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Articlehttp://pubs.acs.org/journal/acsodf

Chemical Modiﬁcation of the siRNA Seed Region Suppresses OﬀTarget Eﬀects by Steric Hindrance to Base-Pairing with Targets
Hanna Iribe,† Kengo Miyamoto,‡ Tomoko Takahashi,§ Yoshiaki Kobayashi,§ Jastina Leo,§,∥Misako Aida,‡ and Kumiko Ui-Tei*,†,§†

Department of Computational Biology and Medical Science, Graduate School of Frontier Sciences, The University of Tokyo, Chiba
277-8581, Japan‡
Center for Quantum Life Sciences and Department of Chemistry, Graduate School of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan§
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan∥
Faculty of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, United States
S Supporting Information*

ABSTRACT: Chemical modiﬁcations of 2′-O-methyl (2′OMe) and locked nucleic acid (LNA) of the nucleotides in theseed region (positions 2−8) of the small interfering RNA(siRNA) guide strand signiﬁcantly reduced seed-matched(SM) oﬀ-target eﬀects. The siRNA with 2′-OMe modiﬁcations
inhibited the expression of a completely-matched (CM) target
gene, whereas that with LNA modiﬁcations did not inhibit theexpression of the CM target. By computational predictions ofconformational changes of siRNA by these modiﬁcations, werevealed that both modiﬁcations in the siRNA seed region
reduce SM oﬀ-target eﬀects by steric hindrance to base-pairingwith target transcripts but LNA modiﬁcations also disturb theassociation of the siRNA guide strand with the Argonaute(AGO) protein by altering RNA conformation. Thus, chemical modiﬁcations of the siRNA guide strand, which alter stericconformation to disturb base-pairing with target transcripts but do not disturb the association with the AGO protein, may
successfully suppress oﬀ-target eﬀects without substantial loss of RNA silencing activity.

■

INTRODUCTION
RNA interference (RNAi) is a prominent tool for functional
genomics because small interfering RNAs (siRNAs) caneﬀectively knock down the target genes with perfect sequence
complementarity to the siRNA. siRNAs are double-stranded
RNAs, 21 nucleotides (nts) in length with 2 nt 3′ overhangs(Figure 1). When an siRNA is transfected into cells, it isincorporated into the RNA-induced silencing complex (RISC),
which includes the core protein Argonaute (AGO), and thisprocess is referred to as RISC loading. Subsequently, the siRNAis unwound into single-stranded RNAs. Then, one RNA strand(guide strand) remains associated with AGO to form active
RISC, whereas the other strand (passenger strand) isdegraded.1 The guide strand forms complete base-pairingwith a target transcript, and AGO cleaves the target to suppress
its function.2
Only a limited fraction of siRNA sequences are functional inmammalian cells. We revealed the guidelines for functional
siRNA sequences in mammalian cells using various types ofexperiments.3 Functional siRNAs satisﬁed the following four
conditions simultaneously: (i) A/U at the 5′-terminus of thesiRNA guide strand, (ii) G/C at the 5′-terminus of the© 2017 American Chemical Society

passenger strand, (iii) AU richness at the 5′ one-third of theguide strand, and (iv) absence of a long GC stretch (>9 nts)
(Figure 1). Results from a number of studies suggest thatasymmetry in the stability of the siRNA termini is essential for
determining the unwinding direction of the siRNA duplex into
single-stranded RNAs.3−5 An siRNA duplex is easily unwoundfrom the more unstable terminus, and the unwound 5′-terminal
nucleotide is anchored in the binding pocket in the mid domainof the AGO protein, in which A or U is preferentially anchoredat an aﬃnity up to 30-fold higher than that with either G or C.6
Thus, an RNA strand with the unstable 5′-terminus acts as afunctional guide RNA. If the passenger strand of siRNA
remains in the RISC, it may induce unintended oﬀ-target
eﬀects. However, as the siRNA selected according to the above
criteria possesses a G or C residue at the 5′-terminus of thepassenger strand, which has a lower aﬃnity with the AGO
pocket, the passenger-strand-dependent oﬀ-target eﬀects areeﬀectively eliminated even when the passenger strand remains
Received: March 11, 2017
Accepted: May 5, 2017
Published: May 12, 2017
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Articlein the RISC. A total of 56 375 087 (100%) 23-mer (19 nt
double-stranded region with 2 nt overhangs at both termini)
subsequences were obtained from human mRNAs registered in
the RefSeq database (release 30). Bioinformatics analysis
revealed that 14.7% of the human siRNA sequences satisﬁed
all four criteria, (i)−(iv), for functional siRNAs; 98% of morethan 100 diﬀerent siRNAs satisfying these sequence rules wereexperimentally conﬁrmed to be functional. Furthermore, atleast one unique functional siRNA was selected for 92 and 99%
of human and mouse genes, respectively.3
Partial complementarity of the siRNA guide strand withnontargeted mRNAs induces unintended oﬀ-target eﬀects7−10(Figure 1). Every backbone phosphate of the seed nucleotides
at positions 2−8 from the anchored 5′-terminal nucleotide
preordered on the AGO protein to make stable base-pairing
between the siRNA seed region and target mRNA in an A-form
helix.11,12 The eﬃciency of the oﬀ-target eﬀect is positively
correlated with the thermodynamic stability of the base pairbetween the guide strand seed region and SM transcripts.10
Thus, siRNAs with low seed−target stability may be a
promising tool for target-speciﬁc RNAi with fewer oﬀ-target
eﬀects. However, the addition of a ﬁfth condition of low
thermodynamic stability in the seed region to the siRNA
selection criteria (i)−(iv) decreased the percentage of
selectable siRNA candidates from 14.7 to 2.1%.13 This meansthat at least one functional siRNA is selected for just 77.2% of
human genes. To overcome such sequence limitations, weFigure 1. Schematic presentation of the siRNA-mediated RNAi
pathway and oﬀ-target pathways. RNAi is induced by the completely
complementary siRNA guide strand, and seed-dependent oﬀ-target
eﬀects are induced by seed-matched (SM) targets of the guide strand.
The completely-matched (CM) or SM oﬀ-target eﬀect of thepassenger strand is also observed. The siRNA satisfying four functional
siRNA rules was used.

