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Comparative Study
. 2003 May 1;31(9):2401-7.
doi: 10.1093/nar/gkg338.

Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway

Torgeir Holen  1 , Mohammed Amarzguioui, Eshrat Babaie, Hans Prydz
Affiliations
Comparative Study

Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway

Torgeir Holen et al. Nucleic Acids Res. .

Abstract

RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, _targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical _target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3'-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of _target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.

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Figures

Figure 1
Figure 1
Antisense and double-strand siRNA show identical positional effects against the same _target sites on Tissue Factor mRNA. (A) Dose dependence of double-strand (ds) and antisense (as) siRNA. Complexation with Lipofectamine 2000 was performed in one batch for all samples and complexes were diluted in medium immediately before addition to cells. (B) Global and (C) local _target position effect. (B and C) Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with various antisense siRNA (200 nM) or double-strand (100 nM) siRNA. GAPDH was used as a control. Arrowheads (B, right panel) indicate cleavage products for antisense and double-strand siRNA.
Figure 1
Figure 1
Antisense and double-strand siRNA show identical positional effects against the same _target sites on Tissue Factor mRNA. (A) Dose dependence of double-strand (ds) and antisense (as) siRNA. Complexation with Lipofectamine 2000 was performed in one batch for all samples and complexes were diluted in medium immediately before addition to cells. (B) Global and (C) local _target position effect. (B and C) Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with various antisense siRNA (200 nM) or double-strand (100 nM) siRNA. GAPDH was used as a control. Arrowheads (B, right panel) indicate cleavage products for antisense and double-strand siRNA.
Figure 1
Figure 1
Antisense and double-strand siRNA show identical positional effects against the same _target sites on Tissue Factor mRNA. (A) Dose dependence of double-strand (ds) and antisense (as) siRNA. Complexation with Lipofectamine 2000 was performed in one batch for all samples and complexes were diluted in medium immediately before addition to cells. (B) Global and (C) local _target position effect. (B and C) Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with various antisense siRNA (200 nM) or double-strand (100 nM) siRNA. GAPDH was used as a control. Arrowheads (B, right panel) indicate cleavage products for antisense and double-strand siRNA.
Figure 2
Figure 2
Mutational inactivation of the antisense siRNA as-167. Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with 200 nM as-167 (wild-type, wt) or single mutated (as-s3, as-s7, as-s10, as-s13 or as-s16; the numerals refer to the position of the mutation, counted from the 5′ end of the siRNA sense strand) versions of antisense siRNA as-167. GAPDH was used as a loading control.
Figure 3
Figure 3
Comparison of the influence of chemical modification on antisense and double-strand siRNA. (A) Inactivation by methylation of antisense siRNA as-167. (B) Inactivation by methylation of double-strand siRNA hTF167i. (C) Inactivation of antisense siRNA by modification of the 3′-OH of the 3′-terminal nucleotide. as-FITC contains a FITC group in the 3′ position whereas in as-3d, 3′-deoxyribose is substituted for the terminal ribose. hTF167-3d is a double-strand siRNA in which as-3d is paired with a wild-type sense strand. (A–C) Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with 200 nM antisense siRNA or 100 nM double-strand siRNA. GAPDH was used as a loading control.
Figure 3
Figure 3
Comparison of the influence of chemical modification on antisense and double-strand siRNA. (A) Inactivation by methylation of antisense siRNA as-167. (B) Inactivation by methylation of double-strand siRNA hTF167i. (C) Inactivation of antisense siRNA by modification of the 3′-OH of the 3′-terminal nucleotide. as-FITC contains a FITC group in the 3′ position whereas in as-3d, 3′-deoxyribose is substituted for the terminal ribose. hTF167-3d is a double-strand siRNA in which as-3d is paired with a wild-type sense strand. (A–C) Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with 200 nM antisense siRNA or 100 nM double-strand siRNA. GAPDH was used as a loading control.
Figure 3
Figure 3
