DNA-modified siRNA-dependent gene silencing with reduced off-target effect is
induced through a pathway parallel to that for siRNA-mediated RNA interference

1

Kumiko Ui-Tei1, Shuhei Zenno1, 2, Yuki Naito1, Fumitaka Takahashi1, Kenji Nishi1,
Aya Juni1, Ai Tanaka1 and Kaoru Saigo1
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan, 2 Present address: Department of Biotechnology, Faculty of Engineering, Maebashi Institute ofTechnology, 460-1 Kamisadori-cho, Maebashi-shi, Gunma 371-0816, Japan

Abstract:
RNA interference (RNAi) is mediated by 21 nucleotides ofshort interfering RNA (siRNA) through sequence-specificcleavage of the cognate transcript. The position-dependentfunction of ribonucleotide residues in siRNA was analyzed bysystematic DNA substitution. The results indicated that eight
nucleotides from the 5' end of the guide strand and its
complementary sequence are replaceable with DNA
counterparts without any substantial loss of gene silencing
activity. However, the remaining duplex including the 3' end of
the guide strand could not be replaced with DNA, probably
because of binding of RNA-binding proteins such as Argonaute
and TRBP2. In addition, due to the reduced stability of a DNA–RNA hybrid other than the RNA duplex, previous studies havereported that the guide strand of the DNA-modified siRNA was,in most cases, incapable of exerting unintended off-target genesilencing (Ui-Tei et al., Nucleic Acids Res. 36, 2136-2151, 2008).
Argonaute and TRBP2 were found to be necessary for inducing
both nonmodified and DNA-modified siRNA-mediated genesilencing. Although the major target cleavage sites bynonmodified and DNA-modified siRNAs were identical to eachother, transcript cleavage at minor sites was prevented by thepresence of the DNA arm. Unlike the 5' end of thenonmodified-siRNA guide strand, the 5' end of the guide strandof DNA-modified siRNA appeared dispensable for seed
positioning on the Argonaute surface.

1. INTRODUCTION
RNA interference (RNAi) is an evolutionarily conserved
process induced by double-stranded RNA (dsRNA) [1]. In
mammals, short interfering RNA (siRNA), 19 bp in lengthdsRNA with 2-nucleotide 3' overhangs, is widely used for
inducing RNAi [2]. In Drosophila cells, a heterodimer ofDicer2 and R2D2 is thought to sense the differential stabilityof siRNA duplex ends in determining the guide strand [3–7].
The Dicer2/R2D2 dimer in the RNA-induced silencing
complex (RISC) loading complex (RLC) is replaced by
Argonaute (Ago) while siRNA undergoes unwinding. The 5'
and 3' ends of the guide strand may be anchored in pockets
formed in PAZ and Mid domains of Ago, respectively [5, 8,
9]. The 5'-proximal “seed” nucleotides [10], which occupy
positions 2–8 measured from the 5' end and are present on
the surface of the Mid-PIWI-L1 lobe in a quasi-helical form,
may serve as the nucleation site for mRNA [8]. In mammals,
target RNA is cleaved by the RNase activity within Ago2
that forms the core of RISC [11,12]. siRNA with only seedsequence homology to the target is also capable of inducing