Figure 2. Chemical modiﬁcations used in this study. (a) Chemical structures of authentic RNA, 2′-OMe, locked nucleic acid (LNA), DNA,
phosphorothioate (PS) linkage, DNA−PS. (b) siRNA sequences and the position of modiﬁed nucleotides. Red indicates DNA-, PS-, and DNA−PSmodiﬁed nucleotides. Blue indicates 2′-OMe-modiﬁed nucleotides. Green indicates LNA-modiﬁed nucleotides. Underlines indicate the nucleotide
positions with each modiﬁcation in the siRNA. The upper strand is the passenger strand. The lower strand is the guide strand.
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Articleevaluated the impact of chemical modiﬁcations in this study.
Many chemical modiﬁcations of various sites in the siRNA were
examined for improvements in siRNA speciﬁcity, stability, andimmunogenicity. Because the oﬀ-target eﬀects of the guidestrand are induced by seed−target complementarity,7−10 theeﬀects of a few types of modiﬁcations in the seed region were
examined.14−16 Among them, 2′-O-methyl (2′-OMe) modiﬁcations of the nucleotide at position 2 of the guide strand
reduced the SM oﬀ-target eﬀects without a severe reduction in
RNAi activity.14 However, because systematic studies on theeﬀects of chemical modiﬁcations in the seed region have notbeen performed, the mechanism of the reduced oﬀ-target
eﬀects is not well-elucidated. In this study, ﬁve diﬀerent
modiﬁcations, shown in Figure 2a, were introduced into theseed region of authentic siRNA, which has 19 nt doublestranded region with 2 nt overhangs in both termini (Figure
2b).

■

RESULTS AND DISCUSSION
To perform dual-luciferase reporter assays, four types of siRNA
reporter plasmids were constructed (Figure 3a). psiCHECKgCM with a CM sequence of the entire siRNA guide strand in
the 3′UTR region of the Renilla luciferase gene was used toinvestigate RNAi activity of the guide strand. psiCHECK-gSM
containing three tandem-repeated SM sequences of the guidestrand was used to examine seed-dependent oﬀ-target eﬀects(Figure 3a). psiCHECK-pCM with three tandem repeats of
CM sequences on the passenger strand and psiCHECK-pSM
with three tandem repeats of SM sequences were used to
examine passenger-strand-dependent oﬀ-target eﬀects (FigureS1a). Each of these plasmids was transfected into human HeLa
cells with pGL3-Control, expressing the control ﬁreﬂy luciferase
gene, and siRNA with or without chemical modiﬁcations in theseed region. The chemical modiﬁcations of deoxyribonucleic
acid (DNA), PS, and DNA−PS were introduced into all of theseven nucleotides of the seed region of the siRNA guide strand(Figure 2b). In addition, 3 (positions 4−6), 5 (positions 3−7),
and 7 (seed positions 2−8) nts in the guide strand were
modiﬁed with 2′-OMe (named 2′-OMe-3, 2′-OMe-5, 2′-OMe7); and 3 (positions 3−5) and 7 (seed positions 2−8) nts were
modiﬁed with LNA (LNA-3, LNA-7) (Figure 2b). siRNAagainst an enhanced green ﬂuorescent protein (siGY441) wasused as a negative control. One day after transfection, ﬁreﬂy
and Renilla luciferase activities were measured and relative
luciferase activity was calculated as an indicator of the CM
RNAi activity and SM oﬀ-target eﬀects of the guide strand andCM and SM oﬀ-target eﬀects of the passenger strand.
Unmodiﬁed siRNA downregulated the Renilla luciferase
activity via the guide strand CM target in a dose-dependent
manner (Figure 3b). siRNA modiﬁed with DNA, PS, 2′-OMe(2′-OMe-3, -5, -7), or LNA (LNA-3) reduced the expression of
the CM target of the guide strand at the equivalent level to thatof unmodiﬁed siRNA, particularly at high concentrations (0.5
or 5 nM). PS is a modiﬁcation in which one of the nonbridging
oxygens is replaced by sulfur,17 which blocks the exonucleaseaction and increases its permeability through the lipid bilayer,
but PS has little eﬀect on base pair formation.18,19 In fact, PSmodiﬁed siRNA did not inhibit RNAi activity against the CMtarget. The various modiﬁcations at the 2′-position of thepentose sugar of siRNA are not required for RNAi20,21 andtarget recognition.15 DNA is modiﬁed with hydrogen at C2′,and the 5′ one-third of each RNA strand is capable ofreplacement with DNA without substantial loss of RNAi

