2136–2151 Nucleic Acids Research, 2008, Vol. 36, No. 7
doi:10.1093/nar/gkn042

Published online 11 February 2008

Functional dissection of siRNA sequence bysystematic DNA substitution: modified siRNAwith a DNA seed arm is a powerful tool for
mammalian gene silencing with significantly
reduced off-target effect1

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033 and 2Molecular Cellular Oncology and Microbiology, Graduate School,
Tokyo Medical and Dental University, Tokyo, Japan

Received December 11, 2007; Revised January 21, 2008; Accepted January 22, 2008

ABSTRACTINTRODUCTION

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target geneknockdown in mammalian cells. To clarify the
position-dependent functions of ribonucleotides in
siRNA, siRNAs with various DNA substitutions
were constructed. The following could be simultaneously replaced with DNA without substantial lossof gene-silencing activity: the seed arm, which
occupies positions 2–8 from the 5’end of theguide strand; its complementary sequence; the5’end of the guide strand and the 3’overhang of
the passenger strand. However, most part of the 3’
two-thirds of the guide strand could not be replacedwith DNA, possibly due to binding of RNArecognition proteins such as TRBP2 and Ago2. The
passenger strand with DNA in the 3’end proximal
region was incapable of inducing off-target
effect. Owing to lesser stability of DNA–RNA hybrid
than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases,
incapable to exert unintended gene silencing due
to seed sequence homology. Thus, it may bepossible to design DNA–RNA chimeras which effectively silence mammalian target genes withoutsilencing unintended genes.

Several nucleic acid-based methods for knockdown oftarget gene activity have become available over the last
several decades, and RNA interference (RNAi) is probably the most prominent. In mammals, short interfering
RNA (siRNA), 19-bp long double-stranded (ds) RNA
with 2-nt 30 overhangs, is widely used for inducing RNAi
(1,2). In Drosophila cells, a heterodimer of Dicer2 andR2D2 is considered to sense the diﬀerential stability ofsiRNA duplex ends in determining the guide strand (3,4).
The Dicer2/R2D2 dimer in RLC [RNA-induced silencing
complex (RISC) loading complex] is gradually replaced by
Argonaute (Ago). Protein-complexed siRNA undergoes
unwinding. Recent studies on the structures of archea andeubacteria Ago-like proteins (5–7) and the PAZ domain ofhuman Ago1 (8) have provided a great deal insight into
the molecular mechanism of RNAi in mammals and otheranimals. The 50 and 30 ends of the guide strand may beanchored in pockets formed in the Mid and PAZ domainsof Ago, respectively (4,7,9). The 50 -proximal ‘seed’
nucleotides (10), occupy position 2–8 measured from the
50 end of the guide strand, and are present on the surfaceof the Mid-PIWI lobe associated with a linker L1 in aquasi-helical form in the RISC, may serve as the entry or
nucleation site for mRNA (7).
In active RISCs, target mRNA is cleaved by the RNase
H-like slicer activity of Ago (Ago2 in the case of
mammals) (11,12). Only siRNA with seed sequence*To whom correspondence should be addressed. Tel: +81 3 5841 3043; Fax: +81 3 5841 3044; Email: ktei@biochem.s.u-tokyo.ac.jp
Present address:
Shuhei Zenno, Department of Biotechnology, Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisadori-cho, Maebashi-shi,
Gunma 371-0816, Japanß 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

Kumiko Ui-Tei1,*, Yuki Naito1, Shuhei Zenno1, Kenji Nishi1, Kenji Yamato2,
Fumitaka Takahashi1, Aya Juni1 and Kaoru Saigo1

Nucleic Acids Research, 2008, Vol. 36, No. 7 2137

MATERIALS AND METHODS
Cell culture and gene-silencing activity assay
Chinese hamster CHO-K1, human HeLa, mouse embryonic stem (ES) cells (E14TG2a) and Drosophila S2 cells
were cultured and subjected to gene-silencing assay as
described previously (20). Brieﬂy, 1-ml cell suspensions of
CHO-K1 (1  105 cells/ml), HeLa (1  105 cells/ml),
E14TG2a (2  105 cells/ml) and S2 (1  106 cells/ml)
were inoculated in a 1.5-cm well 24 h prior to transfection.
Cells were transfected with pGL3-Control (1 mg, Promega)or pGL2-Control (1 mg; Promega), both coding for ﬁreﬂyluciferase (luc) gene, and Renilla-luc-gene encoding pRLSV40 (0.1 mg, Promega) with or without siRNA or thecorresponding DNA-modiﬁed siRNA. Cells were harvested 24 h after transfection and luc activity wasmeasured using the Dual-Luciferase Reporter Assay
System (Promega). RNA, DNA and DNA-modiﬁed
siRNAs, of which the nucleotide sequences used in thisstudy are listed in Supplementary Table S1, werechemically synthesized (Proligo). Fifty percent inhibitory

concentrations (IC50s) of siRNAs and DNA-modiﬁed
siRNAs were estimated using the Pharmacologic
Calculation Program (22).
RT–PCR
Using Lipofectamine 2000 (Invitrogen), E14TG2a or
HeLa cells were cotransfected with 50 nM siRNA or
DNA-modiﬁed siRNA, and pCAGIPuro-EGFP (0.5 mg/ml;20), which encodes EGFP and puromycin-resistant genes.
Puromycin (2 mg/ml; Clontech) was added to the medium
24 h after transfection. RNA was extracted 3 days after
transfection using RNeasy (QIAGEN). Possible change in
relative fraction of target RNA was examined by
RT–PCR using the RNA LA-PCR kit (Takara) andmGapdh mRNA as an internal control. Primers used arelisted in Supplementary Table S2.
Electrophoresis mobility shift analysis
Regions encoding a full-length human PACT (23) andhuman immunodeﬁciency virus trans-activating response
RNA-binding protein 2 (TRBP2) (24) were ampliﬁed froma cDNA mixture of a total RNA extracted from HeLa
cells by PCR. Used primers are as follows: PACT (50 TTTTTTTTTTCATATGTCCCAGAGCAGGCACCGC
GAGGCC and 50 -TTTTTTTTGCGGCCGCTTACTTT
CTTTCTGCTATTATCTTTAAATA) and TRBP2 (50 -T
TTTTTTTTTCATATGCTGGCCGCCAACCCAGGCA
AGA and 50 -TTTTCCTTTTGCGGCCGCTCACTTGC
TGCCTGCCATGATCTTGAGGTA). The ampliﬁed
fragments were digested with NdeI and NotI and cloned
into pET-28a (Novagen). PACT and TRBP2 proteins
were produced in Escherichia coli BL21 codon plus andRosetta (DE3) pLysS, respectively, as amino-terminal
hexa-histidine-fusion proteins, and puriﬁed with NTA
agarose. Binding to siRNA or its DNA counterparts(siDNA) (5 fmol/ml) was carried out in 25 mM Tris–HCl(pH8.0), 150 mM NaCl, 125 mM imidazole, 13% glycerol,
50 ng/ml salmon sperm DNA and 2 U/ml RNasein for
30 min at room temperature. The mobility shift assay was
conducted on a 5% polyacrylamide gel and analyzed
quantitatively using FLA-2000 image analyzer (Fujiﬁlm).
Target cleavage assay
Luc2-153, the target sequence for the ﬁreﬂy luc siRNA,
siLuc2-153, was inserted into the EcoRI/XhoI site ofpTREC (25) to generate pTREC-2-153. The construct(0.5 mg) was introduced by transfection into HeLa cellswith or without cognate siRNA or DNA-modiﬁed siRNAand cells were recovered 24 h after transfection. Total
RNA was prepared using QuickPrep Micro mRNAPuriﬁcation Kit (Amersham). Primer extension was
carried out using 32P-labeled primer (50 -CTCGAAGCA
TTAACCCTCACT-30 ) and the Primer Extension SystemAMV Reverse Transcription Kit (Promega). Reaction
products were size-fractionated by electrophoresis on 6%
polyacrylamide gel containing 7-M urea in TBE buﬀer.
Cleavage sites were determined using parallel sequence gel.
DNA sequence analysis was carried out by using Thermo
Sequence Fluorescent Labeled Primer Cycle Sequencing