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gene silencing [13]. This seed-dependent gene silencing may
be the major cause of the off-target effect [14–16].
Previously, we systematically examined the effects ofdeoxyribonucleotide substitutions in siRNA on genesilencing [17]. The 5' proximal eight nucleotides containing
the seed sequence of the guide strand and thecomplementary nucleotides could be replaced with their
DNA counterparts without substantial loss of gene silencing
activity. Surprisingly, virtually no off-target effects wereassociated with these DNA-modified siRNAs, indicatingthat the DNA seed arm in RISC may exert very few offtarget effects.
Here, we present additional experimental data essential tofurther understand the molecular mechanism of genesilencing due to DNA-modified siRNA. These results, alongwith those of previous experiments [17], suggest that DNAmodified siRNA-dependent gene silencing is induced intransfected cells through a series of molecular reactions very
similar to that for authentic RNAi.
2. EXPERIMENTAL PROCEDURES
2.1 Synthesis of siRNA and DNA–RNA chimeras,
construction of psiCHECK-sm plasmids and seedsequence activity assay
siRNAs and DNA–RNA chimeras were synthesized by andpurchased from Promega. The construction of psiCHECK
plasmids and the seed sequence assay have been described
previously [17].
2.2 RT-PCR
The plasmids pGL3-Control (1 µg) and pRL-SV40 (10 ng)
were introduced into HeLa cells using Lipofectamine 2000
(Invitrogen) with 5 nM siLuc-36 or 50 nM cognate DNAmodified siLuc-36 along with 50 nM siRNAs for humanDicer, Ago1, 2, 3, 4, TRBP2 and PACT. RNA was extracted
24 h after transfection using an RNeasy kit (QIAGEN).
Possible changes in the relative fractions of each RNA were
examined by RT-PCR using an RNA LA-PCR kit (Takara).
2.3 Target cleavage assay
The target sequences for firefly luc siRNAs, siLuc2-153 andsiLuc-36, were inserted into the EcoRI/XhoI sites of pTREC[18] to generate pTREC-2-153 and pTREC-36, respectively.
The constructs (0.5 µg) were introduced by transfection into

Fig. 1 Basic framework of DNA-modified siRNA-dependent gene silencing. (A) Effects of DNA replacement on gene silencing activity. Passenger(PS) and guide (GS) strands are shown on the top and bottom, respectively. Open rectangles, RNA; Filled rectangles; DNA. GS end, the end of thesiRNA containing the 5' end of the guide strand; PS end, siRNA end containing the 5' end of the passenger strand. The gene silencing activity ofmodified siRNA with less than 9 bp of DNA from the GS end is essentially identical to that of nonmodified siRNA. (B) Structure of active DNAmodified siRNA. (C) Reporter-dependent off-target assay. Note that virtually no off-target effect is associated with DNA-modified siRNA treatment.
(D) Microarray profiling of seed-dependent off-target activity. In contrast to nonmodified siRNA treatment, DNA-modified siRNA treatment was
associated with almost no unintended gene silencing. (E) Unstable mRNA/RISC nucleation complex formation by DNA-modified siRNA.

HeLa cells with or without cognate siRNA or DNAmodified siRNA, and cells were recovered 24 h after
transfection. Total RNA was prepared using QuickPrep
Micro mRNA Purification kit (Amersham).
Primer-340-

extension was carried out using 32P-labelled primer (5'CTCGAAGCATTAACCCTCACT-3') and a Primer
Extension System-AMV 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 byparalleled sequence gel electrophoresis. DNA sequence
analysis was performed using a Thermo Sequenace
Fluorescent Labelled Primer Cycle Sequencing kit(Amersham),althoughfluorescent-labelled
dideoxyribonucleotides were replaced with 32P-labelled
dideoxyribonucleotides.
2.4 Electrophoresis gel mobility shift analysis
A DNA fragment corresponding to the PAZ domain ofhuman Ago1 (amino acid residues 222–376) was amplified
from a cDNA mixture of total RNA extracted from HeLa
cells by PCR using the following primers: 5'CCTGGGATCCTTTTATAAGGCACAGCCAGTGATTG3'and
5'GGAGGAGGCGGCCGCAGGCAGCAGCCACCACCAC
CACCACCACTGACTCCTCCTGTCTGTCTGGAGCGGATCTAGC-3'. The amplified fragment was digested with
BamHI and NotI, and inserted into pGEX-6P2 (Amersham).
The PAZ domain was expressed in the Escherichia coli
BL21 codon plus (Stratagene) as a glutathione S-transferase(GST) fusion protein and purified with a glutathione
Sepharose column (Amersham) according to the
manufacturers’ instructions. The region encoding the fulllength human TRBP2 was amplified by PCR using thefollowingprimers:
5'TTTTTTTTTTCATATGCTGGCCGCCAACCCAGGCAA
GA-3'
and
5'TTTTCCTTTTGCGGCCGCTCACTTGCTGCCTGCCATG
ATCTTGAGGTA-3'. The amplified fragments weredigested with NdeI and NotI and cloned into pET-28a(Novagen). TRBP2 protein was produced in Rosetta (DE3)pLysS as an amino-terminal hexa-histidine-fusion protein,and purified with NTA agarose. Binding to siRNA or its
derivatives (5 fmol/µl) was carried out in 25 mM Tris-HCl(pH 8.0), 150 mM NaCl, 125 mM imidazole, 13 % glycerol,
50 ng/µl salmon sperm DNA, and 2 U/µl RNasin for 30min
at room temperature. The mobility shift assay was conducted
on a 5% polyacrylamide gel and analyzed quantitatively
using an FLA-2000 image analyzer (Fujifilm).
3. RESULTS AND DISCUSSION
3.1 Basic framework of DNA-modified siRNA-dependent
gene silencing associated with little off-target effectIn our previous study, we discovered and characterized thebasic properties of DNA-modified siRNA-dependent genesilencing [17]. Here, we first present a summary of ourprevious work and describe its implications to clarify thebasic framework of gene silencing due to DNA-modified
siRNA.
To examine the effects of DNA replacement in siRNA on
gene silencing in mammalian cells, various types of siRNAs
with DNA substitution were constructed based on thesequences of highly functional class I anti-luc siRNAs [17,-341-

