Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing

Abstract

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

FIGURE 1.

Position-specific impact of chemical modification on siRNA specificity. (A) Position-specific effect of 2′-O-methyl modifications on silencing of off-target transcripts. siRNAs to MAPK14-193 were synthesized to contain either a single 2′-O-methyl modification or paired 2′-O-methyl modifications at overlapping consecutive pairs of nucleotides on the antisense (guide) strand. Chemically modified duplexes were phosphorylated on the antisense strand. siRNAs were transfected into HeLa cells, and changes in transcript regulation were analyzed by microarray profiling (Jackson et al. 2003). Shown is a heat map representing the entire signature of transcripts down-regulated by the wild-type MAPK14 siRNA (52 genes, X-axis) in 29 experiments (Y-axis). The transcripts shown were regulated with p ≤ 0.01, with no cuts placed on fold regulation. siRNA transcript regulations were analyzed using a consensus signature list for the unmodified MAPK14-193 duplex. Transcripts down-regulated in siRNA-transfected cells are shown in light blue, and transcripts up-regulated in siRNA-transfected cells are shown in magenta. Black indicates no change in regulation. Two or three independent experiments are shown for each modified or unmodified siRNA. The gold box indicates the location of the siRNA seed region (positions 2–8 of the guide strand). Transcripts are ordered by percent change in down-regulation (normalized mlratio change) across the wild-type signature. The arrow indicates the location of the target transcript MAPK14. (B) Comparison of MAPK14-193 modification walks. The average percent change in down-regulation (normalized mean log ratio change) for off-target transcripts relative to the wild-type siRNA sequence was calculated from the microarray for both base mismatches (Jackson et al. 2006) and chemical modification, analyzed as in A, and plotted as a function of position in the siRNA guide strand, 5′–3′ orientation. Green dashes indicate chemical modification; longer dashes represent paired modifications, and shorter dashes represent single residue modifications. The red line indicates base substitutions. Positions are marked with asterisks where modification reduced down-regulation significantly more than the random reductions seen between repeats of unaltered transfections (p < 1e − 4 with Bonferroni correction, Kolmogorov-Smirnov goodness-of-fit test, negative tail).

FIGURE 2.

Chemical modification reduces off-target silencing for all siRNAs tested. (A) Seven siRNAs were synthesized with (+) and without (−) the 2′-O-methyl modification as described in the text. Transcripts regulated by siRNAs with p ≤ 0.01 in at least one experiment are shown. Replicate experiments are shown for transfections when available. siRNA transcript regulations were clustered using a combined consensus signature list for repeats of each unmodified duplex. A common signature unrelated to duplex sequence was observed and removed from further analysis. Gold boxes indicate the unique transcript signature for each wild-type siRNA duplex. siRNAs from top to bottom: MPHOSPH1-2692, PIK3CA-2629, PRKCE-1295, SOS1-1582, VHL-2651, VHL-2652, MPHOSPH1-202. (B) Quantitation of the effects of chemical modification on off-target transcript regulation. Show is the mean decrease (±SD) of regulation of consensus off-target transcripts by chemical modification for the siRNAs shown in A. Also included are data for three additional siRNAs not shown in A: MAPK14-193, HEC-6346, and STK6–6347.

FIGURE 3.

Chemical modification preferentially reduces silencing of off-target transcripts and proteins. (A) siRNA dose titration analysis of on-target and off-target transcript silencing. Target transcript silencing was quantitated from the microarray. Off-target silencing was quantitated as the mean silencing of all off-target events, defined as those transcripts down-regulated with p < 0.01. No cuts were placed on fold down-regulation. In all cases, there was a greater reduction in off-target gene silencing relative to on-target silencing with the modified siRNA (p = 0.004, Wilcoxon rank-sum). (B) siRNA dose titration analysis of silencing of off-target RNA and protein levels. Transcript silencing was quantitated by TaqMan analysis 24 h following transfection of the PIK3CB siRNA at the indicated concentrations. YY1 protein levels were quantitated by Western analysis 48 h following transfection of the PIK3CB siRNA at the indicated concentrations. The extent of sequence complementarity between the PIK3CB siRNA and the YY1 off-target transcript are indicated. Red text indicates complementary nucleotides.

FIGURE 4.

2′-O-Methyl modification reduces off-target phenotypes. The impact of the modification on target transcript silencing was measured by bDNA for the six siRNAs indicated (upper left panel). The effect of the modification on target transcript silencing was also measured by microarray analysis (upper right panel). The effect of the modification on off-target transcript silencing was measured by microarray, quantitated as the mean percent silencing for the entire off-target signature for each siRNA (lower left panel). The effect of the modification on growth inhibition phenotype was measured for the same six siRNAs by Alamar Blue analysis 72 h post-transfection (lower right panel). The extent of on-target silencing showed no correlation with growth inhibition (R ² = 0.05), while the extent of off-target silencing was highly correlated with growth inhibition (R ² = 0.73).

FIGURE 5.

Chemical modification preferentially reduces silencing of off-target transcripts with weaker free energy in the seed region. (A) Responder and nonresponder transcripts were identified for siRNAs MAPK14-193 and MPHOSPH1-202. Responders (R) and nonresponders (N) were defined as described in the text. Transcripts regulated with p-values between these extremes are indicated as intermediate (I). (−) Indicates transcript regulation in the absence of modification, (+) indicates transcripts regulated in the presence of modification. Within each class (R, I, N), transcripts are sorted by percent change in down-regulation (normalized mlratio change) between modified and unmodified duplex, high (left) to low (right). Five (MAPK14) or four (MPHOSPH1) independent experiments are shown for unmodified and modified duplexes. The arrows indicate the presence of the target transcripts. (B) Free energy analysis of responder and nonresponder transcripts for four siRNAs: MAPK14-193, MPHOSPH1-202, KNTC2, STK6. Shown is the average ΔG for the four siRNAs. Asterisks mark positions where the signature alignment hexamer ΔG is more negative than the background alignment hexamer ΔG with p ≤ 0.01 by Kolmogorov-Smirnov goodness-of-fit test with Bonferroni correction for the number of positions examined.

FIGURE 6.

Position of the 5′ nucleotides of the siRNA guide strand in the complex with Piwi protein from Archaeoglobus fulgidus. (A) Position of the 5′ nucleotides of the siRNA guide (antisense) strand in the complex with Piwi protein from A. fulgidus. The crystal structure coordinates are from Ma et al. (2005). Piwi residues within 4 Å of the 2′-OH of base G2 are shown in white (Asn 155, Leu 156, Gln 159); all other Piwi residues are in purple. The sense RNA strand is not shown. White numbers and arrows show the 2′-OH positions of nucleotides 2–6 of the guide strand where methyl moieties were added in this study. Images were created using PyMol (DeLano Scientific). (B) View of the binding pocket rotated roughly 180° from the view in A. Nucleotides 1–6 are indicated. The divalent cation is shown as a green sphere.