7100–7109 Nucleic Acids Research, 2008, Vol. 36, No. 22
doi:10.1093/nar/gkn902

Published online 6 November 2008

Thermodynamic stability and Watson–Crickbase pairing in the seed duplex are major
determinants of the efficiency of thesiRNA-based off-target effectKumiko Ui-Tei*, Yuki Naito, Kenji Nishi, Aya Juni and Kaoru Saigo

Received September 15, 2008; Revised and Accepted October 27, 2008

ABSTRACT
Short interfering RNA (siRNA) may down-regulate
many unintended genes whose transcripts possess
complementarity to the siRNA seed region, whichcontains 7 nt. The capability of siRNA to induce
this off-target effect was highly correlated with thecalculated melting temperature or standard freeenergy change for formation of protein-free seed
duplex, indicating that thermodynamic stabilityof seed duplex formed between the seed andtarget is one of the major factor in determining thedegree of off-target effects. Furthermore, unlike
intended gene silencing (RNA interference), offtarget effect was completely abolished by introduction of a G:U pair into the seed duplex, and this loss
in activity was completely recovered by a second
mutation regenerating Watson–Crick pairing, indicating that seed duplex Watson–Crick pairing is
also essential for off-target gene silencing. The
off-target effect was more sensitive to siRNA concentration compared to intended gene silencing,which requires a near perfect sequence match
between the siRNA guide strand and target mRNA.
INTRODUCTION
A growing body of evidence from large-scale knockdown
experiments (1–5) suggests that short interfering RNA(siRNA) could generate oﬀ-target eﬀects through amechanism similar to that of target silencing by
microRNAs (miRNAs) (6–8), which inﬂuence the expression levels of many transcripts in a tissue-speciﬁc manner(9). The 30 UTRs of oﬀ-target transcripts or miRNAtargets are complementary to the guide strand (GS)seed region, nucleotide positions 2–8 (Supplementary
Figure S1) (3–6). Within cells, the GS binds to Ago toform an RNA-induced silencing complex (RISC) (10,11).
In RISC, the seed nucleotides are presumed to be present
on the surface of Ago in a quasi-helical form to serve as
the entry or nucleation site for mRNA (12,13). However,
little is known about the molecular basis that determines
the eﬃciency of seed-dependent oﬀ-target gene silencing.
To clarify this point, using a reporter system and microarray proﬁling, we examined the relationship between
stability of the protein-free seed duplex and the capabilityof siRNA to induce oﬀ-target eﬀects.
MATERIALS AND METHODS
Cell culture
Human HeLa cells were cultured and subjected to genesilencing as described previously (14,15). Cells were plated
into an individual well of 24-well culture plate at1  105 cells/ml (1 ml/well) 24 h prior transfection.
Transfection was carried out using Lipofectamine 2000
(Invitrogen). Sequences of siRNAs chemically synthesized(Proligo) are listed in Supplementary Table S1.
Plasmid construction and gene-silencing assay
All plasmids constructed are derivatives of psiCHECK-1(Promega). Chemically synthesized 75-bp-long ds oligonucleotides, each including three tandem repeats of an identical 23 bp (Supplementary Table S2) and cohesive XhoI/
EcoRI ends, were inserted into the corresponding restriction enzyme sites of psiCHECK-1 to generate psiCHECKcm and psiCHECK-sm (Figure 1A and B). Inserted cm or
sm targets, each 21 nt in length, were expressed as partof the 30 UTR of Renilla luc mRNA in transfected cells.
The completely matched target (cm) matches the siRNA
GS completely, whereas the seed-matched (sm) target
consists of two parts. 30 terminal 8 nt of the target arecomplementary in sequence to the 50 end (nucleotide position 1) and the seed (positions 2–8) of the corresponding*To whom correspondence should be addressed. Tel: +81 3 5841 3043; Fax: +81 3 5841 3044; Email: ktei@biochem.s.u-tokyo.ac.jp
ß 2008 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Downloaded from nar.oxfordjournals.org at Ekigaku-Kyoshitsu (UNIV OF TOKYO) on October 17, 2010