19]. In DNA-modified siRNAs 9–11 in Fig. 1A (left panel),ribonucleotides were progressively replaced withdeoxyribonucleotides from the GS end, which contains the 5'
end of the guide strand, while in modified siRNAs 1–6,
deoxyribonucleotide substitutions occurred progressively
from the PS end with the 5' end of the passenger strand.
As shown schematically in Fig. 1A (right panel), nearly
all luc gene activity was virtually completely abolished after
transfection with modified siRNAs with DNA substitution in
the region ≤ 8-bp from the GS end. Gene silencing activityof modified siRNA with 8-bp GS-DNA substitution wasessentially identical to that of nonmodified cognate siRNA(Fig. 1A, 12), indicating that modified siRNA with 8-bp GSDNA substitution can be used as a reagent for gene silencing
with similar to that of nonmodified authentic siRNA. In
contrast, all modified siRNAs with DNA replacement fromthe PS end showed little gene silencing effect (Fig. 1A, 1-6),
indicating that RNA in the vicinity to the PS end is essential
for gene silencing (Fig. 1B). Similar results were obtained
when many other class I DNA-modified siRNAs were used
for transfection in place of anti-luc siRNAs. Furthermore,
our analysis indicated that the effect of DNA replacement ismainly due to changes in the guide strand [17]. Therefore,
we considered that mammalian gene silencing due tosiRNAs with 8-bp GS-DNA substitution is due primarily tointeractions between the 5' proximal guide strand region andRNA-binding protein moieties of RISC and/or RLC (Fig.
1B).
DNA positions 2–8 of the guide strand in active DNAmodified siRNA corresponds to the seed region of
microRNA that has been implicated in gene regulation. In
nonmodified siRNA-dependent gene silencing, thecorresponding “seed” region of siRNA is considered to play
an essential role in the entry or nucleation stage of mRNArecognition and to be responsible for gene silencing due to
off-target effects (see Fig. 1E) [14–16].
A DNA–RNA hybrid is generally thought to be lessstable thermodynamically than an RNA duplex [20-23].
Thus, the seed activity or off-target effect inducing activity
due to the DNA seed arm in the putative RISC whichcontains the guide strand of modified siRNA with 8-bp GSDNA substitution is expected to be considerably weaker
than that of the nonmodified siRNA RISC. To clarify thispoint, the off-target effect due to siRNA with 8-bp GS-DNA
substitution was examined using the psiCHECK-sm
expression system and genome-wide microarray expression
profiling.
In the psiCHECK-sm expression system, threeconsecutive copies of an identical seed matched (sm) target
sequence, which possesses homology only to the seedsequence of nonmodified or DNA-modified siRNAs, wereintroduced into the 3' untranslated region of psiCHECK-1
Renilla luc (Fig. 1C, left panel). Many sm-targets for highly
functional siRNAs were examined as inserts. A considerable
reduction in relative luc activity was observed in sm-target
expressing cells transfected especially with certain