Figure 3. Results of luciferase reporter assays. (a) Structures ofreporter constructs for luciferase reporter assays. psiCHECK-gCM
contains a CM sequence of the siRNA guide strand. psiCHEK-gSM
contains three tandem repeats of the SM sequences of the guidestrand. (b) Results of RNAi activities on the CM target using
psiCHECK-gCM with unmodiﬁed or modiﬁed siRNAs. (c) Results ofseed-only silencing on the SM target using psiCHECK-gSM withunmodiﬁed or modiﬁed siRNAs. The data were averaged from three tofour independent experiments, and the bar indicates the standard
deviation.

activity.15 Consistent with our previous report,15 DNA
replacement at the seed region of the guide strand alone
retained eﬃcient RNAi activity on the CM target. 2′-OMe is awell-known C2′-modiﬁcation that increases serum stability andspeciﬁcity in base-pairing and abrogates immunogenicity.20,22−24 2′-OMe is tolerated at multiple positions in thesiRNA guide strand because of its small size, comparable to the2′-OH of natural RNA.25 Our results also showed that three 2′OMe nucleotide modiﬁcations resulted in similar suppression
levels on the CM target compared with unmodiﬁed RNA,
although the eﬃciency was slightly reduced with increasing 2′OMe modiﬁcations in the seed region (Figure 3b). LNAs
contain a bridge connecting the 2′-oxygen with the 4′-carbon of
the ribose ring, resulting in a greater stability compared to thatof 2′-OMe.26,27 LNA-3 suppressed the expression of the CMtarget, but LNA-7 showed little to no activity.
The oﬀ-target eﬀects on the SM target of the guide strand
were observed for unmodiﬁed siRNAs in a reporter assay using
psiCHECK-gSM (Figure 3c). siRNAs with PS modiﬁcations
exhibited similar, but slightly weaker inhibitory eﬀects
compared to those from unmodiﬁed siRNAs, probably because
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ArticleFigure 4. Computational prediction of unmodiﬁed, LNA-modiﬁed, and 2′-OMe-modiﬁed RNA structures. Structures of unmodiﬁed RNA (a−c) and
LNA-modiﬁed (d−f) and 2′-OMe-modiﬁed (g−i) GC RNAs with formations of W−C face (c, f, i), rigid strand 1 (b, e, h), rigid strand 2 (a, d), andstretch type (g). (j) The relative calibrated energies. Superposition of RNA and modiﬁed RNAs (k−m). The superposition patterns of O−P−O at
the 5′ side of guanine (upper panels), and O−P−O at central phosphate (lower panels) was overlapped.

PS modiﬁcation has little eﬀect on base pair formation.18,19 The
inhibitory eﬀect on the SM target of DNA-modiﬁed siRNAs
was decreased, consistent with our previous results.15 DNA−
PS-modiﬁed siRNA exhibited almost no eﬀects. This may be
because the base-pair stability of the DNA−RNA duplex isweaker than that of the RNA duplex.15 In contrast to DNAmodiﬁed siRNAs, the inhibitory eﬀects on the SM target of the
2′-OMe and LNA modiﬁcations were strikingly decreased, eventhough both result in strong base pairs28 (Figure 3c).

Because we used siRNAs that satisfy the four functional
siRNA sequence criteria,3 the guide strand is easily unwoundfrom the 5′ terminus, but the passenger strand is not. In fact,
almost no or little inhibitory eﬀects on the CM or SM target ofthe passenger strand were observed in the reporter assays using
psiCHECK-pCM or psciCHECK-pSM, respectively (Figure
S1a).
Although DNA duplexes form B-form structures, structural
superposition of the guide DNA−target RNA heteroduplex in
the AGO complex ﬁts better with the A-form helix of RNA
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ArticleFigure 5. Computational prediction of the unmodiﬁed, 2′-OMe-modiﬁed, and LNA-modiﬁed structures loaded on the AGO protein binding pocket.
Unmodiﬁed AAA RNA with the R792 and K566 crystal structures from Schirle et al.12 (a), optimized structure of unmodiﬁed (b), 2′-OMe-modiﬁed(c), and LNA-modiﬁed RNA and (d) RNA with R792 and K566. Superposition at C4′-C3′-O3′ of A(3) of unmodiﬁed RNA with 2′-OMe-modiﬁed
RNA (e) and unmodiﬁed RNA with LNA-modiﬁed RNA (f). The numbers in (f) are the distances (in Å) between C4′ (of A(3)) and Hα (of R792)
atoms in unmodiﬁed RNA (in brown) and in LNA-modiﬁed RNA (in blue).