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan

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Figure 1. Oﬀ-target gene silencing assay using reporter plasmids. Structures of (A) psiCHECK-cm and (B) psiCHECK-sm (the luc transcript ofwhich, respectively, presents three tandem repeats of cm and sm in the 30 UTR, shown with thick arrows). (C) Gene-silencing activities of 26 siRNAs
were examined in HeLa cells as a function of siRNA concentration. In most cases, luc mRNA with cm was eﬀectively inactivated. In contrast,
inactivation of mRNA with sm varied signiﬁcantly, depending on the siRNA concentration used for transfection. Sequences of siRNA, cm targets
and sm targets are shown in Supplementary Tables S1 and S2. (D) Simultaneous reduction in mRNA amount and luc activity in psiCHECKtransfected cells. Ordinate represents relative luc activity (%) in diﬀerent psiCHECK-sm and -cm transfected cells. Abscissa represents relative
amount of luc mRNA (%) in the corresponding psiCHECK-sm and -cm transfected cells. Results strongly suggest that the reduction of luc activity(protein activity) is due mainly to gene-silencing-dependent degradation of the corresponding mRNA.

7102 Nucleic Acids Research, 2008, Vol. 36, No. 22

Calculation of thermodymanic parameters
Standard Gibbs free energy change (G) and Tm were
calculated according to the nearest neighbor model (16)and the thermodynamic values for RNA–RNA (17).
Dissociation constant (Kd) for the seed duplex was determined using the following formula, G = –RTln(1/Kd),
where T was 298.15 K. The calculation formula for Tm
is as follows: Tm = {(1000  H)/[A + S + ln(Ct/4)]}
273.15 + 16.6log[Na+]. H (kcal/mol), sum of nearest
neighbor enthalpy change. A, helix initiation constant(–10.8). S, sum of nearest neighbor entropy change(17). R, gas constant (1.987 cal/deg/mol). Ct, total molecular concentration of strand (100 nM). [Na+] was ﬁxed at
100 mM.
Microarray analysis
HeLa cells (1  105 cells/ml) were inoculated into an individual well culture plate 24 h prior to transfection. Cells
were transfected with 50 nM siRNA. Total RNA (3 mg)
was puriﬁed using RNeasy Kit (Qiagen) 24 h after transfection and hybridized to Human Genome U133 Plus 2.0
GeneChip (Aﬀymetrix) containing about 47 400 humantranscripts according to the manufacturer’s protocol.
RNA from mock-transfected cells, which treated withtransfection reagent in the absence of siRNA, was used
as a control. The transcript expression value was calculated using Microarray Suite 5.0 (MAS5) (18) with quantile normalization (19); those transcripts with strong
enough hybridization signals to be called present (P)were used in this study. To identify transcripts that were
downregulated on the array, the cumulative distributionof expression changes for those messages with the site

