP 416 siRNA are 5 and 1 nM, respectively. TRBP is fixed to 150 nM. The correlation coefficient (R) is −0.589 (P = 1.2 × 10−3).
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Figure 8: FCS measurement of binding affinity of TRBP against various siRNAs. (A) FCS analysis measured translational diffusion velocity of TRBP–siRNA complex passing through a confocal laser spot in a mixture. Human TRBP was titrated in a range of 0–200 nM. Concentration of TAMRA-labeled siRNA, ssRNA or free TAMRA is fixed to 1 nM. (B) Competition analysis using non-labeled siRNAs. Competitor siRNA activities versus diffusion times are plotted with error bars. Competitor siRNA sequences targeting EGFP mRNA start from nt positions 8, 25, 37, 50, 64, 99, 123, 166, 176, 242, 264, 299, 359, 371, 383, 416, 417, 418, 450, 476, 485, 532, 591, 617, 626, 662 and 701. Concentration of competitor siRNA and TAMRA-labeled EGFP 416 siRNA are 5 and 1 nM, respectively. TRBP is fixed to 150 nM. The correlation coefficient (R) is −0.589 (P = 1.2 × 10−3).

Mentions: The periodical RNAi efficacy of every third position in siRNA sequences indicates the molecular interaction of siRNA with a protein component in RNAi machinery. In the cell, siRNA is initially recognized by Dicer with its partner protein to form RLC. We suppose that a part (not all) of siRNA functionality should be determined in this step. According to the biochemical data (43), it was reported that human Dicer itself has a weak affinity to dsRNA, while its partner protein TRBP has a dominant role for dsRNA recognition in Dicer–TRBP complex. We have examined binding affinity of recombinant human TRBP against EGFP siRNAs with various sequences. To measure siRNA–TRBP interaction precisely, we employed a new technique to study molecular interaction by FCS (52). FCS is a high-sensitive technique to observe a single fluorescence (F)-labeled molecule in a micro spot (1 femto-L) in a solution, which is illuminated by a confocal laser. FCS exactly determines the diffusion time of F-labeled molecules passing by the laser spot, which reflects the molecular weight of the labeled molecules. If the F-labeled siRNA interacts with TRBP, its increased molecular weight can be observed by its slow diffusion time. Another merit of this analysis is that FCS can analyze the complex formation under the equilibrium condition in a solution mixture. Different concentrations of recombinant human TRBP were incubated with TAMRA-labeled siRNA or ssRNA, then the diffusion time of the complex was measured (Figure 8A). We prepared two F-labeled siRNAs having equal GC content, which target positions 416 and 64 in EGFP mRNA. EGFP416 (GGCACAAGCUGGAGUACAA) was used as a representative siRNA with the 3-nt periodicity showing high activity (89.95); A or U at every 3n + 1 position (except position 1) and G or C at every 3n + 2 position. EGFP64 (GACGUAAACGGCCACAAGU) is a less active siRNA (53.64) with no 3-nt periodicity. In the absence of TRBP, diffusion time of both TAMRA-labeled siRNAs showed ∼400 μs. When TRBP was titrated, a dose-dependent prolongation of the diffusion time was observed in both EGFP416 and EGFP64 siRNAs, whereas no increase of the diffusion time was seen in their ssRNAs, demonstrating that TRBP specifically recognized double-stranded form of siRNAs. In the presence of 200 nM TRBP, the diffusion time of EGFP416 and EGFP64 siRNAs was found to be ∼1900 and ∼1200 μs, respectively. The result revealed that TRBP prefers to bind to efficacious siRNA having the 3-nt periodicity. To confirm this result, we carried out a competition experiment using 27 EGFP siRNAs having various RNAi activities (Figure 8B). Each non-labeled siRNA (5 nM) as a competitor was mixed with TAMRA-labeled EGFP416 siRNA (1 nM) in the presence of 150 nM TRBP. The FCS analysis monitoring diffusion time of the labeled siRNA revealed that efficacious siRNAs are more likely to compete with the labeled siRNA. The negative correlation between the diffusion time of the labeled siRNA and the RNAi activity of each competitor siRNA was observed with R factor −0.589 (P = 1.2 × 10−3), indicating that functionality of siRNA is associated with the binding affinity to TRBP. The 3-nt periodicity in siRNAs is supposed to originate from siRNA–TRBP interaction in the initial step of RNAi.Figure 8.