duplexes than with the B-form.29 This implies that the duplex
formed between a target transcript and an AGO-preloaded
guide strand is modiﬁed to an A-form structure.
Computationally, we investigated how each chemical
modiﬁcation aﬀects the single-stranded RNA structure that is
not associated with protein. The single-stranded RNA
structures of 5′-GC-3′ modiﬁed by 2′-OMe or LNA were
calculated using density functional theory at the ωB97-XD/631G(d) level. The results of geometry optimization revealed
that unmodiﬁed RNA can form at least three diﬀerent stableconformation types: (i) the “Watson (W)−Crick (C) faceform” in which two neighboring nucleotides are stabilized by
two stacked hydrogen bonds, primed to form W−C base pairs
when the complementary RNA strand is recognized (Figure4a); (ii) “rigid strand 1” in which the OH residue at the C2′
position forms bifurcated hydrogen bonds with the sugar anddownstream phosphate (Figure 4b); and (iii) “rigid strand 2”
with diﬀerent hydrogen bonds compared to those in structure 1(Figure 4c). LNA-modiﬁed RNA had structures corresponding
to structures 1 and 3 of unmodiﬁed RNA (Figure 4d−f), but wecould not detect the corresponding structure 2. 2′-OMemodiﬁed RNA had structures corresponding to structures 1 and
2 of unmodiﬁed RNA (Figure 4g,h). Additionally, structure 4,
containing a stretched backbone, was also observed (Figure 4i).
A comparison of the relative energies of these structures in each
modiﬁcation is shown in Figure 4j. In the case of 2′-OMe
modiﬁcations, the W−C face form (structure 1) was the moststable and the relative energies of the other structures(structures 2 and 4) were rather high, which indicated that
2′-OMe modiﬁcations destabilized largely the other structures.
In the case of unmodiﬁed RNA, even though structure 1 (W−C
face form) is the most stable, other structures can be taken
rather easily because of the hydrogen bonding ability of 2′-OH
with the phosphate region of the backbone (Figure 4j). Thissuggests that 2′-OMe and LNA modiﬁcations inhibit theconformational variation of the RNA backbone and tend tokeep the RNA structure suitable for base-pairing with target

RNA; thus, the 2′-OMe modiﬁcation results in the most stable
W−C base-pairing and the LNA modiﬁcation results in thesecond-most-stable pairing, with unmodiﬁed RNA having thelowest stability. Our previous results demonstrated that SM oﬀtarget eﬀects are high when the stability of the seed−target
duplex is high. 10 However, both 2′-OMe and LNA
modiﬁcations exhibited strong base-pairing stability withweaker oﬀ-target eﬀects, in contrast to our previous results.
The phosphates of the siRNA guide strand interact with theamino acid side chains of the AGO protein.12 Therefore, it was
speculated that 2′-OMe and LNA sugar modiﬁcations did notinhibit the association between the guide RNA and AGO. Our
computational prediction revealed that structure 1 of the 2′OMe-modiﬁed RNA was similar to that of the unmodiﬁed RNA(Figure 4k), consistent with a previous report.30 However, thestructure of LNA-modiﬁed RNA diﬀered from that of 2′-OMemodiﬁed RNA (Figure 4k), probably, because the sugar
puckering may aﬀect the backbone conformation. Structure 2of unmodiﬁed RNA and 2′-OMe-modiﬁed RNA exhibited only
slight diﬀerences (Figure 4l), whereas structure 3 of unmodiﬁed
RNA and LNA-modiﬁed RNA strongly diﬀered in their overall
structures, probably, because the sugar puckering is aﬀected by
the linkage between C2′ and C4′ (Figure 4m). Thus, the LNA
modiﬁcation aﬀects the RNA structure more severely comparedto the 2′-OMe modiﬁcation.
We further examined 2′-OMe- and LNA-modiﬁed singlestranded RNA structures on the AGO protein. The unmodiﬁed
5′-AAA-3′ RNA structure (nucleotides 2−4 (A(2), A(3), andA(4)) of the guide RNA) on the human AGO protein reported
by Schirle et al.12 (Figure 5a) was used after optimization(Figure 5b). The side chains of the Arg (R792) and Lys (K566)
residues in AGO form hydrogen bonds with the phosphates of
the guide RNA (Figure 5a). Therefore, the full geometry
optimization was achieved including R792 and K566, withcapping each of the amino acid main chains with a methylgroup (Figure 5b). The RNA structure in which the sugar ofA(3) was modiﬁed with 2′-OMe (Figure 5c) or LNA (Figure2059