versus those with no canonical site were compared. The
statistical signiﬁcance of their dissimilarity was quantiﬁed
based on the P-value using Wilcoxon’s rank-sum test (6).
Motif analysis of 3’ UTR and CDS
We mapped the probe sequences, which were takenfrom the annotation table provided on the Aﬀymetrix
Web site (http://www.aﬀymetrix.com), to the RefSeq
human mRNA sequences (release 24) to identify thetarget transcripts. We found 67 220 annotations forhuman transcripts to be corresponded to 54 675 of theprobe sets. In microarray analyses using siVIM-270 and
siVIM-812, the number of transcripts called P by MAS5
was estimated at 19 856. Of 19 856, 16 783 that represented
RefSeq entries (25%) with the 30 UTR were consideredhere. In the experiments using siCLTC-2416 andsiCLTC-4819, 17 321 of 20 673 transcripts deﬁned as
P were analyzed. Seven nucleotide sequences matching
the seed region (nucleotides 2–8) of GS were assigned as
extracted 30 UTR or CDS sequences.
Quantitative RT–PCR (qRT–PCR)
qRT–PCR was carried out using an aliquot of total
RNA analyzed using the microarray. RNA was reversetranscribed using the SuperScript First-Strand Synthesis
System for RT–PCR (Invitrogen). The mixture of resultant
cDNA and SYBR Green PCR Master Mix (Applied
Biosystems) were incubated at 958C for 10 min before the
PCR reaction. The levels of PCR products were monitoredwith ABI PRISM 7000 sequence detection system and analyzed with ABI PRISM 7000 SDS software (Applied
Biosystems). Each reaction ran in triplicate. The expression
level of each sample was ﬁrst normalized to the amount ofb-actin and then to the mock transfection control. Used
primer sets are listed in Supplementary Table S3.
RESULTS AND DISCUSSION
Variation in the efficiency of off-target effect according
to the seed sequence
To determine the relationship between siRNA sequences
and oﬀ-target eﬀects, we introduced 26 pairs of cm and sm
target sequences into an expression reporter plasmid,
psiCHECK (Figure 1A and B) (14) and examined thechange in luc activity in transfected HeLa cells as a function of siRNA concentration (Figure 1C). psiCHECK
encodes the Renilla luc gene. Three tandem repeats of
cm or sm target sequences were introduced into theregion corresponding to the 30 UTR of the luc mRNAto generate psiCHECK-cm or psiCHECK-sm, respectively. The cm targets, 21 nt in length, were completely
complementary to the GS of siRNAs (Figure 1A), whichwere arbitrarily chosen from the highly functional class I
siRNA (15), and used for determining the eﬃciencies ofintended gene silencing, basically RNA interference(RNAi). Class I siRNA possesses A or U residues at position 1, three to six A/U residues in nucleotide positions
2–7 and G/C at position 19 (15). The GS of any class I
siRNA is expected to be incorporated into RISC very

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siRNA GS, while the remaining 13 nt are totally unhomologous to the GS (Supplementary Table S2).
HeLa cells in a well of 24-well culture plate were transfected simultaneously with one of psiCHECK derivatives
constructed above (10 ng), pGL3-Control (Promega, 1 mg)
or phLuc-Control (0.5 mg) (14) and siRNA (0.05–50 nM).
Cells were harvested 24 h after transfection and relative
luc activity (Renilla luc activity/ﬁreﬂy luc activity) was
determined using the Dual-Luciferase Reporter Assay
System (Promega). pGL3-Control encoding ﬁreﬂy luc
also served as a control for relative luc assay for
siRNAs against endogenous genes. Used siRNAs
against mammalian endogenous genes are: siVIM[human vimentin]-269, -270, -596, -805, -812, -1128 and-1261; siOct [mouse Oct4]-670, -797 and -821; siGRK4[human G protein-coupled receptor kinase 4]-934;
siPLS3 [human plastin3, T-isoform]-1310, -1528 and-1657; siCTLC [human clathrin heavy chain]-2416, -3114
and -4819; siKIF23 [human kinesin family member23]-430; siTUBA2 [human tubulin alpha]-714; siITGA10[human integrin alpha2]-2803; siMC4R [human melanocortin 4 receptor]-490; siCCNC [human cyclin C]-571.
Gene silencing by siRNAs against ﬁreﬂy luc (siLuc-36,-309, -774 and 2-153) was carried out using phLucControl, which encodes luc diﬀerent from ﬁreﬂy luc.
siGY-441, an siRNA for GFP knockdown, used as ansiRNA control.