Fig. 2 Requirement of Ago2 and TRBP2 for gene silencing due tononmodified siRNA and DNA-modified siRNA in HeLa cells. (A)HeLa cells were transfected with corresponding siRNA (50 nM). RNA
expression was detected 24 h after transfection by RT-PCR. The
number of amplification cycles were 17, 26, 25, 27, 25, 26, 25 and 25
for GAPDH, Ago1–Ago4, Dicer, TRBP2 and PACT RNA,
respectively. Guide strand and passenger strand sequences of siRNAs
are as follow: siDicer (5'-GCUUGAGUUGAUUACAAGACU (guidestrand) and 5'-UCUUGUAAUCAACUCAAGCAU (passenger
strand)), siAgo1 (5'-GGCAGCGCUGUAUUAAAAAGC and 5'UUUUUAAUACAGCGCUGCCCA),
siAgo2(5'CGAUCGGCAAGAAGAGAUUAG
and5'AAUCUCUUCUUGCCGAUCGGG),
siAgo3(5'CCUACCUGCCACUAGAAGUCU
and5'ACUUCUAGUGGCAGGUAGGUG),
siAgo4(5'CGAAAGGCAUGUAUUAGCUUG
and5'AGCUAAUACAUGCCUUUCGAA),siTRBP2(5'CGUCAGCUACCUGGAUAUUGA
and5'AAUAUCCAGGUAGCUGACGUG)
andsiPACT(5'GCCAAUGCAAGUAUUUGCUUU
and5'AGCAAAUACUUGCAUUGGCUU). The following primers wereused for RT-PCR: GAPDH (5'- CAAGGCTGAGAACGGGAAGCT
and5'GACACGGAAGGCCATGCCAGT),
Dicer(5'CGCTCTGGAGAGGTTACCAT
and5'CAGGTTTTTCAAGCCGAAGA),
Ago1(5'GGCCAGGAACAAAAGCATAC
and5'TCTGGAGCGGATCTAGCTGT),
Ago 2(5'ACATTGTGGCAGGACAAAGA
and5'GAAACTTGCACTTCGCATCA),
Ago 3(5'ACCTTCCCTGTCTGCAAGTC
and5'CAGATCTTGCTGTTGCCTTG),
Ago 4(5'TGTGGTTGGCAGTATGGATG
and
5'TGGAATAGAGACTGAGCGAGTG),
TRBP2(5'AGGTGGAGCCTGATGATGAC
and5'GCAGAGCCATGACACACAGT)and
PACT(5'CCTGCACAGGTGAAGGTACA
and5’TGGAAGGGTCAGGCATTAAG).
(B)
HeLa
cells
were
simultaneously transfected with pGL3-Control DNA, pRL-SV40 DNA
and siRNA (50 nM) against Dicer, Ago1-4, TRBP2 or PACT. In
addition, either nonmodified siLuc2-153 (5 nM) or its modified siRNAcounterpart (50nM) was used for luc gene silencing. As an siRNA
control, siGY441 (50nM), an siRNA for EGFP knockdown, was used.
Gene silencing activity was measu red 24 h after transfection andnormalized with the activity of siGY441. (C) Binding of the PAZ
domain of Ago1 and TRBP2 to siRNA (siLuc-36), modified siRNAand siDNA. The 5' ends of the guide strand and the passenger strand
were phosphorylated with [γ-32P] ATP and with cold ATP,
respectively. A mobility shift assay was carried out using PAZ and
TRBP2 proteins purified from E. coli cells. Relative binding activity
was determined densitometrically.

nonmodified siRNAs with a higher G/C content in the seedregion (U.-T. et al., in preparation). However, virtually no
sm target knockdown was observed by treatment with DNAmodified siRNAs irrespective of G/C content in the seedregion (Fig. 1C, right panel). The seed activity of authentic
siRNA increased with increasing copy number of the sm
target, but no such effect was observed in the case of DNAmodified siRNA treatment [17], supporting the suggestionthat most, if not all, functional siRNAs with 8-bp GS-DNA
substitution induce virtually no seed sequence-mediated offtarget effect.