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

Katoh T, Suzuki T - Nucleic Acids Res. (2007)

FCS measurement of binding affinity of TRBP against various siRNAs. (A) FCS analysis measured translational diffusion velocity of TRBP–siRNA complex passing through a confocal laser spot in a mixture. Human TRBP was titrated in a range of 0–200 nM. Concentration of TAMRA-labeled siRNA, ssRNA or free TAMRA is fixed to 1 nM. (B) Competition analysis using non-labeled siRNAs. Competitor siRNA activities versus diffusion times are plotted with error bars. Competitor siRNA sequences targeting EGFP mRNA start from nt positions 8, 25, 37, 50, 64, 99, 123, 166, 176, 242, 264, 299, 359, 371, 383, 416, 417, 418, 450, 476, 485, 532, 591, 617, 626, 662 and 701. Concentration of competitor siRNA and TAMRA-labeled EGFP 416 siRNA are 5 and 1 nM, respectively. TRBP is fixed to 150 nM. The correlation coefficient (R) is −0.589 (P = 1.2 × 10−3).
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Figure 8: FCS measurement of binding affinity of TRBP against various siRNAs. (A) FCS analysis measured translational diffusion velocity of TRBP–siRNA complex passing through a confocal laser spot in a mixture. Human TRBP was titrated in a range of 0–200 nM. Concentration of TAMRA-labeled siRNA, ssRNA or free TAMRA is fixed to 1 nM. (B) Competition analysis using non-labeled siRNAs. Competitor siRNA activities versus diffusion times are plotted with error bars. Competitor siRNA sequences targeting EGFP mRNA start from nt positions 8, 25, 37, 50, 64, 99, 123, 166, 176, 242, 264, 299, 359, 371, 383, 416, 417, 418, 450, 476, 485, 532, 591, 617, 626, 662 and 701. Concentration of competitor siRNA and TAMRA-labeled EGFP 416 siRNA are 5 and 1 nM, respectively. TRBP is fixed to 150 nM. The correlation coefficient (R) is −0.589 (P = 1.2 × 10−3).
Mentions: The periodical RNAi efficacy of every third position in siRNA sequences indicates the molecular interaction of siRNA with a protein component in RNAi machinery. In the cell, siRNA is initially recognized by Dicer with its partner protein to form RLC. We suppose that a part (not all) of siRNA functionality should be determined in this step. According to the biochemical data (43), it was reported that human Dicer itself has a weak affinity to dsRNA, while its partner protein TRBP has a dominant role for dsRNA recognition in Dicer–TRBP complex. We have examined binding affinity of recombinant human TRBP against EGFP siRNAs with various sequences. To measure siRNA–TRBP interaction precisely, we employed a new technique to study molecular interaction by FCS (52). FCS is a high-sensitive technique to observe a single fluorescence (F)-labeled molecule in a micro spot (1 femto-L) in a solution, which is illuminated by a confocal laser. FCS exactly determines the diffusion time of F-labeled molecules passing by the laser spot, which reflects the molecular weight of the labeled molecules. If the F-labeled siRNA interacts with TRBP, its increased molecular weight can be observed by its slow diffusion time. Another merit of this analysis is that FCS can analyze the complex formation under the equilibrium condition in a solution mixture. Different concentrations of recombinant human TRBP were incubated with TAMRA-labeled siRNA or ssRNA, then the diffusion time of the complex was measured (Figure 8A). We prepared two F-labeled siRNAs having equal GC content, which target positions 416 and 64 in EGFP mRNA. EGFP416 (GGCACAAGCUGGAGUACAA) was used as a representative siRNA with the 3-nt periodicity showing high activity (89.95); A or U at every 3n + 1 position (except position 1) and G or C at every 3n + 2 position. EGFP64 (GACGUAAACGGCCACAAGU) is a less active siRNA (53.64) with no 3-nt periodicity. In the absence of TRBP, diffusion time of both TAMRA-labeled siRNAs showed ∼400 μs. When TRBP was titrated, a dose-dependent prolongation of the diffusion time was observed in both EGFP416 and EGFP64 siRNAs, whereas no increase of the diffusion time was seen in their ssRNAs, demonstrating that TRBP specifically recognized double-stranded form of siRNAs. In the presence of 200 nM TRBP, the diffusion time of EGFP416 and EGFP64 siRNAs was found to be ∼1900 and ∼1200 μs, respectively. The result revealed that TRBP prefers to bind to efficacious siRNA having the 3-nt periodicity. To confirm this result, we carried out a competition experiment using 27 EGFP siRNAs having various RNAi activities (Figure 8B). Each non-labeled siRNA (5 nM) as a competitor was mixed with TAMRA-labeled EGFP416 siRNA (1 nM) in the presence of 150 nM TRBP. The FCS analysis monitoring diffusion time of the labeled siRNA revealed that efficacious siRNAs are more likely to compete with the labeled siRNA. The negative correlation between the diffusion time of the labeled siRNA and the RNAi activity of each competitor siRNA was observed with R factor −0.589 (P = 1.2 × 10−3), indicating that functionality of siRNA is associated with the binding affinity to TRBP. The 3-nt periodicity in siRNAs is supposed to originate from siRNA–TRBP interaction in the initial step of RNAi.Figure 8.

Bottom Line: Different efficacy of each siRNA is considered to result from sequence preference by protein components in RNAi.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

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.

Show MeSH
Related in: MedlinePlus