Nucleic Acids Research, 2008, Vol. 36, No. 22 7103

Apparent correlation between seed-dependent
off-target effect and thermodynamic stability of thesiRNA seed duplex
Melting temperature (Tm) and standard free energychange (G) for the formation of the seed duplex
may be good measures for the thermodynamic stabilityof the protein-free seed duplex. We ﬁrst clariﬁed theirrelationship. As shown in Figure 2A, the calculated Tm
values of 26 seed duplexes in 100 mM NaCl, which
varied from 108C to 368C, were negatively correlatedwith the calculated G values, which ranged from 16
to 7 kcal/mol. The correlation coeﬃcient was estimatedat 0.95, indicating a very strong correlation. Therefore,
a seed duplex with a high Tm is generally considered to
be associated with a low G value, and vice versa.
Figure 2B demonstrates the positive correlation between
relative luc activity compromised by the oﬀ-target eﬀect
and the calculated G of the protein-free seed duplex.
Shown in Figure 2C are the calculated Tm values of theprotein-free seed duplex, which were negatively correlated with the compromised luc activity. Correlationcoeﬃcients of the former and the latter, respectively,
were 0.69 and 0.72, indicating a close relationshipbetween the seed-dependent oﬀ-target eﬀect and theseed-duplex G or Tm. The value for G may be converted to the dissociation constant using the formulaG = –RTln(1/Kd) (24). Thereby, 15 Kd values were
calculated and are listed in Figure 2D. The highest Kd
value (CLTC-4819 seed-duplex with the highest G,
7.2 kcal/mol) was found to be more than 106 times
greater than the lowest one (CLTC-2416 seed-duplex
with the lowest G, 15.5 kcal/mol). This indicates awide range of seed-duplex stability, which may accountfor the strong siRNA-concentration dependency of theoﬀ-target eﬀect (Figure 1C). Thus, it may follow thatthe degree of oﬀ-target eﬀects is primarily governed by
the thermodynamic stability of duplex, 7 bp in length,
which is formed between the GS seed region and its
mRNA counterpart.
A considerable deviation was observed in luc activity
measurements shown in Figure 2B and C. This may be
due in part to diﬀerences in the non-seed sequence and/or its counterpart in target mRNA, since the target
sequences that correspond to the non-seed region makean appreciable contribution to target recognition by
miRNAs and/or siRNAs in microarray proﬁling (1,6,8)(Supplementary Figure S1). The reporter assay usingeight diﬀerent targets with common seed and diﬀerent
non-seed sequences showed that oﬀ-target eﬀects arerather various, indicating possible involvement of siRNA
non-seed region and/or its target counterpart in oﬀ-target
eﬀect (Supplementary Figure S3). However, correlation
between seed-dependent gene silencing activity of siRNA
used in this study (Figure 1C) and calculated Tm of protein-free seed duplex of them aligned in all possible 7-nt
sequences (47 = 16 384) may suggest that 21.58C serves
as a kind of benchmark Tm (Figure 3A), which may
discriminate almost oﬀ-target-free seed sequences fromoﬀ-target-positive ones.