Downloaded from nar.oxfordjournals.org at Ekigaku-Kyoshitsu (UNIV OF TOKYO) on October 17, 2010homology to the target may serve as microRNA andinduce gene silencing via translational repression rather
than mRNA cleavage (13). In addition, recent microarray
proﬁling experiments revealed that unintended reduction
in expression of a large number of transcripts can also be
observed following the transfection of siRNA withoutcomplete sequence complementarity to the transcripts,
reminiscent of microRNA-dependent reduction in gene
expression (10,14,15).
Certain ribonucleotides in siRNAs undergo substitution
with deoxyribonucleotide counterparts without substantial loss of gene-silencing activity. The 30 overhangs are
frequently replaced with deoxythymidine to prevent thedegradation of siRNA in cells (1,16,17). The passenger
strand of siRNA may be substituted totally with the DNA
counterpart (18). Chiu and Rana (19) introduced various
chemically modiﬁed nucleotides into siRNAs and foundthat the 20 OH of the guide strand near the mRNA
cleavage site may not be required for catalytic ribonuclease activity of RISC.
Here, we examined the eﬀects of systematic deoxyribonucleotide substitutions of highly functional siRNAs(20) on gene silencing. The 50 end and the 50 proximal ‘seed’
arm of the guide strand were found capable of being
completely replaced with cognate deoxyribonucleotides
with little or no loss of gene-silencing activity. In contrast,
replacing the 30 proximal RNA sequence of the guidestrand with its DNA counterpart resulted in almost
complete loss of gene-silencing activity. Unlike thenonmodiﬁed siRNAs, DNA-modiﬁed siRNAs, in which
the seed region is DNA and the remainder is RNA, could
hardly exert reduction of the activity of genes other than
the target gene (oﬀ-target eﬀect) (21). These ﬁndings may
indicate most mammalian genes to be eﬀectively knocked
down without substantial oﬀ-target eﬀect by treating
cells with a class of DNA-modiﬁed siRNAs possessing a
DNA-seed-arm sequence.

2138 Nucleic Acids Research, 2008, Vol. 36, No. 7

Kit (Amersham) but ﬂuorescent-labeled dideoxyribonucleotides were replaced with 32P-labeled dideoxyribonucleotides.
Construction of psiCHECK derivatives and the assay
of seed-sequence- and passenger-strand-based
off-target activity

Calculation of melting temperature
Melting temperature (Tm) was calculated based on thenearest-neighbor model (26) and the thermodynamic
values for RNA–RNA (27) and RNA–DNA (28).
Oligonucleotide and sodium chloride concentrations
were assumed to be 100 nM and 100 mM, respectively.
Microarray analysis
HeLa cells (1  105 cells/ml) were inoculated in a 1.5-cm
well 24 h prior to transfection. Cells were transfected with
50 nM siVIM-270 or chiVIM-270. Total RNA (3 mg)
puriﬁed using RNeasy Kit (Qiagen) 1 day after transfection was used for hybridization to Human Genome U133
Plus 2.0 GeneChip (Aﬀymetrix) containing 47 400
human transcripts according to the manufacturer’s protocol. RNA from mock-transfected cells, which weretreated with transfection reagent in the absence ofnonmodiﬁed or DNA-modiﬁed siRNAs, was used as acontrol. To calculate transcript expression values,
Microarray Suite 5.0 (MAS5, ref. 29) was used withquantile normalization (30), and transcritps with suﬃcienthybridization signals to be called present (P) were used inthis study. To identify transcripts that were downregulated on the array, we compared the cumulative distribution of expression changes for messages with the site
versus those with no canonical site, and calculated the
maximum positive cumulative diﬀerence between the two
distributions. The statistical signiﬁcance of their dissimilarity was quantiﬁed using the Wilcoxon’s rank-sum test
(31,32) as P-value.
Motif analysis of 3’UTR
The probe sequences were taken from the latest
annotation table provided on the Aﬀymetrix Web site(http://www.aﬀymetrix.com). We mapped them to the
RefSeq human mRNA sequences (release 24), and thenidentiﬁed the target transcripts. We found 67 220

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The plasmids psiCHECK-sm-X1, X2 and X3, respectively, included one, two and three copies of seed-matched(sm) target sequences while psiCHECK-cm-X1-X3,
included one to three copies of completely matched (cm)target sequences (see Figure 4, Supplementary Figures S6,
S8 and S9). They were constructed as follows. Chemicallysynthesized oligodeoxynucleotides (29–75 bp) including
one to three copies of the same 23-bp sm or cm target
sequences with cohesive XhoI/EcoRI ends were inserted
into the psiCHECK-1 (Promega) XhoI/EcoRI site, which
is situated in the region encoding the 30 UTR of Renillaluciferase mRNA. Human vimentin and mouse Oct4
sequences were used as targets (Supplementary Table S3).
The cm sequence is completely matched that of the guidestrand of nonmodiﬁed or DNA-modiﬁed siRNAs, and isexpressed as a part of the target mRNA in transfected cells(see Figure 4). In contrast, the sm sequence in the target
mRNA consists of two parts. Its 30 -terminal one-third iscomplementary in sequence to the 8-bp long 50 proximal
region of the guide strand of siRNA or DNA-modiﬁed
siRNA, while the remaining two-thirds, totally nonhomologous (see Figure 4). To construct psiCHECK-sm-X6,
which includes six copies of Oct-797 and Luc-36 target
sequences (see Supplementary Figure S9), 144-bp of ds
insert DNA was synthesized by enzymatic ligation of 71and 73-bp long chemically synthesized dsDNA fragmentswith 4-nt long 50 protrusions, and inserted into the XhoI/
EcoRI site of psiCHECK-1. psiCHECK-GS andpsiCHECK-PS, respectively, were constructed by insertinga PCR-ampliﬁed fragment containing target region(nucleotide position 97–856) of HPV16 E6E7 (accession
number K02718) into the NotI site of psiCHECK-1 (seeFigure 3). Used primers are as follows: 50 -GCGGCCGC
AACTGCAATGTTTCAGGACC and 50 -GCGGCCGC
TTATGGTTTCTGAGAACAGA. As GS/PS inserts,
HPV-16 sequences along with three copies of 23-bp longtarget sequence of VIM-270 and Oct-797 (Supplementary
Table S3) were used. The orientation of the insert wasdetermined by nucleotide sequence analysis and plasmids
with diﬀerent insert orientations were selected. Plasmids
expressing the GS and PS targets, respectively, used as
psiCHECK-GS and psiCHECK-PS. HeLa cells in a wellof 24-well culture plate were transfected simultaneouslywith one of psiCHECK derivatives (0.1 mg each) andpGL3-Control (Promega, 1 mg), phLuc-Control (seebelow, 1 mg) and siRNA or DNA-modiﬁed siRNA. Cells
were harvested 24 h after transfection and luc activity wasmeasured using the Dual-Luciferase Reporter Assay
System (Promega). For assay using HVP16 target
sequences, HeLa cells in a well of 96-well culture
plate were transfected with a psiCHECK derivative(1.6 ng), pZeoSV2-hLuc (0.4 ng) and nonmodiﬁed
or DNA-modiﬁed siRNAs and analyzed 48 h after

transfection. pGL3-Control, encoding the ﬁreﬂy luc,
served as a control for Renilla luc silencing assay for thesiRNAs (siVIM-270, -596, -812 and -1128, siOct-797, and-821, siGRK4-934) along with cognate DNA-modiﬁed
siRNAs (chiVIM-270, -596, -812 and -1128, chiOct-797,
and -821, chiGRK4-934). The gene-silencing assay for
siRNAs (siLuc-36, -49, -309, -774 and 2-153) and cognate
DNA-modiﬁed siRNAs (chiLuc-36, -49, -309, -774 and 2153) was carried out using as a control phLuc-Control
coding for a modiﬁed ﬁreﬂy luciferase gene. The assay forﬁve siRNAs (siHPV16-497, 573, -698, -707 and -752) andcognate DNA-modiﬁed siRNAs (chiHPV16-497, 573,
-698, -707 and -752) was carried out using pZeoSV2hLuc. phLuc-Control is a derivative of pGL3-Control(Promega) in which the 1.7-kb XbaI/HindIII region
encoding ﬁreﬂy luc is replaced with the XbaI/HindIII
hLuc fragment of psiCHECK-2 (Promega). pZeoSV2hLuc is constructed by introducing hLuc fragment into
pZeoSV2 (Invitrogen). The siGCY416 is an siRNA forknockdown of GFP and serves as an siRNA control. The
nucleotide sequences for the target described above areshown in Supplementary Table S3.