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The off-target effect due to DNA-modified-siRNA wasalso examined by microarray analysis using RNA extracted
from cells transfected with 8-bp GS-DNA-modified andnonmodified anti-vimentin siRNAs (siVIM-270 and
chiVIM-270, respectively), both recognizing the CDS ofvimentin mRNA [17]. A total of 16,783 transcripts, 6 % ofwhich showed similarity in the 3'UTR to the seed sequenceof siVIM-270, were examined. As shown schematically in
Fig. 1D, unintended gene silencing due to chiVIM-270 was
significantly less prominent than that due to siVIM-270,
whereas intended gene silencing due to chiVIM-270 was aseffective as that due to siVIM-270.

Fig. 3 DNA-modified siRNA-mediated target mRNA cleavage. (A) HeLa
cells were co-transfected with pTREC-2-153 and siLuc-1085 (negative
control siRNA), siLuc2-153 (target-cleavable siRNA) or its DNA-modified
cognate siRNA. RNA was extracted 24 h after transfection. Cleavage sites
were determined by primer extension. Sequence ladder was prepared using
the same pTREC construct. The panels on the right show enlarged
photographs of the regions enclosed by rectangles in the left panel. Notethat the position of the main cleavage site is identical between nonmodified
siRNA-dependent RNAi and DNA-modified siRNA-dependent genesilencing. (B) HeLa cells were co-transfected with pTREC-36 andnonmodified or DNA-modified versions of siLuc36. Triangles 2 and 3,
major cleavage sites; Triangle 1 and 4, minor cleavage sites.

Taken together, these results suggest that replacement of
the seed arm of highly functional class I siRNAs withcognate DNA sequences leads to almost complete loss ofoff-target effects with no substantial effect on gene silencing
activity in mammalian cells. Thus, it is quite likely thatsiRNA with DNA seed requires not only DNA seed/target
mRNA interactions but non-seed RNA/target mRNAinteractions for stable “mRNA-RISC complex” formation(Fig. 1E).
3.2 Involvement of interactions between Ago/TRBP2 andthe PS terminal RNA half in gene silencing due to DNAmodified siRNA
Gene silencing through RNAi is carried out by RISC. The
human Ago family has eight members, four of which(Ago1–4) are closely related in sequence and co-expressed-343-

in many cell types [24]. TRBP2 [25] and PACT [26] areputative mammalian counterparts of the Drosophila RLC
protein, R2D2. The Dicer–R2D2 complex is considered tosense the differential stability of siRNA duplex ends todetermine which strand will be loaded into RISC as theguide strand [3]. We examined whether these proteins are
responsible for gene silencing due to transfection withnonmodified siRNA and modified siRNA with an 8-bp
DNA seed arm in HeLa cells by reducing their activities
using highly functional class I siRNAs (Fig. 2A). As shown
previously [17] and in Fig. 2B, reduction of Ago2 and
TRBP2 siRNAs clearly prevented luc gene silencing due toDNA-modified and nonmodified siRNAs (siLuc2-153).
Ago2 and TRBP2 siRNA treatment increased relative luc
activity by 5- and 2-fold when nonmodified and DNAmodified siRNAs were used for target gene silencing,respectively. No appreciable prevention of target genesilencing was observed with reduction of other factors.
These results suggested that Ago2 and TRBP2 are involvedin gene silencing not only due to nonmodified siRNA but
also to DNA-modified siRNA. The absence of a genesilencing prevention effect by siRNAs for factors other than
Ago2 and TRBP2 may indicate that these factors are not
involved in gene silencing or that they are functionally
redundant in the cells examined. This remains to be clarified
in future studies.
TRBP2 and PAZ domain of Ago contain dsRNA binding
domains [24, 25] and, as anticipated, are capable of binding
to dsRNA but not dsDNA (Fig. 2C). Fig. 2C also shows thatboth PAZ and TRBP2 are capable of binding to siRNA with
8-bp GS-DNA, indicating that the PS terminal RNA regionof siRNA includes binding sites for TRBP2 and Ago.
However, TRBP2 and Ago differ from each other in themanner of binding to the RNA portion of DNA-modified
siRNA. TRBP2 requires binding sites situated in a more
central region for effective binding to the chimera. Ago may
also require some additional terminal sites for effective
binding (Fig. 2C).
Thus, as with functional siRNA, modified siRNA with
DNA seed appears to be capable of forming RLC with
TRBP2 and RISC with Ago2 in mammalian cells and thesecomplexes are required for gene silencing due to modified
siRNA with an 8-bp GS-DNA substitution as in the case ofthat due to nonmodified siRNA.
3.3 Prevention of target mRNA cleavage at minor
cleavage sites in DNA-modified siRNA-dependent genesilencingIn RNAi, the target mRNA is cleaved at a point
corresponding to the center of the guide strand via RNase Hlike activity of the PIWI domain of Ago2 [11, 12, 27, 28].
Previously [17], we determined the major cleavage site in
the target luc mRNA, which was induced by transfection of
DNA-modified siRNA (siLuc2-153) with 8-bp GS-DNA
substitution. HeLa cells were transfected with a targetexpression plasmid, pTREC-2-153 and either the DNA–