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eﬀectively (15,20,21), such that RISC formation may not
be a rate-limiting step in class I-siRNA-dependent unintended gene silencing. The sm sequence possesses complete
complementarity to the entire seed region (positions 2–8)
but not to the remaining non-seed region, positions 9–21
(Figure 1B), and was used for determining the eﬃciency ofseed-dependent unintended oﬀ-target eﬀect. The sm andcm sequences, along with homology to the corresponding
siRNA GS are shown in Supplementary Tables S1 and S2.
Under our experimental conditions, the content of luc
mRNA produced within cells was estimated at about
300 copies per ng of the total RNA, a value onehundredth of that found for b-actin mRNA. The luc
activities measured using diﬀerent psiCHECK-sm and-cm constructs targeted by the corresponding siRNAs
were almost proportional to the levels of mRNA
(Figure 1D), indicating that at least under these conditions
most, if not all, of the luc activity reduction due to not
only RNAi but also oﬀ-target eﬀect is attributable tosiRNA-dependent luc mRNA degradation.
As anticipated, all 26 siRNAs examined exhibited highactivity for intended gene silencing at 50 nM (Figure 1C).
Even at 0.5 nM most, if not all, siRNAs used reduced theactivity of the luc gene with the cm target to less than
20%. In contrast, the oﬀ-target gene silencing calculatedusing sm target was much less eﬀective and more susceptible to changes in siRNA concentration, with the exception of siVIM-805. Virtually no oﬀ-target eﬀects were
induced by transfection with any of the 26 siRNAs at
0.05 nM. Only 5 of the 26 siRNAs applied at 0.5 nM
reduced luc activity to less than 35%. More than ﬁve
siRNAs did not bring about any appreciable oﬀ-target
eﬀects even when the siRNA concentration for transfection was increased to 50 nM. The same RISC is considered
to be capable of causing both intended RNAi and unintended oﬀ-target gene silencing (22). Thus, above ﬁndings
may indicate that variations in the eﬃciency of unintended
oﬀ-target gene silencing are due to a diﬀerence in theinteractions between the GS entrapped in RISC andmRNA, i.e. a diﬀerence in the eﬃciency of seed duplex
formation. High complementarity in the non-seed regionin addition to that in the seed region is necessary for
intended gene silencing. A seed/non-seed swapping experiment (Supplementary Figure S2) has shown that unlike
seed complementarity to target mRNA, complementarity
to the non-seed region is incapable of inducing any genesilencing eﬀects. Notably, siVIM-805 might be an exceptional siRNA that exerts intended gene silencing by amechanism similar to that for oﬀ-target gene silencing(Figure 1C).
In our experiments, the base at the 50 end (position 1)
was always A or U, and complementary to its mRNA
counterpart. However, A/U pairing at position 1 may
not be important for unintended gene silencing. First,
the 50 end of the GS is considered to be embedded in a
pocket of Ago in the RISC (12,13). Second, little or nocontribution of A/U-pairing at position 1 to the seed
activity or RISC formation has been conﬁrmed by various
experiments, including microarray analysis (15,23)(Supplementary Figure S1).

7104 Nucleic Acids Research, 2008, Vol. 36, No. 22

Genome-wide analysis of off-target gene silencing
determined by the siRNA seed duplex stability
To further conﬁrm the notion that oﬀ-target gene silencing is determined primarily by seed duplex stability,
genome-wide expression proﬁling was carried out using
four class I siRNAs targeting human vimentin (siVIMs)
or clathrin heavy chain (siCLTCs) (Figure 4;
Supplementary Figures S4 and S5). The seed duplexes
generated by siVIM-270 and siCLTC-2416 have Tmvalues in 100 mM NaCl of 26.28C and 33.28C, respectively, while those generated by siVIM-812 and siCLTC4819 are 8.88C and 10.38C, respectively. HeLa cells weretransfected with 50 nM siRNA and the changes in theexpression level of transcripts were analyzed after 24 h.
The reporter assay described above predicted thatsiRNAs with high seed duplex Tm values (siVIM-270
and siCLTC-2416) may be good inducers, while thosewith low Tm values (siVIM-812 and siCLTC-4819) may
be poor inducers of the oﬀ-target eﬀect.
As anticipated, all four siRNAs used eﬀectively reduced
the amount of vimentin and clathrin heavy chain
mRNA, to less than 20% (arrows in Figure 4A and B;
Supplementary Figure S5) as a result of intended RNAieﬀect. In contrast, the oﬀ-target-dependent reductionin the amount of mRNA depended signiﬁcantly on the
Tm of the seed-duplex. A high level of oﬀ-target eﬀects

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Figure 2. Close relationship between eﬃciency of seed-dependent oﬀ-target gene silencing and the thermodynamic stability of the protein-free seed
duplex. (A) The calculated Tm for the seed duplex decreases with increasing changes in standard free-energy (G) for seed duplex formation(correlation coeﬃcient, 0.95). (B) luc activity compromised by seed-dependent oﬀ-target gene silencing at 50 nM siRNA concentration
was positively correlated with G (correlation coeﬃcient, 0.69). The luc activity data were collected from Figure 1C. (C) Correlation between
seed-dependent gene silencing activity (luc activity) and the calculated Tm of the protein-free seed duplex. A set of luc activities compromisedby seed-dependent gene silencing at 50 nM siRNA concentration were collected from Figure 1C. Tm value of the protein-free seed region (positions2–8) was calculated using the nearest neighbor method. Relative luc activity and calculated Tm were correlated with each other and had acoeﬃcient of 0.72. (D) List of calculated dissociation constants for protein-free seed duplexes. G was converted to Kd using the formulaG = –RTln(1/Kd).