Nucleic Acids Research, 2008, Vol. 36, No. 7 2139

annotations of the human transcripts, which corresponded
to 54 675 of the probesets. Among these, 19 856 transcripts
called P by MAS5, of which 16 783 RefSeq entries (25%)
with the 30 UTR were considered in this study. sm
sequences at positions 2–8, 1–7 and 1–8 from the 50 endof the guide strand were assigned to the extracted 30 UTR
sequences.
Quantitative RT–PCR

RESULTS
Asymmetric requirement of ribonucleotides
in siRNA for effective gene silencing
In the present article, the ends of siRNA possessing the
50 ends of the guide and passenger strands, respectively, arereferred to as GS and PS ends (Figure 1A). To examine
the eﬀects of DNA replacement on gene silencing systematically, six diﬀerent types of partially DNA-substituted
siRNAs, whose structures are illustrated in Figure 1A,
were constructed based on the sequences of siLuc-36(Figure 1) and siLuc2-153 (Supplementary Figure S1).
These siRNAs have been shown to be highly functional in
ﬁreﬂy luc-gene silencing (20). In three left DNA-modiﬁed
siRNAs in Figure 1A, ribonucleotides were progressively
replaced with cognate deoxyribonucleotides from the PS
end, while, in three right ones, deoxyribonucleotide
substitutions occurred progressively from the GS end.
In replacement with dsDNA, paired ribonucleotides were
simultaneously replaced with cognate deoxyribonucleotides. In siRNAs with DNA replacement only in theguide and passenger strand, respectively, guide andpassenger strands are DNA–RNA chimeras, with theremaining being RNA. Using these diﬀerent types of
DNA-modiﬁed siRNAs, examination was made of possible ﬁreﬂy luc gene activity reduction in three mammaliancells, CHO-K1, HeLa and E14TG2a and Drosophila S2cells (Figure 1B–G and Supplementary Figure S1A–F).
DNA replacement of the two siRNA sequences gave
results nearly the same with each other in all threemammalian cells. In the case of dsDNA replacement fromthe PS end, all modiﬁed siRNAs except those containing
DNA replacement only in the vicinity of the PS end had
little, if any, gene-silencing eﬀect on luc target (Figure1B,
Supplementary Figure S1A). In contrast, as with nonmodiﬁed siRNAs, nearly all luc gene activity was

ES-to-differentiated cell fate transition induced
by functional DNA-modified siRNA-dependent
inactivation of Oct4
Oct4 is a gene-encoding POU transcription factor that
regulates ES cell pluripotency (34). A reduction in Oct4
expression leads to the loss of pluripotency and diﬀerentiates ES cells into trophectoderms, which are characterized by ﬂat morphology (34). Indeed, bothmorphological changes and reduction in Oct4 expression
were observed in mouse ES cells transfected with siOct797, an siRNA speciﬁc to Oct4 (Figure 2A1 and 2; B1
and 2). As shown in Figure 2A4 and B4, chiOct-797-GS,
a DNA-modiﬁed siRNA version of siOct-797, of which

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Quantitative RT–PCR was carried out using total RNA
puriﬁed, whose properties were also analyzed using the
microarray. RNA was reverse-transcribed using theSuperScript First-Strand Synthesis System for RT–PCR(Invitrogen). The resultant cDNA and SYBR Green PCR
Master Mix (Applied Biosystems) were mixed andincubated at 958C for 10 min before the PCR reaction.
The levels of PCR products were monitored with ABIPRISM 7000 sequence detection system and analyzed withABI PRISM 7000 SDS software (Applied Biosystems).
Each reaction was run in triplicate. The expression level ofeach sample was ﬁrst normalized to the amount of b-actin
and then to the mock-transfection control. The usedprimer sets are listed in Supplementary Table S4.

abolished subsequent to transfection with modiﬁed
siRNAs with dsDNA substitution 10 bp (Figure 1C) or
8 bp (Figure S1B) in length from the 50 end of the guidestrand. Furthermore, in mammalian cells, gene-silencing
eﬀects due to transfection with modiﬁed siRNAs with
dsDNA substitution from the GS end were noted strongly
correlated with those induced by that with modiﬁed
siRNAs with DNA substitution only in the guide strand(Figure 1B–E, Supplementary Figures S1A–D and S2A).
In contrast, transfection with modiﬁed siRNAs with DNA
substitution only in the passenger strand gave a small, if
any, eﬀect on luc target gene silencing in mammalian cells(Figure 1F and G, and Supplementary Figures S1E and F
and S2B). Mammalian gene silencing due to treatment
with siRNAs with dsDNA substitution may thus beconcluded due primarily to interactions between the guidestrand and target mRNA and/or those between the guidestrand and protein moieties involved in gene silencing. The
contribution of the passenger strand to gene silencing ismuch greater in Drosophila than mammalian cells, sinceconsiderable reduction of luc gene activity was evident in
S2 cells transfected with modiﬁed siRNAs with DNA
substitution only in the passenger strand (Figure 1F andG, and Supplementary Figure S1E and F).
As described below, ribonucleotide residues in the PSend proximal region are essential to RLC- and/or RISCprotein binding. The 8-bp long GS-end proximal region,
which can be replaced with the DNA counterpart without
substantial loss of gene-silencing activity, includes the seedregion situated from nucleotide position 2 to 8, measured
from the 50 end of the guide strand.
DNA-replacement/gene-silencing proﬁles of the siLuc2153 sequence were unexpectedly found to shift a few bases
to the side of the 50 end of the guide strand from thoseof siLuc-36 sequence (Supplementary Figure S3). The
apparent proﬁle shift in the vicinity of the PS end may
be due in part to diﬀerent sequence-dependent eﬀects ofdeoxyribonucleotide replacement in the 30 overhang.
The presence or the absence of 50 terminal phosphategave no signiﬁcant diﬀerence on gene silencing(Supplementary Figure S4), suggesting that, as withnonmodiﬁed siRNA (33), the 50 end of DNA-modiﬁed
siRNA is phosphorylated within cells. Interferon response
due to DNA-modiﬁed siRNA was not more profoundthan that due to nonmodiﬁed siRNA (Supplementary
Figure S5).

2140 Nucleic Acids Research, 2008, Vol. 36, No. 7

A

dsDNA replacement

PS endpassenger strand
guide strand

guide strand

5′

3′

3′

GS end

5′

5′

3′

3′

5′

Guide-strand-specific DNA replacement
5′
3′
5′
3′

5′
3′

3′
5′

Passenger-strand-specific DNA replacement
passenger strand

5′

3′

3′

5′

5′
PS end↓5′
GS end↓siLuc-36

21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 −1 −2

Relative luc activity(%)
Relative luc activity(%)

F passenger strand DNA
5′
3′
3′
5′

3′
5′

3′
5′

dsDNA

dsDNA
5′
3′

3′
5′

E

5′
3′
guide strand DNA

5′
3′

3′
5′

guide strand DNA

Gpassenger strand DNA
5′
3′

3′
5′

Relative luc activity(%)

D

5′
3′

Relative luc activity(%)

C

Relative luc activity(%)

B

Relative luc activity(%)

5′-C A U U C U A U C C G C U G G A A G A U G -3′3′-C G G U A A G A U A G G C G A C C U U C U-5′

Position of DNA/RNA boundary from 5′ end of guide strand
S2

CHO-K1HeLa

E14TG2a

Figure 1. Eﬀects of the size and position of deoxyribonucleotide substitutions on gene silencing. (A) siRNAs with various DNA substitutions wereconstructed based on the siLuc-36 sequence, which is shown in the lower margin with nucleotide position measured from the 50 end of the guidestrand, and were subjected to the luc gene assay. Six diﬀerent types of DNA-modiﬁed siRNAs are schematically shown. In three left constructs, RNAsequences were progressively replaced with DNA counterparts from the PS end, which includes 50 and 30 ends of passenger and guide strands,
respectively. In three right constructs, RNA was replaced with DNA from the GS end, including the 5 0 end of the guide strand and 30 end of thepassenger strand. Both left and right constructs include three types of DNA replacement, that is, double-strand replacement, and guide andpassenger-speciﬁc replacement. DNA and RNA portions are colored in blue and orange, respectively, and arrows indicate the direction of DNA
replacement. (B–G) Ribonucleotides of double (B, C), guide (D, E) and passenger (F, G) strands were progressively replaced withdeoxyribonucleotide counterparts from the PS end (B, D, F) or the GS end (C, E, G). Numerals from -2 to 21, which are situated below eachgraph indicate nucleotide position from the 50 end (position 1) of the guide strand. (–1, –2) and (20, 21), respectively, correspond to 30 overhangs at
the GS and PS ends. Relative luc activity at position ‘x’ indicates the activity due to authentic siRNA (siLuc-36). S2, CHO-K1, HeLa and E14TG2acells were transfected with pGL3-Control DNA (1 mg) and pRL-SV40 DNA (0.1 mg) with or without authentic siRNA or the corresponding modiﬁed
siRNAs with various DNA substitution (50 nM each). Relative luc activity was measured 24 h after transfection. Note that modiﬁed siRNA with
10-bp or <10-bp long single or double-stranded DNA substitution from the GS end was capable of inactivating the target luc gene as eﬀectively
as authentic siRNA.

