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. 2001 Dec 3;20(23):6877-88.
doi: 10.1093/emboj/20.23.6877.

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate

S M Elbashir  1 , J Martinez, A Patkaniowska, W Lendeckel, T Tuschl
Affiliations

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate

S M Elbashir et al. EMBO J. .

Abstract

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

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Figures

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Fig. 1. Variation of the 3′ overhang of duplexes of 21 nt siRNAs. (A) Outline of the experimental strategy. The capped and polyadenylated sense target mRNA is depicted and the relative positions of sense and antisense siRNAs are shown. Eight series of duplexes according to the eight different antisense strands were prepared. The siRNA sequences and the number of overhanging nucleotides were changed in 1 nt steps. (B) Normalized relative luminescence of target luciferase (Photinus pyralis, Pp-luc) to control luciferase (Renilla reniformis, Rr-luc) in D.melanogaster embryo lysate in the presence of 5 nM blunt-ended dsRNAs (Elbashir et al., 2001b). The luminescence ratios determined in the presence of dsRNA were normalized to the ratio obtained for a buffer control (bu; black bar). Normalized ratios less than 1 indicate specific interference. (C–J) Normalized interference ratios for eight series of 21 nt siRNA duplexes. The sequences of siRNA duplexes are depicted above the bar graphs. Each part shows the interference ratio for a set of duplexes formed with a given antisense guide siRNA and five different sense siRNAs. The number of overhanging nucleotides (3′ overhang, positive numbers; 5′ overhang, negative numbers) is indicated on the x-axis. Data points were averaged from at least three independent experiments. Error bars represent standard deviations.
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Fig. 2. Variation of the length of the sense strand of siRNA duplexes. (A) Representation of the experiment. Three 21 nt antisense strands were paired with eight sense siRNAs. The siRNAs were changed in length at their 3′ end. The 3′ overhang of the antisense siRNA was 1 nt (B), 2 nt (C) or 3 nt (D), while the sense siRNA overhang was varied for each series. The sequences of the siRNA duplexes and the corresponding interference ratios are indicated.
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Fig. 3. Variation of the length of siRNA duplexes with preserved 2 nt 3′ overhangs. (A) Graphic representation of the experiment. The 21 nt siRNA duplex is identical in sequence to the one shown in Figures 1H and 2C. The siRNA duplexes were extended to the 3′ side of the sense siRNA (B) or the 5′ side of the sense siRNA (C). The siRNA duplex sequences and the respective interference ratios are indicated.
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Fig. 4. Substitution of the 2′-hydroxyl groups of the siRNA ribose residues. The 2′-hydroxyl groups (OH) in the strands of siRNA duplexes were replaced by 2′-deoxy (d) or 2′-O-methyl (Me). 2 and 4 nt 2′-deoxy substitutions at the 3′ ends are indicated as 2- and 4-nt d, respectively. Uridine residues were replaced by 2′-deoxythymidine.
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Fig. 5. Mapping of sense and antisense target RNA cleavage by 21 nt siRNA duplexes with 2 nt 3′ overhangs. (A) Representation of 32P (asterisk) cap-labelled sense and antisense target RNAs and siRNA duplexes. The position of sense and antisense target RNA cleavage is indicated by triangles on top and below the siRNA duplexes, respectively. (B) Mapping of target RNA cleavage sites. After 2 h incubation of 10 nM target RNA with 100 nM siRNA duplex in D.melanogaster embryo lysate, the 5′ cap-labelled substrate and the 5′ cleavage products were resolved on 6% sequencing gels. Length markers were generated by partial RNase T1 digestion (T1) and partial alkaline hydrolysis (OH) of the target RNAs. The bold lines to the left of the images indicate the region covered by the siRNA strands 1 and 5 of the same orientation as the target.
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Fig. 6. The 5′ end of a guide siRNA defines the position of target RNA cleavage. (A and B) Representation of the experimental strategy. The antisense siRNA was the same in all siRNA duplexes, but the sense strand was varied between 18 and 25 nt by changing the 3′ end (A) or 18 and 23 nt by changing the 5′ end (B). The position of sense and antisense target RNA cleavage is indicated by triangles on top and below the siRNA duplexes, respectively. (C and D) Analysis of target RNA cleavage using cap-labelled sense (top) or antisense (bottom) target RNAs. The residual amount of targeted substrate and the cap-labelled 5′ cleavage products are shown. The sequences of the siRNA duplexes are indicated and the length of the sense siRNA strands is marked on top. The control lane, marked with a dash in (C), shows target RNA incubated in absence of siRNAs. Markers were as described in Figure 5. The arrows in (D), bottom, indicate the target RNA cleavage sites that differ by 1 nt.
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Fig. 7. Sequence variation of the 3′ overhang of siRNA duplexes. The 2 nt 3′ overhang (NN, in grey) was changed in sequence and composition as indicated (T, 2′-deoxythymidine; dG, 2′-deoxyguanosine; asterisk, wild-type siRNA duplex). Normalized interference ratios were determined as described in Figure 1. The wild-type sequence is the same as depicted in Figure 4.
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Fig. 8. Sequence specificity of target recognition. The sequences of the mismatched siRNA duplexes are shown, modified sequence segments or single nucleotides are shaded in grey. The reference duplex (ref) and the siRNA duplexes 1–7 contain 2′-deoxythymidine 2 nt overhangs. The silencing efficiency of the thymidine-modified reference duplex was comparable to the wild-type sequence (Figure 7). Normalized interference ratios were determined as described in Figure 1.
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