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ArticleFigure 6. Microarray analysis of the expression levels of the target vimentin gene and SM oﬀ-target genes. (a) MA plot. The vertical bar indicates themean log 2 of signal intensities relative to those of mock transfection (M value), and the horizontal bar indicates the averaged log 10 signal intensities
of mock and siRNA transfection (A value). The dark blue dots indicate the transcripts with SM sequences, and the light blue dots indicate the othertranscripts. (b) Cumulative distribution. The horizontal axis indicates the M value of (a), and the vertical axis indicates the cumulative fraction oftranscripts. The blue line indicates the cumulative curve of SM transcripts, and the black line indicates the cumulative curve of the other non-SMtranscripts. The downregulation of SM transcripts is shown by the fold-change in the expression of SM transcripts compared to that of the othernon-SM transcripts. (c) Expression levels of the target vimentin gene by the transfection of authentic siRNA or modiﬁed siRNAs. Seed-dependentoﬀ-target eﬀects of the siRNA guide strand (d) and passenger strand (e). The vertical axis indicates the fold-change of oﬀ-target transcripts in thecells transfected with unmodiﬁed siRNA and modiﬁed siRNAs.

5d) was also optimized including R792 and K566. Overlappingof unmodiﬁed RNA and 2′-OMe-modiﬁed RNA andunmodiﬁed RNA and LNA-modiﬁed RNA on the AGO
protein is shown in Figure 5e,f, respectively. As shown in
Figure 5e, the 2′-OMe modiﬁcation of A(3) led to little
conformational change in A(2), A(3), R792, and K566.
However, the 2′-OMe modiﬁcation of the sugar in A(3)caused the repulsion of the methyl group with the nucleotide(A(4)) on the 3′ side and the conformation of the third
adenine residues (A(4)) of 2′-OMe-modiﬁed RNA became

diﬀerent from that of unmodiﬁed RNA (Figure 5e). On theother hand, the LNA modiﬁcation induced a diﬀerent eﬀect:
the methylene group of the LNA caused steric hindrance to Hαof R792 and pushed R792 away, which would result in a large
structural change of the AGO binding site, even though astacked structure of LNA-modiﬁed RNA was almost kept
(Figure 5f).
Thus, although both 2′-OMe and LNA are well-known
modiﬁcations that enhance the base-pairing stability underprotein-free conditions, our results strongly suggest that these2060

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Articlethe annotated transcripts, and the cumulative distribution
indicated the averaged fold-changes of the SM targets andcontrol non-SM transcripts (Figures 6a,b, S2, and S3). The
expression level of a CM target of vimentin gene wasunambiguously downregulated by the unmodiﬁed siRNA to
11%, and the siRNAs modiﬁed with DNA, PS, 2′-OMe-3, 2′OMe-5, 2′-OMe-7, and LNA-3 exhibited similar levels of RNAi
activity as that of the unmodiﬁed siRNA, but LNA-7
modiﬁcations showed little to no activity (Figure 6c). The
diﬀerence in the mean log 2 fold-changes of SM or non-SMtranscripts was calculated as an indicator of the degree of oﬀtarget eﬀects (Figure 6d,e). PS modiﬁcations had no eﬀect onoﬀ-target eﬀects, similar to the reporter assay results, comparedto those of unmodiﬁed siRNA (Figure 3b), and DNA
modiﬁcations reduced the oﬀ-target eﬀects as reported
previously.15 In the microarray experiments, oﬀ-target eﬀects
caused by the passenger strand were also observed. Unmodiﬁedand DNA-modiﬁed siRNAs showed similar levels of oﬀ-target
eﬀects as those of the passenger strand, but PS-modiﬁed siRNA
exhibited stronger passenger-strand-dependent oﬀ-target eﬀects(Figure 6e). Three 2′-OMe-modiﬁed siRNAs (2′-OMe-3, -5,
and -7) and two LNA-modiﬁed siRNAs (LNA-3 and -7)
exhibited reduced SM oﬀ-target eﬀects in both strands
compared to those of unmodiﬁed siRNA. These results also
support the importance of steric hindrance in association with
the AGO protein and base pair with target RNA. If asymmetry
in siRNA terminal stability is the exclusive determinant of thesiRNA unwinding eﬃciency, simultaneous reductions in the oﬀtarget eﬀects of both strands cannot be explained. In this study,
it was suggested that siRNAs modiﬁed by LNA in the seedregion did not successfully associate with AGO. When thenumber of nucleotides with LNA modiﬁcations is small, a target
gene could be successfully repressed, but when all of the seed
nucleotides were modiﬁed, a CM target as well as SM target(s)should not be repressed. However, it was suggested thatsiRNAs modiﬁed by 2′-OMe in the seed region did not aﬀect
the association with the AGO protein but formed defective basepairs with target transcripts. This means that the 2′-OMe
modiﬁcation may exhibit the similar eﬀect of siRNA with seedsequences composed of nucleotides with low thermodynamic
stabilities, as shown in our previous report,15 because bothcommonly disturb the stable base pair between the siRNA seedregion and its SM targets.
The oﬀ-target eﬀects of the guide strand of 2′-OMe-modiﬁed
siRNAs were greatest for the 2′-OMe-5 modiﬁcation comparedto those for the 2′-OMe-3 and 2′-OMe-7 modiﬁcations (Figure6d). A similar pattern was also observed in the reporter assay(Figure 3c). These results may be explained by the counterbalance between two opposing factors regulating the seeddependent oﬀ-target eﬀects: base-pair stability in the seed−target duplex and steric hindrance in the base pair with the SMtargets on the AGO protein. The 2′-OMe-7 modiﬁcation may
result in a more stable base pair in the seed−target duplex
compared to that from the 2′-OMe-3 modiﬁcation underprotein-free conditions. However, the base pair with the SMtarget RNAs on AGO should be more sterically distorted with
2′-OMe-7 compared to that from 2′-OMe-3. With regard to 2′OMe-5, the balance between seed−target base-pairing stability
and steric hindrance may induce greatest SM oﬀ-target eﬀects
among the three 2′-OMe-modiﬁed siRNAs.
However, unlike those of the guide strand, the oﬀ-target
eﬀects of the passenger strand gradually increased with thenumber of 2′-OMe modiﬁcations in the guide strand seed

