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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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Related in: MedlinePlus

Prediction of siRNA activities for endogenous gene. (A) Scatter plot for 50 GAPDH–siRNA activities versus their prediction values. The correlation coefficient is 0.776 (P = 3.4 × 10−11). Number near the spot corresponds to the passenger strand 5′ end position of each siRNA in GAPDH mRNA. (B) Prediction of sequential siRNA activities for GAPDH. The nt sequences show the sense strand of GAPDH–siRNAs for positions 950–974. The color code for bases represent bases that have positive or negative effects on the activity as described in the legend of Figure 3B. siRNA activities (%) for GAPDH are shown in bars. The prediction value calculated for each siRNA is plotted in red.
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Figure 7: Prediction of siRNA activities for endogenous gene. (A) Scatter plot for 50 GAPDH–siRNA activities versus their prediction values. The correlation coefficient is 0.776 (P = 3.4 × 10−11). Number near the spot corresponds to the passenger strand 5′ end position of each siRNA in GAPDH mRNA. (B) Prediction of sequential siRNA activities for GAPDH. The nt sequences show the sense strand of GAPDH–siRNAs for positions 950–974. The color code for bases represent bases that have positive or negative effects on the activity as described in the legend of Figure 3B. siRNA activities (%) for GAPDH are shown in bars. The prediction value calculated for each siRNA is plotted in red.

Mentions: shRNA was transcribed by T7 RNA polymerase and then converted into siRNA by limited digestion with ribonuclease T1 (47) (see supporting information). We prepared 702 species of siRNAs that target all positions of the 720 nt EGFP coding region. The siRNA sequences targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (1008 nt) start from nucleotide positions 8, 23, 33, 43, 53, 65, 93, 109, 120, 132, 145, 167, 198, 231, 241, 304, 337, 349, 368, 383, 402, 409, 448, 473, 491, 519, 539, 587, 595, 613, 636, 655, 676, 710, 734, 746, 761, 777, 789, 798, 802, 817, 845, 863, 889, 916, 941, 950, 974 and 989 for the experiment shown in Figure 7A, and sequential nucleotide positions 950–974 for the experiment shown in Figure 7B. The siRNA sequences targeting β-catenin mRNA start from nucleotide positions 579, 598, 654, 1224, 1694, 1681, 1898, 2101, 2219 and 2254 (Figure S4). For fluorescence correlation spectroscopy (FCS), 3′-TAMRA (6-carboxy-tetramethylrodamine)-labeled passenger strands for EGFP416 and EGFP64 were chemically synthesized and purified by HPLC (Hokkaido System Science, Inc.). Four nanomoles of TAMRA-labeled RNA was annealed with equal amount of its non-labeled guide strand at 37°C for 30 min in 20 μl of 30 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate and 2 mM magnesium acetate, which was then purified by non-denaturing gel.


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

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

Prediction of siRNA activities for endogenous gene. (A) Scatter plot for 50 GAPDH–siRNA activities versus their prediction values. The correlation coefficient is 0.776 (P = 3.4 × 10−11). Number near the spot corresponds to the passenger strand 5′ end position of each siRNA in GAPDH mRNA. (B) Prediction of sequential siRNA activities for GAPDH. The nt sequences show the sense strand of GAPDH–siRNAs for positions 950–974. The color code for bases represent bases that have positive or negative effects on the activity as described in the legend of Figure 3B. siRNA activities (%) for GAPDH are shown in bars. The prediction value calculated for each siRNA is plotted in red.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851635&req=5

Figure 7: Prediction of siRNA activities for endogenous gene. (A) Scatter plot for 50 GAPDH–siRNA activities versus their prediction values. The correlation coefficient is 0.776 (P = 3.4 × 10−11). Number near the spot corresponds to the passenger strand 5′ end position of each siRNA in GAPDH mRNA. (B) Prediction of sequential siRNA activities for GAPDH. The nt sequences show the sense strand of GAPDH–siRNAs for positions 950–974. The color code for bases represent bases that have positive or negative effects on the activity as described in the legend of Figure 3B. siRNA activities (%) for GAPDH are shown in bars. The prediction value calculated for each siRNA is plotted in red.
Mentions: shRNA was transcribed by T7 RNA polymerase and then converted into siRNA by limited digestion with ribonuclease T1 (47) (see supporting information). We prepared 702 species of siRNAs that target all positions of the 720 nt EGFP coding region. The siRNA sequences targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (1008 nt) start from nucleotide positions 8, 23, 33, 43, 53, 65, 93, 109, 120, 132, 145, 167, 198, 231, 241, 304, 337, 349, 368, 383, 402, 409, 448, 473, 491, 519, 539, 587, 595, 613, 636, 655, 676, 710, 734, 746, 761, 777, 789, 798, 802, 817, 845, 863, 889, 916, 941, 950, 974 and 989 for the experiment shown in Figure 7A, and sequential nucleotide positions 950–974 for the experiment shown in Figure 7B. The siRNA sequences targeting β-catenin mRNA start from nucleotide positions 579, 598, 654, 1224, 1694, 1681, 1898, 2101, 2219 and 2254 (Figure S4). For fluorescence correlation spectroscopy (FCS), 3′-TAMRA (6-carboxy-tetramethylrodamine)-labeled passenger strands for EGFP416 and EGFP64 were chemically synthesized and purified by HPLC (Hokkaido System Science, Inc.). Four nanomoles of TAMRA-labeled RNA was annealed with equal amount of its non-labeled guide strand at 37°C for 30 min in 20 μl of 30 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate and 2 mM magnesium acetate, which was then purified by non-denaturing gel.

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