Nucleic Acids Research, 2008, Vol. 36, No. 22 7105

was evident in the case of transfection with siVIM-270(Figure 4C) and siCLTC-2416 (Supplementary
Figure S4A), which were predicted as good oﬀ-target
inducers. The expression levels of the transcripts withseed complementary sequences (positions 2–8 of siRNA
guide strand) in 30 UTRs were clearly reduced byboth siRNAs in common, although the transcripts
with complementarities in the other positions were
not changed at least coincidentally (Supplementary
Figure S1), in a manner consistent with the previous
reports (3–6). Transfection with siVIM-812 (Figure 4D)

Elimination of seed-dependent off-target gene silencing
by G:U pairing
G:U pairing is not typical of Watson–Crick pairing, which
consists of G:C and A:U pairs in the case of RNA (28).
G:U pairs were frequently incorporated into RNA secondary structures, probably because the standard freeenergy change for G:U pair formation is almost equivalent
to that for A:U pair formation (28) (Supplementary
Table S4). However, array analysis may suggest that
G:U pairing causes a virtually complete elimination of
the oﬀ-target eﬀect (Figure 5) (8). To extend this point,
G:U pairing mutations were introduced into the VIM-270
seed-duplex (Figure 6). Both target sequence and siRNAmutations were introduced by chemical oligonucleotide
synthesis. The luc mRNA with a mutated target was
expressed using psiCHECK. Note that all mutations producing G:U pairing at positions 2, 3 and 7, are target
sequence mutations, which may not inﬂuence the eﬃciency of RISC formation (22). Figure 6 shows clearly
that introduction of any G:U pairing to the seed duplex
results in an almost complete loss of the oﬀ-target eﬀect.
The second mutation was introduced into the opposite
strand to generate a phenotypic revertant of Watson–
Crick pairing (Figure 6C–G). The oﬀ-target eﬀect wasalmost completely recovered, irrespective of diﬀerencesin location of the mutation within the seed duplex.

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Figure 3. Correlation between seed-dependent gene-silencing activity
and calculated Tm of protein-free seed duplex. Gene-silencing activity
was measured using relative luc activity in HeLa cells transfected withpsiCHECK-sm and cognate siRNAs at 50 nM as shown in Figure 1C.
Tm of the protein-free seed region (positions 2–8) was determined using
nearest neighbor method (Figure 2C). (A) All possible 7-nt seedsequences (47 = 16 384) were ordered as a function of GC content
and Tm values of their ds counterparts. Note that, because of its deﬁnition, class I siRNA cannot possess more than four GC in the seedregion. Blue: combinations of target and siRNA, giving less than
50% relative luc activity. Red: combinations of target and siRNAwith little or no oﬀ-target eﬀect (luc activity >50%). Dotted line at
21.58C may correspond to 50% luc activity reduction. (B) Tm distribution of 733 human microRNAs registered in miRbase. Ordinate represents calculated seed-duplex Tm. Abscissa represents percentage.
Seventy-ﬁve percent of 565 siRNAs registered was estimated to be
associated with Tm more than 21.58C.