Comparison of the influence of chemical modification on antisense and double-strand siRNA. (A) Inactivation by methylation of antisense siRNA as-167. (B) Inactivation by methylation of double-strand siRNA hTF167i. (C) Inactivation of antisense siRNA by modification of the 3′-OH of the 3′-terminal nucleotide. as-FITC contains a FITC group in the 3′ position whereas in as-3d, 3′-deoxyribose is substituted for the terminal ribose. hTF167-3d is a double-strand siRNA in which as-3d is paired with a wild-type sense strand. (A–C) Northern analysis of Tissue Factor mRNA after transfection of HaCaT cells with 200 nM antisense siRNA or 100 nM double-strand siRNA. GAPDH was used as a loading control.
Figure 4
Figure 4
Competition between double-strand and antisense siRNA. Results in the black, grey and white columns are from three separate experiments. Experiments were performed in HaCaT cells as previously described, unless otherwise stated, and relative Tissue Factor/GAPDH expression normalised to levels in mock-transfected cells in each experiment. Column 1, mock; column 2, 50 nM antisense siRNA as-167; columns 3–5, 50 nM as-167 + 75 nM various inactive (column 3, hTF167-ds10/16) and irrelevant (column 4, PSK229i; column 5, BCR-ABL-1i) double-strand siRNA; column 6, 200 nM as-167; column 7, 50 nM as-167; columns 8–11, 50 nM as-167 + increasing concentrations (75, 100, 150 and 200 nM) of irrelevant double-strand siRNA (PSK208i); column 12, 200 nM as-167; column 13, 200 nM as-167 + 2000 nM PSK208i; column 14, 2000 nM PSK208i. In columns 12–14, cells were transfected with the less toxic agent Oligofectamine (Gibco BRL), enabling the use of higher total concentrations of RNA and excess of competitor, at the expense of reduced transfection efficiency.
Figure 5
Figure 5
Time-courses of the efficacy of double-strand and antisense siRNA. (A) Actinomycin D time-course. Cells were transfected with double-strand siRNA (100 nM) hTF167i or mock control as indicated above each lane. Actinomycin D (10 µg/ml) was added to the medium 2 h later and cells harvested at 4, 6 and 8 h post-transfection as indicated. The Tissue Factor signal was standardised to GAPDH and normalized to levels in the control at each time point. The results are representative for two independent experiments. (B) Long-term effect of antisense (as) and double-strand (ds) siRNA _targeting hTF167. Tissue Factor mRNA was measured at days 1 and 3 post-transfection and standardized to a GAPDH control. Data are from one of two independent experiments. (C) Short-term time-course of antisense and double-strand siRNA mRNA depletion. Cells were transfected as described and harvested at the indicated time points. Tissue Factor/GAPDH expression was normalised to levels in mock-transfected cells. The results are representative of two independent experiments.
Figure 5
Figure 5
Time-courses of the efficacy of double-strand and antisense siRNA. (A) Actinomycin D time-course. Cells were transfected with double-strand siRNA (100 nM) hTF167i or mock control as indicated above each lane. Actinomycin D (10 µg/ml) was added to the medium 2 h later and cells harvested at 4, 6 and 8 h post-transfection as indicated. The Tissue Factor signal was standardised to GAPDH and normalized to levels in the control at each time point. The results are representative for two independent experiments. (B) Long-term effect of antisense (as) and double-strand (ds) siRNA _targeting hTF167. Tissue Factor mRNA was measured at days 1 and 3 post-transfection and standardized to a GAPDH control. Data are from one of two independent experiments. (C) Short-term time-course of antisense and double-strand siRNA mRNA depletion. Cells were transfected as described and harvested at the indicated time points. Tissue Factor/GAPDH expression was normalised to levels in mock-transfected cells. The results are representative of two independent experiments.
Figure 5
Figure 5
Time-courses of the efficacy of double-strand and antisense siRNA. (A) Actinomycin D time-course. Cells were transfected with double-strand siRNA (100 nM) hTF167i or mock control as indicated above each lane. Actinomycin D (10 µg/ml) was added to the medium 2 h later and cells harvested at 4, 6 and 8 h post-transfection as indicated. The Tissue Factor signal was standardised to GAPDH and normalized to levels in the control at each time point. The results are representative for two independent experiments. (B) Long-term effect of antisense (as) and double-strand (ds) siRNA _targeting hTF167. Tissue Factor mRNA was measured at days 1 and 3 post-transfection and standardized to a GAPDH control. Data are from one of two independent experiments. (C) Short-term time-course of antisense and double-strand siRNA mRNA depletion. Cells were transfected as described and harvested at the indicated time points. Tissue Factor/GAPDH expression was normalised to levels in mock-transfected cells. The results are representative of two independent experiments.
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