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nucleotide positionpassenger strand
guide strand

3′

3′

Nucleic Acids Research, 2008, Vol. 36, No. 7 2141

siLuc-12(control)A

siOct-797

1

2

chiOct-797-PS

chiOct-797-GS
4

3(days)

siLuc-12(control)0

siOct-797
2

3

chiOct-797PS
2
3

chiOct-797GS
2
3

mGapdh
Oct4
Hand1
Psx1

Requirement of 7-bp long GS-end proximal AT-rich DNA
sequence for effective gene silencing due to transfection withmodified siRNA with 8-bp GS-DNA substitution

Cdx2
Fgf5
1

C

2

3

Class-I siRNA (active)
5′3′-

modified siRNA with 8bpPS-DNA substituion(inactive)

4
RNA
-3′
-5′

DNA
AU richmodified siRNA with 8bpGS-DNA substituion(active)

Figure 2. Functional assay of modiﬁed siRNAs with DNA substitution
in mouse E14TG2a cells. The siOct-797 is a highly eﬀective class-I
siRNA for Oct4 gene silencing (20). siLuc-12 is an siRNA forknockdown of unrelated luc gene and serves as a control. chiOct-797PS is a DNA-modiﬁed siOct-797, of which the 11-bp long PS-endproximal region is DNA, while chiOct-797-GS is a DNA-modiﬁed
siOct-797, of which the 8-bp long GS-end-proximal region is DNA.
DNA and RNA are colored in blue and orange, respectively, while the
7-bp long AU- or AT-rich region is indicated by the red horizontal bar.
Arrows indicate the guide strands. (A) Morphological change inducedby transfection of nonmodiﬁed and cognate DNA-modiﬁed siRNAs.
The structures of nonmodiﬁed siRNA or two types of DNA-modiﬁed
siRNAs are schematically shown in the lower margin of each panel.
E14TG2a cells were simultaneously transfected with pCAGIPuroEGFP (0.5 mg/ml), encoding Enhanced Green Fluorescent Protein(EGFP) and puromycin-resistant genes, and siLuc-12 (negative
control), siOct-797 (positive control), chiOct-797-PS or chiOct-797GS, 50 nM each. Puromycin (2 mg/ml) was added to the medium 24 hafter transfection, and possible morphological change was observed
under a phase contrast microscope 3 days after transfection. As with
the nonmodiﬁed functional siRNA (siOct-797), chiOct-797-GS but not
chiOct-797-PS induced ﬂat morphology, typical of trophectoderm(34,35). (B) Expression of trophectoderm diﬀerentiation marker genes.
The mGapdh was used as an internal control. Results ofunrelated siLuc-12 transfection is shown in lane 1. As with thenonmodiﬁed eﬀective siRNA (siOct-797; lane 2), chiOct-797-GS

In a previous work (20), we classiﬁed siRNAs into threegroups, class-I, -II and -III and showed class-I siRNAs tobe highly functional. Class-I siRNAs satisfy the threefollowing sequence-conditions simultaneously: A/U at the
50 terminus of the guide strand; G/C at the 50 terminus of
the passenger strand; at least four A/U residues in the 50
terminal 7 bp of the guide strand. siRNAs opposite inthese features give rise to little or no gene silencing in
mammalian cells, and are grouped as class-III. In
addition, more than 9-bp long G/C stretch is absent
from class-I siRNAs. siRNAs other than class-I and III
are deﬁned as class-II. This guideline for highly eﬀective orfunctional siRNAs has been used widely along with those
proposed by other investigators (36,37). Modiﬁed siRNAs
with DNA substitution described above were all designed
based on sequences of siRNAs belonging to class-I.
To further examine whether modiﬁed siRNAs with 8-bp
or less than 8-bp GS-DNA substitution are capable ofinducing eﬀective target gene silencing provided that
their nonmodiﬁed siRNA counterparts belong to class-I,(lane 4) induced Hand1, Psx1 and Cdx1 expression and repressed
Oct4 and Fgf5 expression, indicating that chiOct-797-GS is capable ofeﬀectively inactivating Oct4. In contrast, no appreciable gene expression change was induced by chiOct-797-PS (lane 3), indicating
chiOct797-PS is an inactive modiﬁed siRNA. (C) Structures offunctional DNA-modiﬁed siRNA. Highly eﬀective siRNAs (class-I
siRNAs) can be converted into either active or inactive DNA-modiﬁed
siRNAs depending on whether DNA arms are situated in the PS or GS
terminal halves. Modiﬁed siRNA associated with an 8-bp long DNA in
the GS-end-proximal region is as active as the parental authentic
siRNA. In contrast, modiﬁed siRNA with DNA substitution in the
PS-end proximal region is inactive.

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

the 8-bp long GS-end-proximal region is occupied by
DNA, also induced a very similar morphological change
and a signiﬁcant reduction in Oct4 expression. Reduction
in Oct4 activity is also known to induce the expression ofheart- and neural crest derivatives-expressed protein 1(Hand1), placenta-speciﬁc homeobox 1 (Psx1) and caudal
type homeobox 2 (Cdx2) genes and represses Fgf5
expression (34,35). Figure 2B shows that, as with siOct797, chiOct-797-GS induced Hand1, Psx1 and Cdx2
expression and repressed Fgf5 expression as well. In
contrast, changes in neither morphological nor gene
expression were observed in ES cells transfected with
chiOct-797-PS possessing DNA substitution in the PS-end
proximal third (Figure 2A3 and B3).
Taken together, results described above may indicate
8-bp long dsRNA from the 50 guide strand end offunctional siRNAs to be replaceable with DNA counterparts without substantial loss of gene silencing activity.
siRNAs in which all ribonucleotides in the 8-bp long ds
region from the GS end and those in the 30 overhang of thepassenger strand are replaced with cognate deoxyribonucleotides (Figure 2C) are referred to as modiﬁed siRNAs
with 8-bp GS-DNA substitution from here on andvice versa.

2142 Nucleic Acids Research, 2008, Vol. 36, No. 7

psiCHECK-PS
LucORF

PS target mRNA
5′ -

- 3′

PS guide
5′ PS
GS 3′ -

PS target

Relative luc activity (%)

3′UTR

B

- 3′
- 5′

GS guide
- 5′

3′ -

LucORF

3′UTR

psiCHECK-GS

authentic siRNA
modified siRNA
GS target

Figure 3. The absence of passenger-strand-dependent oﬀ-target eﬀects from gene silencing due to transfection with modiﬁed siRNA with GS-DNA
substitution. The action of authentic and modiﬁed siRNAs to the guide strand targets (GS targets, lower graph) and passenger strand targets (PStarget, upper graph) were examined using ﬁve HPV16, one human vimentin and one mouse Oct4 sites. Modiﬁed siRNAs with 6-bp GS-DNA
substitution were used for ﬁve HPV16 targets, while those with 8-bp GS-DNA substitution, for vimentin (VIM) and Oct4 (Oct) targets.
(A) Structures of psiCHECK-PS and GS along with PS and GS target recognition mechanisms by modiﬁed siRNA with 8-bp GS-DNA substitution
are schematically shown. DNA sequence in DNA-modiﬁed siRNA is colored in blue. The passenger strand (PS) is completely complementary to the
PS target sequence situated in the 30 UTR of luc mRNA transcribed from psiCHECK-PS. The guide strand (GS) completely matches the GS target
sequence in luc mRNA transcribed from psiCHECK-GS. (B) HeLa cells were transfected with psiCHECK-PS or -GS and plasmids for luc activity
control as shown in ‘Materials and methods’ section. The lower graph shows silencing eﬀects on GS-targets, while the upper graph, those on
PS-targets. Results shown in the lower graph indicate that not only all authentic siRNAs but also all seven modiﬁed siRNAs with GS-DNA
substitution are highly active in silencing GS-targets, indicating that the guide strand (GS) in which a 6- or 8-bp region from the 50 end is DNA is asactive as that of cognate authentic siRNA. In contrast, virtually no reduction in PS-target expression can be seen in the case of transfection with
modiﬁed siRNAs with GS-DNA substitution, while occasional reduction in PS target expression occurred in the case of non-modiﬁed siRNAtreatment, indicating that, unlike nonmodiﬁed-siRNA-based RNAi, gene silencing due to transfection of modiﬁed siRNAs with GS-DNA
substitution is associated with no or little PS-dependent oﬀ-target eﬀect.