and siCLTC-4819 (Supplementary Figure S4B) that werepredicted as poor oﬀ-target inducers exhibited little oﬀtarget eﬀects (Figure 4E, Supplementary Figure S4C).
Results from microarray quantitative analysis were
essentially identical to those obtained by qRT–PCR(Supplementary Figure S6). We conclude that the levelof oﬀ-target gene silencing is determined by the thermodynamic stability of the seed duplex formed between thesiRNA GS and the target mRNA.
Furthermore, in accordance with the recent papers
described that not only 30 UTR but also CDS is miRNAtarget (25,26), our (Supplementary Figure S1) and others’(6) microarray analyses indicated that seed-dependentoﬀ-target silencing eﬀects are also observed in transcripts
containing seed complementary sequences in CDSs,
although the eﬀects were marginal. As in the case of 30
UTR, the oﬀ-target eﬀects by the transfection of siRNAs
with low thermodynamic stabilities of seed duplexes(siVIM-812 and siCLTC-4819) were reduced in the transcripts with seed complementarity in CDSs (data notshown) compared to the results of siRNAs with high stabilities (siVIM-270 and siCLTC-2416; Supplementary
Figure S1D and F). Thus, the thermodynamic stabilityof the seed duplex might be basic determinant of oﬀtarget silencing in both CDSs and 30 UTRs, although
the mechanism of marginal seed-dependent silencing
eﬀects in CDSs remains unknown.
The miRbase includes 733 human miRNA sequences(27). Our calculation indicated that 546 of the 733 werecapable of providing seed duplexes of which Tm values are
greater than 21.58C (Figure 3B), suggesting that about
75% of miRbase miRNAs may exert relatively strong
seed-dependent gene silencing.

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In contrast to the oﬀ-target eﬀect, G:U pairing aﬀorded
virtually no eﬀect on intended gene silencing (Figure 6),
indicating an apparent diﬀerence between the molecularmechanisms of intended RNAi and unintended oﬀ-target
gene silencing.
As shown in Supplementary Table S4, the diﬀerence inG values between the G:U pair-containing seed duplex

and its cognate with an A:U pair is very small, if at all,indicating that a diﬀerence in G, or thermodynamic stability of seed duplex, cannot account for a catastrophic
change in oﬀ-target eﬀects due to a G:U/A:U replacement.
The substitution of any Watson–Crick pair by a G:U pairhas been reported to result in structural perturbation,
regardless of the sequence (28). Structural analysis of the

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Figure 4. Microarray-based oﬀ-target-eﬀect proﬁling and data analysis. Proﬁles of transcript downregulation by (A) siVIM-270 and (B) siVIM-812.
The cumulative distribution of transcripts from cells treated with (C) siVIM-270 and (D) siVIM-812, are shown as log2 of fold change to mocktransfection. The blue line (C, D) indicates the cumulative fraction of transcripts with one or more sequence complementary to the siRNA seed in the
30 UTR. The gray line (C, D) shows transcripts with no seed complementarity. Distributions of log fold change, deﬁned as the log2 of expression in
siVIM-270- or siVIM-812-transfected cells over that in mock-transfected cells, for mRNAs lacking seed complementarity are 0.221  0.001 (C) and
0.016  0.005 (D), respectively. (E) Comparison of microarray proﬁles of transcripts downregulated by siVIM-270 and siVIM-812. Only transcripts
with 30 UTR complementarity to two or more than two siRNA seed sequences were examined. Ordinate represents fraction. Abscissa represents
signal intensity obtained after siVIM-270 treatment divided by that after siVIM-812 treatment. The blue and red bars denote the distribution oftranscripts with 30 UTR complementary to the VIM-812 and VIM-270 seed sequence, respectively. The black bar represents transcripts with seed
complementarity to neither siVIM-270 nor siVIM-812.

Nucleic Acids Research, 2008, Vol. 36, No. 22 7107

Aquifex aeolicus Ago has shown that the phosphodiester
backbone of ﬁve consecutive residues corresponding
to position 2–6 of the GS, which adopts a helical pitch,
has extensive surface and charge complementarity withthe side chains in the Mid and PIWI domains of Ago(12,13). Thus, these structural constraints on the GS
seed may make it diﬃcult to form any G:U pairing
between target mRNA and the GS seed binding to
Ago in RISC.