modiﬁcations induce diﬀerent steric hindrance, which would
result in diﬀerent eﬀects on RNA silencing eﬃciency on CM
and SM targets in terms of steric hindrance in association with
the AGO protein or base-pairing with the target RNA. The 2′OMe modiﬁcation in the seed region of the siRNA guide strand
was essentially stable on the AGO protein, as shown in the
crystal structure by Schirle et al.,31 but it was revealed that the
2′-OMe modiﬁcation may disturb base-pairing with the target
RNA because of the conformational change in the 3′-side
nucleotide (Figure 5e). Schirle et al. also showed that 2′-OMeof a speciﬁc nucleotide in 2′-OMe-modiﬁed guide RNA leads toa major positional shift of ∼6 Å (31). Such a positional shift of
2′-OMe-modiﬁed nucleotides propagates further and disorders
the modiﬁed siRNA structure. Certainly, the suppression
activity on the SM target was greatly reduced by the guidestrand with 2′-OMe modiﬁcations in all of the sevennucleotides compared to that from those in three or ﬁve
nucleotides, although the eﬀect of siRNA with three 2′-OMe
modiﬁcations was slightly weaker than that with ﬁve (Figure3c). However, all of the 2′-OMe-modiﬁed siRNAs showedstrong gene repression eﬀects on the CM target (Figure 3b),although the inhibitory eﬀects were slightly reduced according
to the increase of the 2′-OMe modiﬁcations. The 5′-half of thesiRNA guide strand is more sensitive sterically to chemical
modiﬁcations than is the 3′-half with regard to RNAi activity
against the CM target.32 Then, the nonseed region may play
important roles in RNAi against the CM target. Recent singlemolecule imaging revealed that the AGO protein reshapes thebinding properties of the microRNA (miRNA) guide strand
and serves as a speciﬁcity determinant with thermodynamic andkinetic properties typical of RNA-binding proteins.33,34 They
showed that when RISC binds a target transcript through bothmiRNA seed and its 3′ supplementary nonseed region it
dissociates nearly as rapidly as for seed-only binding and therates of association and dissociation are very similar for these
two binding modes. Our results clearly suggested that theseed−target base pair regulates the SM target expression.
However, even when the expression of the SM target was notinhibited by siRNA with 2′-OMe or LNA modiﬁcations in theseed region, the expression of its CM target was signiﬁcantly
inhibited (Figure 3b,c). Thus, our result suggests that thenonseed region may compensate the incomplete seed−target
base pair for inducing RNAi on the CM target.
The guide RNA structure with LNA modiﬁcations on the
AGO protein was kinked by the sugar puckering by the linkage
between C2′ and C4′ (Figure 4k−m), indicating that the basepairing with target RNA is disturbed to some extent.
Furthermore, the LNA-modiﬁed guide strand repulsed within
the AGO binding site (Figure 5f). Thus, it was considered thatsiRNA modiﬁed with LNA in 7 nts at the seed region, LNA-7,
did not associate with the AGO protein. Then, the expressionof both the CM and SM targets may not be inhibited (Figure3b,c). However, LNA-3 can associate with the AGO protein.
Then, the SM oﬀ-target eﬀects were reduced by the disturbanceof base-pairing with the target RNA (Figure 3c) without
substantial eﬀects on the CM target (Figure 3b).
Microarray analysis was performed to examine the genomewide oﬀ-target eﬀects of siRNAs modiﬁed with DNA, PS, 2′OMe, and LNA (Figure 2b). Unmodiﬁed and modiﬁed siRNAs
were each transfected into HeLa cells. Total RNA was puriﬁed
from cells 1 day later and subjected to microarray analyses. The
MA plots (M = intensity ratio, A = average intensity) of the
microarray data indicated changes in the expression levels of
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ArticleGAACTCTCCTGGTGCATCATTTTGAACTCTCCTGGTGCATCATTTTGAACTCTCCTGGTGCATCATT-3′) and the passenger