gene-silencing activities of 16 modiﬁed siRNAs with 6- or
8-bp GS-DNA substitution were compared with those of
cognate authentic siRNAs. Results at 16 target sites areshown in the lower graphs of Figures 3 and 4. Not only all
16 nonmodiﬁed siRNAs but also16 DNA-modiﬁed
siRNAs caused more than 70% of reduction in target
gene activity at siRNA concentration of 50 nM.
Target-gene knockdown eﬀect varied depending on
siRNA concentration (Supplementary Figure S6). DNAmodiﬁed siRNAs exhibited gene-silencing activity virtually similar to that due to the nonmodiﬁed cognatesiRNAs when the siRNA concentration was 0.5–50 nM(Figures 3 and 4, Supplementary Figure S6), although, at
0.05 nM, gene silencing induced by DNA-modiﬁed siRNAwas less profound than that induced by nonmodiﬁed
siRNA counterparts possibly due to weaker seed activityof the DNA seed arm (see below). Calculated IC50 valuesof the nonmodiﬁed siRNAs varied from 1 to 318 pM
depending on sequences, while those of DNA-modiﬁed
siRNA counterparts were from 47 to 5463 pM (Table 1;
fold IC50 changes, 1–35).
It may thus follow that modiﬁed siRNAs with 8-bp or<8-bp GS-DNA substitution are capable of inducing
eﬀective gene silencing if the concentration is properlychosen and their nonmodiﬁed siRNA counterparts belong
to class-I.

Involvement of Ago2, TRBP2, PACT and sequence-specificmRNA cleavage in gene silencing due to transfection
with modified siRNAs with 8-bp GS-DNA substitution
in mammalian cells
TRBP2 (24) and PACT (22) are putative mammaliancounterparts of Drosophila RLC protein, R2D2, which
senses the diﬀerential stability of siRNA duplex ends todetermine which strand will be loaded into RISC as theguide strand (3). R2D2 appears to bind more stable RNA
end (3). TRBP2, PACT and R2D2 contain dsRNAbinding domains (23,24,38) and, as shown in Figure 5A,
are capable of binding to dsRNA but not dsDNA (39,40),
suggesting that, in the case of modiﬁed siRNAs with 8-bp
GS-DNA substitution, these RLC proteins bind only tocertain ribonucleotides present in PS terminal two-thirds.
Ago2 is a key RNA-binding protein for nonmodiﬁed
siRNA-based RNAi in mammals (41,42).
We demonstrate that these proteins are essential tofunctional mammalian gene silencing due to transfection
with modiﬁed siRNAs with 8-bp GS-DNA substitution in
HeLa cells. The activity of TRBP2 and Ago2 were reduced
signiﬁcantly through RNAi with TRBP2 and Ago2-seciﬁc
siRNAs, respectively (Figure 5B). As shown in Figure 5C,
Ago2 and TRBP2 siRNA treatment of HeLa cells not
only speciﬁcally reduced Ago2 and TRBP2 expression but

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

GS target mRNA

Relative luc activity(%)

A

Nucleic Acids Research, 2008, Vol. 36, No. 7 2143

A
3′UTR

3′-

- 5′
- 3′
- 5′
- 5′

LucORF

3′UTR

psiCHECK-cm

authentic siRNA
modified siRNAcm target

Figure 4. Comparison of seed-sequence-dependent oﬀ-target eﬀects due to RNA and DNA seed arms. Oﬀ-target eﬀects of 12 sets of functional
siRNA and cognate modiﬁed siRNA with 8-bp GS-DNA substitution were examined using psiCHECK-sm and -cm plasmids with various sm andcm targets and the dual luciferase assay. (A) Structures of psiCHECK-sm and -cm plasmids and modiﬁed-siRNA-dependent mechanisms of sm andcm target recognition are schematically shown. Three thick green and orange arrows, respectively, indicate sm and cm target sequences inserted.
Black thick horizontal bar, seed arm sequence (position 2–8) of the guide strand. DNA sequence in modiﬁed siRNA is colored in blue. Only the seedsequence-including DNA portion of the guide strand possesses a complete complementarity to the sm target sequence which is situated in the 30 UTR
of luc mRNA transcribed from psiCHECK-sm. The entire guide strand completely matches the cm sequence in luc mRNA transcribed frompsiCHECK-cm. Luc, VIM and Oct, respectively, indicate to contain sequences related to ﬁreﬂy luc, human vimentin and mouse Oct4. (B) HeLa cells
were transfected with psiCHECK-1 containing three repeats of sm or cm target sequences (0.1 mg) and either phLuc-Control or pGL3-Control (1 mg)
was used as a control. Cells were simultaneously transfected with either nonmodiﬁed or DNA-modiﬁed siRNA at 50 n M. The lower graph showssilencing eﬀects on cm targets, while the upper graph, those on sm targets. Results shown in the lower graph indicate that not only all nonmodiﬁed
siRNAs but also modiﬁed siRNAs with 8-bp GS-DNA substitution are highly active in silencing cm target sequences, indicating that the guide strand
in which an 8-bp region from the 50 end is DNA is as active in gene silencing as that of cognate authentic siRNA. The upper graph shows that
virtually no reduction in sm target expression can be seen in the case of transfection with modiﬁed siRNAs with 8-bp GS-DNA substitution. In
contrast, occasional reduction in sm target expression occurred in the case of authentic siRNA treatment. These ﬁndings may indicate that genesilencing due to transfection of modiﬁed siRNAs with 8-bp GS-DNA substitution is associated less frequently, if any, with seed-sequence-dependentoﬀ-target eﬀect than classical RNAi.
Table 1. IC50s of non-modiﬁed and DNA-modiﬁed siRNAs
Luc-309 VIM-812 GRK4-934 Oct-821 Luc-774 VIM-1128 Luc2-153 VIM-596 Oct-797 VIM-270 Luc-36 Luc-49
Non-modiﬁed siRNA(pM)
7DNA-modiﬁed siRNA(pM)
48
IC50 fold change(DNA-modiﬁed
siRNA/non-modiﬁed
siRNA)
7.0

25

34

318

13

9

18

17

1

156

167

5

146

744

247

85

75

179

131

47

5463

270

16

5.8

21.9

0.8

6.4

8.0

10.2

7.9

33.3

35.0

1.6

3.2

also prevented siLuc-36 or siLuc-774-based luc genesilencing. By comparison with control siRNA treatment(treatment with siGY441, a siRNA speciﬁc for GFP),
Ago2 or TRBP2 siRNA treatment was found to increase
relative luc activity by 30–50 points in DNA-modiﬁed
siRNA-dependent gene silencing as in the caseof nonmodiﬁed-siRNA-dependent RNAi (Figure 5C).
A separate experiment showed PACT siRNA treatment
prevented DNA-modiﬁed-siRNA-based gene silencing tosome extent (Supplementary Figure S7). Ago2, PACT and
TRBP2 may thus be involved in gene silencing due tomodiﬁed siRNA with 8-bp GS-DNA substitution as in thecase of RNAi with nonmodiﬁed siRNAs. However, notethat siGY441, a control siRNA, much more strongly
prevented DNA-modiﬁed-siRNA-dependent gene silencing than RNAi, indicating that certain critical RISC and/or RLC proteins may have less aﬃnity toward modiﬁed
siRNAs with 8-bp GS DNA substitution than toward
nonmodiﬁed siRNAs.
In RNAi, target mRNA is cleaved at the point
corresponding to the center of the guide strand via
RNase H-like activity of the PIWI domain of Ago2

Downloaded from nar.oxfordjournals.org at Ekigaku-Kyoshitsu (UNIV OF TOKYO) on October 17, 20103′-

Relative luc activity (%)

5′-

Guide strand 3′RNAi(on-target
effect)Relative luc activity (%)

LucORF

Off-target
effectsm target

B
psiCHECK-sm

2144 Nucleic Acids Research, 2008, Vol. 36, No. 7

His-PACT100110 (µl)

1

1

(µl)

