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. 2008 May;14(5):853-61.
doi: 10.1261/rna.704708. Epub 2008 Mar 26.

Experimental validation of the importance of seed complement frequency to siRNA specificity

Emily M Anderson  1 , Amanda Birmingham, Scott Baskerville, Angela Reynolds, Elena Maksimova, Devin Leake, Yuriy Fedorov, Jon Karpilow, Anastasia Khvorova
Affiliations

Experimental validation of the importance of seed complement frequency to siRNA specificity

Emily M Anderson et al. RNA. 2008 May.

Abstract

Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.

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Figures

FIGURE 1.
FIGURE 1.
Degradation of message RNA can occur by two separate pathways in RNAi. (A) Perfect or near-perfect complementarity of the siRNA guide strand with a target site leads to RISC-mediated endonucleolytic cleavage and degradation of the message. (B) The seed region (nucleotides ∼2–7) of the siRNA guide strand binds to the 3′ UTR of the mRNA and inhibits translation and/or promotes mRNA degradation.
FIGURE 2.
FIGURE 2.
3′ UTR hexamer frequency is widely distributed in the human genome. A plot of all possible 4096 hexamers (X-axis) versus the number of 3′ UTRs that contain a given hexamer (Y-axis) is presented. 3′ UTR sequences were derived from Refseq 15. The approximate seed frequency ranges from which low, medium, and high-frequency seeds were chosen is indicated by red circles.
FIGURE 3.
FIGURE 3.
Microarray signatures of GAPDH- and PPIB-targeting siRNAs identify a relationship between seed complement frequency and the magnitude of off-target signatures. (A) Heatmaps of off-target signatures generated by PPIB- and GAPDH-targeting siRNA having low (L), medium (M), or high (H) seed complement frequencies. Level of target knockdown is represented by zoomed-in bars above the heatmap. Dashed black boxes outline the nearly identical signatures of PPIB H17 and GAPDH H15, two siRNAs targeting unrelated genes but having identical seeds. A solid black box outlines signatures of GAPDH M1 and M8, two siRNAs that differ by a 1-nt shift in the target site. (A) Saturation on the color scale reflects fourfold up (red) and fourfold down (green) with respect to mock lipid transfection. (B,C) Box plots quantitating the number of off-targets induced by each class of siRNA targeting (B) GAPDH, and (C) PPIB.
FIGURE 4.
FIGURE 4.
Chimeric siRNAs demonstrate that the seed plays a dominant role in determining off-target signature size. (A) Diagram of the chimeric siRNA having seeds with low (L), medium (M), or high (H) SCFs associated with constant regions (position 1 and positions 8–19) targeting GAPDH. (B) Heatmaps generated from HeLa cells transfected with chimeric siRNAs. The knockdown and signature of the siRNA from which the scaffold was taken (G4 OT) is in the last lane. (C) Box plot showing the relationship between off-target signature size and SCFs.
FIGURE 5.
FIGURE 5.
A strong association exists between siRNA SCF and off-target induced phenotypes. (A) One hundred forty-four duplexes targeting PPIB, GAPDH, or ACTB were transfected into HeLa cells and assessed at 48 h using the APO-One assay. Sequences were divided into five seed complement frequency (SCF) ranges (91–588, 613–2382, 2385–2657, 2660–3301, and 3313–5377, 29 siRNAs per SCF range group with the exception of the highest group, which had 28 siRNAs) and plotted as the fraction of siRNAs in each group that induce positive phenotypes (scored as 1.5-fold or higher caspase induction over background). (B) Two hundred ninety duplexes targeting GAPDH or PPIB were individually transfected into cells as described by Federov et al. (2006). Following transfection, cell viability was assessed by alamarBlue according to the manufacturer's instructions. All measurements were performed in triplicate. Sequences were divided into five SCF ranges (187–3485, 3485–4556, 4561–5566, 5593–6606, and 6629–17844, 58 siRNAs per SCF group) and plotted as the fraction of siRNAs in each group that induced positive phenotypes (scored as inducing 50% or greater reduction in culture viability). SCF ranges used in both studies are derived from RefSeq 15 distributions.
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