strand (5′-tcga AATGATGCACCAGGAGTTGATGGAATGATGCACCAGGAGTTGATGGAATGATGCACCAGGAGTTGATGG-3′ and 5′-aattCCATCAACTCCTGGTGCATCATTCCATCAACTCCTGGTGCATCATTCCATCAACTCCTGGTGCATCATT-3′), each of which hascomplementarity with the 8 nt long seed-containing sequence
but not with the nonseed region, were also generated andnamed psiCHECK-gSM and psiCHECK-pSM, respectively.
Each of the inserted targets was expressed as part of the 3′UTR region of Renilla luciferase mRNA in the transfected cells.
Cell Culture and the RNA Silencing Activity Assay
Using the Fireﬂy Luciferase Reporter System. Human
HeLa cells were cultured in Dulbecco’s modiﬁed Eagle’s
medium (Gibco BRL) supplemented with 10% heat-inactivatedfetal bovine serum (Mitsubishi Kagaku) at 37 °C. The cells
inoculated in each well of 24-well plates at 1 × 105 cells/mL
were transfected simultaneously with psiCHECK-gCM, -gSM,-pCM, and -pSM target constructs (100 ng), pGL3-Control(100 ng; Promega), and siRNA duplex (0.0005, 0.005, 0.05, 0.5,
and 5 nM) using 2 μL of lipofectamine 2000 per well (LifeTechnologies). siGY441 was used as the control. The cells were
harvested 24 h post-transfection, and the relative luciferase
activity (Renilla luciferase activity/ﬁreﬂy luciferase activity) was
measured using a dual-luciferase reporter assay system(Promega). The pGL3-Control encoding ﬁreﬂy luciferase
served as a control for the calculation of the relative luciferase
activity.
Computational Prediction of the Structure of
Modiﬁed RNA. All of the geometries of single-stranded 5′GC-3′ RNA structures [unmodiﬁed and modiﬁed (2′-OMe or
LNA for both G and C)] were optimized at the theoretical levelof ωB97-XD/6-31G(d). We conﬁrmed that all of the optimized
geometries are local minima with all positive harmonicfrequencies. Zero-point energy (ZPE) was calculated using
the harmonic frequencies for each of the local minima. The
optimized geometries are shown in Figure 4, and the energies
are listed in Table S1, together with all of the Cartesian
coordinates.
The Cartesian coordinates of the crystal structure12 (PDB
4OLA) were used as an initial geometry for 5′-AAA-3′ RNAloaded on the AGO protein. Arg792 and Lys566 from the AGO
protein were taken into the calculations, where the main chainsof these amino acids were replaced by methyl groups (FigureS4). For 5′-AAA-3′ RNA, we used three types: unmodiﬁed andmodiﬁed (2′-OMe or LNA for the central A). The fullgeometry optimization was performed for each of the models of
the three types (5′-AAA-3′ RNA with Arg792 and Lys566) at
the theoretical level of ωB97-XD/6-31G(d). The optimized
geometries are shown in Figure 5, and the energies are listed inTable S2, together with all of the Cartesian coordinates. The
Gaussian 0935 program package was used for all of thecalculations. The calculations were carried out at the Center for
Quantum Life Sciences (QuLiS) and at the Research Center for
Computational Science, Okazaki National Research Institutes.
Microarray Analysis. The cells inoculated in each of 2
wells of 24-well plates at 1 × 105 cells/mL were transfectedwith 50 nM of siRNA duplex using 2 μL of Lipofectamine
2000. At 24 h post-transfection, total RNA was puriﬁed with an
RNeasy kit (Qiagen) and RNA quality was assessed using a
NanoDrop 2000 spectrophotometer (Thermo Scientiﬁc) and aregion (Figure 6e). The results suggest that only the unwinding
eﬃciency of each terminus may aﬀect the oﬀ-target eﬀect of thepassenger strand. The 5′-terminal base-pairing stability in
siRNA duplexes was highest for the 2′-OMe-7 modiﬁcation andlowest for the 2′-OMe-3 modiﬁcation, indicating that
unwinding from the 5′-terminus of the guide strand is mostfavorable for 2′-OMe-3 but unfavorable for 2′-OMe-7. Thus,
unwinding from the 5′-terminus of the passenger strand is easyfor 2′-OMe-7 but diﬃcult for 2′-OMe-3, leading to a stronger
induction of oﬀ-target eﬀects by the former compared to thatby the latter.