-ControlsiRNA

1+ControlsiRNA

0

siTRBP2

B

His-TRBP2

siAgo2

His-PACT

A

hGapdh
Ago2

siDNA

120

120

100

100

80

80

60

60

40

40

20

20

siLuc-36 chiLuc-36

Sequence
ladderG A T C

00

D

siLuc-774

chiLuc774-GS

chiLuc774-PS

Target5′-CACGUACGCGGAAUACUUCGA-3′

Relative luc activity (%)−siRNA control (−siGY441, −siAgo2, −siTRBP2)+siRNA control (siGY441)+siAgo2+siTRBP2

chiLuc2-153

siRNA

siLuc2-153

C

siDNA

Figure 5. Involvement of Ago2, TRBP2 and PACT in modiﬁed-siRNA-dependent gene silencing. (A) PACT and TRBP2 binding to siRNA (siLuc36) and cognate siDNA. The 50 end of the guide strand of siRNA or siDNA was phosphorylated with [g-32P] ATP and the 50 end of the passenger
strand was phosphorylated with cold ATP. A mobility shift assay was carried out using PACT and TRBP2 proteins puriﬁed from E. coli cells. Notethat TRBP2 and PACT are capable of binding to dsRNA but not dsDNA. Arrows indicate the position of protein–nucleic acid complexes.
(B) Reduced expression of Ago2 and TRBP2 by RNAi. HeLa cells were transfected with siGY441 (control siRNA), siAgo2 or siTRBP2 (50 nM)
along with pCAGIPuro-EGFP (0.5 mg) and subjected to RT–PCR 3 days after transfection. Note that Ago2 and TRBP2 RNA were speciﬁcallyknocked down by siAgo2 and siTRBP2, respectively. (C) Requirement of Ago2 and TRBP2 for gene silencing due to transfection with modiﬁed
siRNA with 8-bp GS-DNA substitution. HeLa cells were simultaneously transfected with pGL3-Control DNA (1 mg) and pRL-SV40 DNA (0.1 mg),and a mixture of siRNA and modiﬁed siRNA with DNA substitution. Two siRNAs, siLuc-36 (left panel) and siLuc-774 (right panel), and their
modiﬁed siRNA counterparts, 5 nM each, were used for luc gene silencing. chiLuc-36 and chiLuc-774-GS are functional DNA-modiﬁed siRNAs
speciﬁc to luc knockdown, whereas chiLuc-774-PS is a modiﬁed siRNA with DNA replacement in the PS-end-proximal region. Ago2 and TRBP2
were knocked down using 50 nM siAgo2 and siTRBP2, respectively. As an siRNA concentration control, siGY441 (50 nM), an siRNA for EGFP
knockdown, was used. Gene-silencing activity was measured 24 h after transfection. Note both nonmodiﬁed and DNA-modiﬁed siRNA-dependent
luc gene-silencing activity to be reduced by 30–50 points by knocking down Ago2 and TRBP2 activity through siAgo2 and siTRBP2 RNAi,
respectively. (D) Functional DNA-modiﬁed siRNA-dependent target mRNA cleavage. HeLa cells were cotransfected with pTREC-2-153 and siLuc1085 (a negative control siRNA), siLuc2-153 (target-cleavable siRNA) or its cognate modiﬁed siRNA with 8-bp GS-DNA substitution, each 5 nM.
RNA was extracted 24 h after transfection, and cleavage sites were determined by primer extension. Sequence ladder was prepared using the samepTREC construct (25). Note that the position of the main cleavage site is precisely identical between RNAi and gene silencing due to transfection
with modiﬁed siRNA with 8-bp GS-DNA substitution.

(11,12,16,43). Real-time PCR experiments indicatedthat the mRNA target is eﬀectively degraded throughDNA-modiﬁed siRNA treatment (data not shown).
Examination was thus made to determine at what point
target mRNA is cleaved in DNA-modiﬁed-siRNA-dependent gene silencing using chiLuc2-153, a modiﬁed siRNAof siLuc2-153 with 8-bp GS-DNA substitution(Figure 5D). The position of the main cleavage site wasfound to be precisely the same to that of RNAi. In bothcases, the main cleavage point was situated between
position 10 and 11, as measured from the 50 end of theguide strand. A similar coincidence of target cleavage site
was also found when siLuc-36 sequence was examined(data not shown).
These results presented so far may indicate that, as withfunctional siRNA, functional modiﬁed siRNA with 8-bp

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

siRNA

siLuc-1085(Control)

TRBP2

Nucleic Acids Research, 2008, Vol. 36, No. 7 2145

GS-DNA substitution is loaded into Ago2-including
RISC and that the guide strand in its DNA portion
serves as guide for target mRNA nucleation andsubsequent cleavage. As in the case of nonmodiﬁedsiRNA-based RNAi, TRBP2 and PACT are likely to be
involved in the formation of RLC including modiﬁed
siRNA with 8-bp GS-DNA substitution in mammaliancells.
Absence of gene-silencing activity from the passenger
strand with DNA substitution in the 3’terminal region

Reduction in seed-sequence-based off-target effect ingene silencing due to transfection with functional
DNA-modified siRNAs
The 8-bp long DNA of the guide strand in DNA-modiﬁed
siRNA corresponds in position to the ‘seed’ region of
microRNA that is implicated in gene regulation
by translational inhibition and/or mRNA degradation(45–49). In nonmodiﬁed siRNA, the region of nucleotide
position 2–8 measured from the 50 end of the guide strand
may play an essential role in the initial stage of mRNArecognition. The DNA–RNA hybrid is generally lessstable thermodynamically than RNA duplex (27,28,
50,51). Thus, the seed activity or capability of inducing
inhibition of translation and/or mRNA degradation due
to the DNA seed arm in the putative RISC containing the

Genome-wide analysis of off-target effects due toDNA-modified-siRNA-dependent gene silencing
To further clarify that gene silencing due to modiﬁed
siRNAs with 8-bp GS-DNA substitution is less frequently
associated with oﬀ-target eﬀect, genome-wide expression
proﬁles were examined using microarray analysis. siVIM270 is a functional nonmodiﬁed siRNA for humanvimentin knockdown (see Figures 3 and 4), while
chiVIM-270 is cognate modiﬁed siRNA with 8-bp GSDNA substitution. As a total, 16 783 transcripts were
examined by microarray proﬁling (Figure 6C and D and
Supplementary Figure S10).
Homology between the seed region of siRNA and the
30 UTR but not coding sequence of mRNA has beenshown to be important for seed-dependent oﬀ-target eﬀect(10) and, as described above, the position 1–8 region of theguide strand, consisting of the 50 very end (position 1) andthe seed sequence (position 2–8), is replaceable with DNA
without appreciable reduction of gene silencing. The 50 end-proximal, 7-bp region of class-I siRNA is A/U-rich.
Thus, 16 783 transcripts were classiﬁed into four groups
based on homology between 30 UTR and the guide-strand
seed region (Figure 6A). About 5% transcripts (n = 695)
possessed homology to the entire position 1–8 region of
the guide strand of siVIM-270 (group-1). About 7%
transcripts (n = 1195) were completely complementary
to the region 1–7 but not position 8 (group-2).
Approximately 6% transcripts (n = 1039) had homology

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

In nonmodiﬁed-siRNA-based gene silencing, it may be
diﬃcult to evade the possible passenger-strand-dependentsilencing of unintended genes (44). As described above (seee.g. Figure 1), if the 30 terminal region of the guide strandof siLuc-36 or siLuc2-153 is substituted with DNA, no or
little target gene silencing occurred. The 30 terminal regionof the passenger strand of modiﬁed siRNA with GS-DNA
substitution is always occupied by DNA (see Figure 2C),
suggesting that gene silencing due to transfection withfunctional modiﬁed siRNA with GS-DNA substitution is
not associated with any activity of inducing passengerstrand-dependent oﬀ-target eﬀect.
To further conﬁrm this point, one copy of cm target
sequence for guide or passenger strands (GS or PS targets,
respectively) were introduced into the 30 UTR ofpsiCHECK-1 to generate psiCHECK-GS and -PS plasmids, and nonmodiﬁed and DNA-modiﬁed siRNAdependent reporter gene silencing eﬀects were examined.
All siRNAs used here belong to highly functional class-I.
As described above and can be seen in the lower graph of
Figure 3B, all 7 DNA-modiﬁed siRNAs used here appear
to possess a highly functional guide-strand activity. The
upper graph of Figure 3B showed that target genesilencing subsequent to transfection with at least four ofseven nonmodiﬁed siRNAs is associated with apparent
passenger-strand-dependent oﬀ-target activity. Note that,in all four cases, more than 80% reduction in PS target
activity was evident. In contrast, none of seven modiﬁed
siRNAs with GS-DNA substitution could induce eﬀective
silencing of luc gene fused with PS targets. Thus, weconclude that gene silencing due to transfection withfunctional DNA-modiﬁed siRNAs may not be associatedwith passenger-strand-dependent oﬀ-target eﬀects.