We therefore consider that for RNAi, which requires anear perfect match between target mRNA and thesiRNA GS, the ﬁrst recognition of mRNA by the GS
seed arm and subsequent target recognition by the nonseed region of GS are so highly cooperative that thesecond proof-reading process can easily surmount the
G:U pairing problem in the initial mRNA nucleation
process.
SUPPLEMENTARY DATACONCLUSION
We found that the seed-dependent oﬀ-target eﬀect ishighly correlated with the thermodynamic stability in the
duplex formed between the seed region of the siRNA
guide strand and its target mRNA. G and Tm for theformation of the seed duplex, respectively, are positively
and negatively correlated with seed-dependent gene silencing activity. The seed-dependent oﬀ-target gene silencing
eﬀect is almost completely eliminated with G:U pairing inthe seed duplex. In this case, oﬀ-target eﬀect is avoided
probably because of the structural perturbation of seed
duplex but not seed duplex stability.
Unlike the oﬀ-target eﬀect, intended gene silencing,RNAi, was signiﬁcantly tolerant to not only the presenceof a G:U pair in the seed region (see e.g. Figure 6A and B),
but also to changes in siRNA concentration (Figure 1C).

Supplementary Data are available at NAR Online.
FUNDING
Grant-in-Aid for priority area and Genome Network
Project from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (to K.U.-T., partial).
Funding for open access charge: Ministry of Education,
Culture, Sports, Science and Technology.
Conﬂict of interest statement. None declared.
REFERENCES
1. Jackson,A.L., Bartz,S.R., Schelter,J., Kobayashi,S.V., Burchard,J.,
Mao,M., Li,B., Cavet,G. and Linsley,P.S. (2003) Expression

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Figure 5. Microarray analysis of the eﬀect of G:U pairing in the seed duplex. Two siRNAs, (A) siVIM-270 and (B) siCLTC-2416, with high Tm
values (26.28C and 33.28C, respectively) were used at 50 nM for transfection. Together, (A) and (B) indicate that transcripts in which the 30 UTR isforming the seed duplex consisting only of Watson–Crick pairing are capable of exerting a signiﬁcant oﬀ-target eﬀect. Transcripts forming a G:U
pair-containing seed duplex are associated with virtually no oﬀ-target eﬀect. Nucleotide sequences of transcripts analyzed are shown in the lower
margin. Red denotes transcripts forming a seed duplex consisting only of Watson–Crick base pairs [1043 transcripts in (A), 97 in (B)]. Other colors
represent transcript groups that are capable of forming a G:U pair-containing seed duplex in the 30 UTR. The numbers of transcripts are as follows:in (A), 1076 blue, 104 sky blue, 331 green and 931 yellow; in (B), 114 blue, 1226 sky blue and 157 green. Solid vertical line denotes Watson–Crickpairing. Dotted line denotes G:U pairing.

7108 Nucleic Acids Research, 2008, Vol. 36, No. 22

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Figure 6. Reporter plasmid analysis of the eﬀect of G:U pairing in the seed duplex. Three copies of cm or sm targets were introduced into
psiCHECK. Blue bars represent silencing of cm targets. Red bars refer to oﬀ-target gene (sm-target) silencing. Bases replaced by the ﬁrst mutation
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aﬀected by the mutation, sm-target silencing (oﬀ-target eﬀect) was almost completely abolished. (C) G!A and C!U substitutions were introducedat position 3 of siVIM-270 GS and its counterpart in the passenger strand (PS), respectively, as the second mutations to generate a siRNA mutant,siVIM-270m3. Thus, U:A pairing was introduced at ds position including nucleotide 3 of the GS to generate a phenotypic Watson–Crick pairing
revertant. A signiﬁcant recovery of oﬀ-target eﬀect is evident. (D–G) Four additional examples of G:U pairing mutation and phenotypic reversion.
Mutations were introduced into VIM270-cm, VIM270-sm or siVIM-270 to generate pairing mutants of the seed duplex at GS positions (D) 6, (E) 5,(F) 2 and (G) 7. In all cases examined, the ﬁrst mutation (G:U pair introduction in the seed region) abolished nearly all oﬀ-target silencing activity,
which was almost completely recovered by the second mutation, which regenerated Watson–Crick pairing.

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