■

CONCLUSIONSIn conclusion, the chemical modiﬁcations of DNA, PS, DNA−PS, 2′-OMe, and LNA in the seed region of the siRNA guidestrand reduced the oﬀ-target eﬀects of the guide strand SM
targets and may also reduce those of the passenger strand CM
and SM targets. Such oﬀ-target eﬀects were greatly reduced
without aﬀecting the RNAi eﬀects on the CM target usingsiRNA containing 2′-OMe modiﬁcations in the guide strand
seed region. Our results suggest that appropriate introductionof 2′-OMe into siRNA can reduce oﬀ-target eﬀects by inducing
steric hindrance in duplex formation on the AGO protein. Sucha chemical modiﬁcation may function in a similar manner to the
nucleotide sequences in the siRNA seed region with low
thermodynamic stability. Because the oﬀ-target eﬀects areserious problems that need to be addressed, the modiﬁcations
like 2′-OMe in the seed region may overcome the limitations ofsiRNA sequences by reducing the oﬀ-target eﬀects and may be
useful as a potential therapeutic in the future.

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EXPERIMENTAL SECTION
Preparation of Chemically Synthesized siRNA Duplexes. In this study, we used an siVIM-270,3 which satisﬁes
four functional sequence conditions, to examine the seeddependent oﬀ-target eﬀects. The guide strand sequence ofsiRNA against human vimentin (siVIM-270) is 5′-UUGAACUCGGUGUUGAUGGCG-3′ and the passenger strand
sequence is 5′-CCAUCAACACCGAGUUCAAGA-3′. RNA
oligonucleotides of siRNA duplexes with and without
modiﬁcations, shown in Figure 2a, were chemically synthesized(Genepharma).
Construction of Luciferase Reporters with CM and SM
Sequences. All of the reporter plasmids were constructedfrom psiCHECK-1 (Promega). Oligonucleotides with thetarget sequence that is CM to the siRNA guide strand (5′tcgaCGCCATCAACACCGAGTTCAAGA-3′ and 5′aattTCTTGAACTCGGTGTTGATGGCG-3′) and three tandem repeats of the passenger strand CM target sequences (5′t c g a TCT T GA A C T C G GT GT TG A T GG C GAA TC T TGAACTCGGTGTTGATGGCGAATCTTGAACTCGGTGTTGATGGCGAA-3′ and 5′-aattTTCGCCATCAACACCGAGTTCAAGATTCGCCATCAACACCGAGTTCAAGATTCGCCATCAACACCGAGTTCAAGA3′) were chemically synthesized with cohesive XhoI/EcoRI
ends. They were annealed and inserted into psiCHECK-1 at
the corresponding restriction enzyme sites and named
psiCHECK-gCM and psiCHECK-pCM, respectively. Similarly,
psciCHECK with three tandem repeats of SM sequences to thesiRNA guide strand (5′-tcga AATGATGCACCAGGAGAGTTCAAAATGATGCACCAGGAGAGTTCAAAATGATGCACCAGGAGAGTTCAA-3′ and 5′-aattTT2062

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Bioanalyzer (Agilent). cDNA was synthesized from each total
RNA sample using an Agilent one-color spike mix kit (Agilent)and used for hybridization to an Agilent SurePrint G3 human
GE microarray (8 × 60 K, ver. 2.0, 3.0) according to the
manufacturer’s protocol. RNA from mock-transfected cells
treated with the transfection reagent in the absence of siRNAwas used as a control, and the distributions of the signal
intensities of transcripts were normalized across all samples byquantile normalization.36 They were shown in MA plots andcumulative accumulations.

■

ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsomega.7b00291.
Results of luciferase reporter assays of the passenger
strand; additional information on microarray analyses,
MA plots and cumulative distributions of the SM oﬀtarget transcripts of the siRNA guide strand; additional
information on microarray analyses, MA plots andcumulative distributions of the SM oﬀ-target transcriptsof the siRNA passenger strand; supplementary computational data, total energy and ZPE-corrected energy of 5′GC-3′ RNA; total energy of 5′-AAA-3′ RNA with the
AGO protein (PDF)■

AUTHOR INFORMATION

Corresponding Author*E-mail: ktei@bs.s.u-tokyo.ac.jp.
ORCID

Misako Aida: 0000-0001-8788-1071
Kumiko Ui-Tei: 0000-0001-8695-9025
Author Contributions

H.I., T.T., Y.K., and J.L. performed the reporter assays.
Microarray experiments were carried out by T.T., and H.I.
and T.T. analyzed the data. Computational predictions of
nucleotide structures were performed by K.M. and M.A. M.A.
and K.U.-T. discussed the experimental and computational
data. The manuscript was written by K.U.-T. and reviewed by
all authors. K.U.-T. designed the study and is responsible forscientiﬁc correspondence. All authors have given approval tothe ﬁnal version of the manuscript.
Funding

This work was ﬁnancially supported by the Grants-in-Aid for
Scientiﬁc Research (B) (No. 15H04319) and on Innovative
Areas (No. 26102713) from the Ministry of Education, Culture,
Sports, Science and Technology and Japan Society for the
Promotion of Science, and by the grant from the Suzuken
Memorial Foundation to K.U.-T.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
Theoretical calculations were partly performed using the
Research Center for Computational Science, Okazaki, Japan.
The English in this document has been checked by at least two
professional editors, both native speakers of English.

■

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