guide strand of modiﬁed siRNA with 8-bp GS-DNA
substitution is expected to be considerably weaker thanthat of the nonmodiﬁed-siRNA–RISC. In fact, in all cases
examined, calculated Tm in the ‘seed’ region of DNAmodiﬁed siRNA was considerably lower than that of
cognate nonmodiﬁed siRNA (Supplementary Table S5).
For further conﬁrmation of this point, three consecutive
copies of an identical sm target sequence, which possesses
homology only to the seed sequence of nonmodiﬁed or
DNA-modiﬁed siRNAs to be examined, were introduced
into the 30 untranslated region of psiCHECK-1 Renilla luc(Figure 4). As a total, 12 sm-targets for highly functional
siRNAs were examined as inserts (see Figure 4,
Supplementary Figure S8 and Table S3). As describedabove (see also Figure 3, Supplementary Figure S6), at
50 nM, all DNA-modiﬁed siRNAs along with cognate
nonmodiﬁed siRNAs eﬀectively silenced cm-target genes.
A considerable reduction in relative luc activity wasobserved in sm-target expressing cells transfected especially with certain nonmodiﬁed siRNAs possessing ahigher G/C content in the seed region (Supplementary
Figure S8). However, virtually no sm target knockdown
was induced by treatment with DNA-modiﬁed siRNAs at
least 11 of 12 cases (Figure 4B). The seed activity ofauthentic siRNA increased with increasing the copy
number of sm targets but no such eﬀect was observedin the case of DNA-modiﬁed siRNA treatment(Supplementary Figure S9). It may thus follow that most,
if not all, functional modiﬁed siRNAs with 8-bp GS-DNA
substitution may hardly induce seed-sequence-based oﬀtarget eﬀects.

B

A
Homologous
position

Group
1

CDS
guide strand
CDS
guide strand

2

CDS
guide strand

3

3′UTR

87654321

3′UTR

1–8

3′

1–7

87654321
5′3′UTR87654321

3′

2–8

5′
No site

4
3
2
1
0

1–8
(533)

1–7
(985)

2–8
(870)

others1
Fold change (log2)
−2
−1
0
−3

1
0

1.0

F
2–8
1–7
1–8
others4

5
6
7
Mean log10 intensity

8

2–8
1–7
1–8
others

0.0

0.0

0.2

0.4

0.6

Cumulative fraction
0.2 0.4 0.6 0.8

−2

−1

3

12.5

8

25

−3

50

1.0

100

vimentin
2–8
1–7
1–8
othersmRNA level (%)

0.8

200

5
6
7
Mean log10 intensity

12.5

4

mRNA level (%)
100
50
25

Fold change (log2)

siVIM-270
chiVIM-270

5

Number of seed region site = 1 ≥ 2

200

vimentin
2–8
1–7
1–8
others3

Cumulative fraction

Log2 ≤ −1.0

D

C

E

6

−2.0

−1.5

−1.0

−0.5

0.0

Fold change (log2)

0.5

1.0

−2.0

−1.5

−1.0

−0.5

0.0

0.5

1.0

Fold change (log2)

Figure 6. Microarray-based oﬀ-target-eﬀect proﬁling. HeLa cells were transfected with siVIM-270, a nonmodiﬁed siRNA targeting the vimentin
transcript, and cognate modiﬁed siRNA with 8-bp GS-DNA substitution, and microarray-based expression proﬁles were examined 24 h after
transfection. Change in gene expression is shown by log2 of fold change ratio to the mock transfection. As a total, 16 783 transcripts were examined.
(A) Classiﬁcation of transcripts into four groups based on 30 UTR homology to the position 1–8 region of the guide strand of siRNA. 30 UTRs ofgroup-1 transcripts (n = 695) possess homology to the entire position 1–8 region of the guide strand. 30 UTRs of group-2 transcripts (n = 1195) arecompletely complementary to the region 1–7 but not position 8. 30 UTRs of Group-3 transcripts (n = 1039) have homology to the entire seed region(region 2–8) but not position 1. Group-4 transcripts (n = 13 965) are transcripts other than those belonging to group 1–3. They consist of transcripts
possessing no homology at nucleotide position 1 and 8, and those possessing 1- or more than 1-bp mismatch in the central seed region (position 2–7).
(B) Percentage of transcripts of which activity was reduced to more than 50% are shown. 1–8, 1–7 and 2–8, respectively, group-1, -2 and -3
transcripts with a single site in the 30 UTR. Greater than two, transcripts of which the 30 UTR contains two or more than seed region sites (1–7 or
2–8 or 1–8). Seed activity increased with increasing the number of sites possessing seed homology. Note that, in all cases, DNA-modiﬁed siRNAs
appear much less eﬀective in inducing oﬀ-target eﬀect than cognate nonmodiﬁed siRNAs. (C, D) Microarray proﬁling of gene expression subsequent
to transfection of siVIM-270 (C) and chiVIM-270 (D). Each point represents the average of 11 observations. Signals of group 1–4 are shownusing four diﬀerent colors. Red, green and blue dots, respectively, represent group-1, -2 and -3 transcripts. Group-4 transcripts or those with nohomology site are colored in gray. Group 1–4 signals are separately shown in Supplementary Figure S10. Signals for vimentin belonging to group-4
are colored in black and labeled with arrows. Vertical axis, log2 of fold change ratio to the mock transfection or gene activity levels (%). Horizontal
axis, log10 of the multiplied ﬂuorescence intensity of mock transfection. The dotted lines show the level of 50% reduction (log2  –1) inducedby siVIM-270 or chiVIM-270 treatment. (E, F) Cumulative distribution of transcripts downregulated by siVIM-270 (E) and chiVIM-270 (F).
Red, green and blue lines, respectively, represent the cumulative fraction of group 1–3 transcripts, respectively. The line for group-4 transcripts
is colored in gray.

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Others43′5′Percentage of repressed genes

2146 Nucleic Acids Research, 2008, Vol. 36, No. 7

Nucleic Acids Research, 2008, Vol. 36, No. 7 2147

levels estimated by quantitative RT–PCR analysis were
essentially identical to those obtained by the microarray
analysis within a limit of error. The correlation coeﬃcient
was estimated at 0.96. Based on these observations, weconclude that gene silencing by modiﬁed siRNA with 8-bp
GS-DNA substitution is associated with oﬀ-target
eﬀect much less frequently than that due to nonmodiﬁedsiRNA-based RNAi.
DISCUSSION
The present study clearly demonstrated that the GS-end
proximal region of siRNA, which includes the seedsequence of the guide strand can be replaced with a
DNA counterpart without substantial loss of genesilencing activity, although most part of the remaining
should be RNA. Our results (see Figures 5 and S7) also
indicated that gene silencing due to transfection of
modiﬁed siRNA with 8-bp GS-DNA substitution not
only requires the activities of RLC-proteins, TRBP2 and
PACT and a RISC-protein, Ago2, but is also associatedwith target mRNA cleavage. Thus, the molecular mechanism of gene silencing due to transfection of modiﬁed
siRNA with 8-bp GS-DNA substitution appears very
similar, if not identical, to that of nonmodiﬁed-siRNAbased RNAi (2,4). In RNAi, siRNA unwinding occurs
from the GS end by an unknown unwinding enzyme(20,52–55). Thus, our ﬁnding of eﬀective gene silencing
induced by DNA-modiﬁed siRNAs in mammalian cells
may indicate that this enzyme is not speciﬁc to dsRNA.
Both dsRNA and dsDNA should serve as substrates forthe enzyme.
Structural analysis (see Figure 5D) indicated that, as in
the case of siRNA-dependent mRNA cleavage, target
mRNA is cleaved by modiﬁed siRNA with 8-bp GS-DNA
substitution mainly at position 10–11, as measured fromthe 50 end of the guide strand, which apparently functions
as a ruler zero-point in RNAi (43). Although one turnof dsDNA (10.5 bp) is shorter than that of RNA duplex(11–12 bp) by 0.5–1.5 bp (56), no diﬀerence in cleavage
site could be detected between nonmodiﬁed andDNA-modiﬁed siRNA-dependent cleavage reactions(Figure 5D). This supports the notion that the helix
pitch in the seed region is determined not by polynucleotides but by Ago protein surface properties (7). Results in
Figure 4 and Supplementary Figure S8 show that the seed
activity of the DNA arm is considerably less than that of
the RNA counterpart. Our results indicated that DNAmodiﬁed-siRNA-dependent gene silencing is associatedwith not only little seed-dependent oﬀ-target eﬀects but
also virtually no passenger-strand-dependent oﬀ-target
eﬀects, provided that DNA-modiﬁed siRNAs possess an
8-bp long DNA guide strand and cognate siRNAs belong
to class-I. In contrast, class-I siRNAs possessing a higher
Tm of the seed sequence occasionally induced seeddependent oﬀ-target eﬀect (our unpublished data; seealso Supplementary Figures S8 and S9).
Since it is possible to design class-I siRNAs for almost
all human or mouse genes (20,25), the above ﬁnding may
indicate that almost all mammalian genes are eﬀectively

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to region 2–8 but not position 1 (group-3). All othertranscripts (n = 13 965), that is, those with no homology
site belong to group-4. Note that a small fraction oftranscripts (0.7%; n = 111) with two or more than two
homologous regions are doubly counted. In Figure 6C–F,
changes in abundance of these four group transcripts
monitored with microarrays are shown using four colors.
In the case of siVIM-270 treatment, repression from
UTRs of group-3 transcripts with complete seed match
was signiﬁcantly more than those from UTRs of group-1,-2 and -4 (P < 10–3, P < 10–25 and P < 10–154, respectively,
Wilcoxon’s rank-sum test) (Figure 6E). Similarly, in thecase of chiVIM-270 treatment, repression from UTRs ofgroup-3 was signiﬁcantly more than that those from
UTRs of group-1, -2 and -4 (P < 10–5, P < 10–3 andP < 10–54 respectively) (Figure 6F). These results are
consistent with previous ﬁnding (31) of the importanceof the seed homology for microRNA-type targeting
speciﬁcity.
Results in Figure 6E and F also show that authentic
siRNA-dependent repression from UTRs of transcripts
with complete seed homology was signiﬁcantly more thanthat due to modiﬁed siRNA with 8-bp GS-DNA
substitution (in the case of group-1 and -3 transcripts,
respectively, P < 10–5, and P < 10–24).
The expression of about 20% (n = 184) of group 3transcripts was reduced to <71% [fold change (log2)
 –0.5] subsequent to siVIM-270 treatment (Figure 6C
and E), while only 5% (n = 54) of group-3 transcripts
were associated with a more than 29% reduction in
expression subsequent to chiVIM-270 treatment(Figure 6D and F). The 30 UTRs of group-1 and -3
transcripts are completely homologous to the seed region(positions 2–8) (see Figure 6A). Figure 6C and D showsthat the number of group-1/3 transcripts of which theexpression levels reduced to more than 50% (fold changeof log2  –1.0) was 47 and 5, respectively, by siVIM-270
and chiVIM-270 treatment. These 47 and 3 transcripts aretranscriptional products of 28 and three genes, respectively. As shown in Supplementary Figure S12, 30 UTRs ofthese gene transcripts did not possess any high homology
to the position 9–19 region of the guide strand. In
addition, as has been reported previously (31), increased
eﬀectiveness of dual sites was evident not only in thecase of siVIM-270 treatment but also in the case of
chiVIM-270 treatment (Figures 6B, 7A–D and
Supplementary Figure S11). The reduction of endogenous
signals of vimentin RNA was 79 and 71% by thetreatment of siVIM-270 and chiVIM-270, respectively(see the arrows in Figures 6C and D, 7A and B), indicatingthat chiVIM-270 was slightly less potent compared with
siVIM-270. Taken together, these ﬁndings may support
the notion that gene silencing due to modiﬁed siRNA with
8-bp GS-DNA substitution is associated with oﬀ-target
eﬀect much less frequently, if any, than the nonmodiﬁedsiRNA-based RNAi.
To conﬁrm reliability of microarray data, we performed
the quantitative RT–PCR analysis for vimentin mRNAand 14 transcripts (genes) selected from a region enclosedwith a rectangle in Figure 7A and B and enlarged in
Figure 7E and F. As shown in Figure 7G, transcription

2148 Nucleic Acids Research, 2008, Vol. 36, No. 7

A

B
1
0
−1

Fold change (log2)

−2
−3

−3

1.0

−1.5

Cumulative fraction
0.2
0.4
0.6
0.8
−1.0

−0.5

0.0

0.5

−2.0

1.0

−1.5

6.5

7.0

7.5

8.0

0.5

1.0

0.0
5.5

6.0

Mean log10 intensity

G

0.0

−0.5
−1.5

6.0

−0.5

−1.0

Fold change (log2)

0.0

F

−0.5
5.5

−1.0

Fold change (log2)−1.0

Fold change (log2)8

seed = 1
seed ≥ 2othersFold change (log2)

−1.5

7

0.0

Cumulative fraction
0.2
0.4
0.6
0.8

0.0
−2.0

E

5
6
Mean log10 intensity

6.5

7.0

7.5

8.0

Mean log10 intensity
120mRNA level measured
by qRT-PCR (%)

100
80
60
40
20
(r = 0.96)
0

0

20

40

60

80

100

120

mRNA level measured by microarray (%)

Figure 7. Microarray analysis of oﬀ-target eﬀects due to transfection of transcripts with plural sites for seed-homology within the 30 UTR. (A, B)
Microarray proﬁles of transcripts extracted from cells treated with siVIM-270 (A) and chi-VIM-270 (B). Vertical axis, log2 of fold change ratio to themock transfection. Horizontal axis, ﬂuorescence intensity relative to that of mock transfection. Fluorescence intensity is expressed using log10. Blue
dots represent transcripts of which the 30 UTR contains only one site complementary in sequence to the position 2–8 region of the guide strand. Red
dots represent transcripts of the 30 UTR with two or more than two sites completely matching the 2–8 position of the guide strand (seeSupplementary Figure S11). The positions of vimentin are indicated as arrows. (C, D) Cumulative fraction of genes or transcripts downregulated by
siVIM-270 (C) and chiVIM-270 treatment (D). In the case of siVIM-270 treatment, repression from UTRs of transcripts with two or more than two

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D

4

1.0

Fold change (log2)
−2
−1
0

1

3

12.5

vimentinseed = 1
seed ≥ 2others8

seed = 1
seed ≥ 2others

25

7

50

5
6
Mean log10 intensity

100

4mRNA level (%)

12.5

C

mRNA level (%)
100
50
25

3

200

200

vimentinseed = 1
seed ≥ 2othersNucleic Acids Research, 2008, Vol. 36, No. 7 2149
that the phosphorylated 50 end of the guide strand isanchored in a highly conserved deep basic pocket
positioned on the surface of the Mid domain of Ago
protein, and that the ﬁrst base pair of the duplex isunwound, separating the 50 nt of the guide strand from thecomplementary target mRNA (5). Recently, Jackson et al.
(57) also have shown that position-speciﬁc, sequenceindependent chemical modiﬁcations within the seed region
reduced silencing of most oﬀ-target transcripts complementary to the seed region of the siRNA guide strand.
Especially, they showed that the 20 -O-methyl ribosyl
modiﬁcation of nucleotide at the position 2 but not 1
from the 50 GS end decreased sharp position-dependentoﬀ-target silencing eﬀect (57). Thus, it is feasible that mostof the 50 terminal nucleotides in the guide strand areessential for establishing conformation of the RLC– and/or RISC–RNA complex and/or executing RNAi. The ﬁrst
7–8-bp region from the 50 end of the guide strand might
have dual functions. That is, nucleotides in the region of
position 1–7 might be closely linked to unwinding ofsiRNA or possible activation of RISC complex and thosein the region of position 2–8 appeared involved in target
mRNA recognition.
In conclusion, we found that replacement of the seedarm of class-I siRNAs with cognate DNA sequences led toalmost complete loss of oﬀ-target eﬀects without losing
substantial gene-silencing activity in mammalian cells.

ACKNOWLEDGEMENTS
We thank A. Tanaka and N. Fukunishi for technicalassistance. This work was supported in part by Special
Coordination Fund for promoting Science andTechnology to K.S., and grants from the Ministry of
Education, Culture, Sports, Science and Technology ofJapan to K.U-T. and K.S. Funding to pay the Open
Access publication charges for this article was provided by
the University of Tokyo Foundation.
Conﬂict of interest statement. None declared.

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