Fig. 4 Seed positioning in DNA-modified siRNA-containing RISC isdetermined by the RNA terminal half of the guide strand. Cells weretransfected with pGL3-Control DNA, pRL-SV40 and siLuc-36
derivatives (50 nM). LDNAn-siRNA is a version of DNA-modified
siLuc-36 with (n-8)-nucleotide DNA extension. Relative luc activity wasmeasured 24 h after transfection using CHO-K1, E14TG2a and HeLa
cells. The guide and passenger strand sequences of DNA-modified
siRNAs are as follows (upper case, RNA; lower case, DNA): (1) 5'CAUUCUAUCCGctggaagatg-3'(guidestrand)and5'tcttccagCGGAUAGAAUGGC-3' (passenger strand), (2) LDNA13siRNA;
5'-CAUUCUAUCCGctggaagatggaacc-3'
and5'ttccatcttccagCGGAUAGAAUGGC-3' ,(3)
LDNA19-siRNA;
5'CAUUCUAUCCGctggaagatggaaccgctgga-3'
and5'cagcggttccatcttccagCGGAUAGAAUGGC-3', (4) LDNA26-siRNA; 5'CAUUCUAUCCGctggaagatggaaccgctggagagcaac-3'
and5'tgctctccagcggttccatcttccagCGGAUAGAAUGGC-3',
(5)
5'and
5'CAUUCUAUCCGctggaagagttccaatagttc-3'(6)5'actattggaactcttccagCGGAUAGAAUGGC-3' ,and
5'ACGGAGCGACGctggaagatggaaccgctgga-3'
cagcggttccatcttccagCGUCGCUCCGUUA-3'. Mismatched sequences areunderlined. Note that modified siRNA (5) but not (6) recognized the luc
target correctly.

modified or nonmodified siRNA (siLuc2-153). Target
mRNA cleavage was examined by primer extension. The
position of the major cleavage site due to the DNA-modified
siRNA was identical to that of RNAi induced by theauthentic siRNA counterpart (Fig. 3A) [17]. In both cases,
the main cleavage point was situated between positions 10
and 11, as measured from the 5' end of the guide strand.
As shown in Fig. 3B, densitometric analysis of primer
extension products generated by nonmodified siRNAtreatment indicated that target mRNA may be cleaved not
only at the major cleavage sites (sites 2 and 3 in Fig.3B, b)
but also at two minor sites (sites 1 and 4) separated by about
ten nucleotides, or one turn of the Watson-Crick helix.
Although neither an increase in siRNA concentration nor
passenger-strand replacement with DNA had a marked
effect on the cleavage pattern (data not shown), this pattern
was quite sensitive to changes in the DNA arm length of
DNA-modified siRNA (Fig. 3B). The cleavage of mRNAtarget at multiple sites therefore may not be a primer
extension artifact but an intrinsic characteristic. The
presence of the 8-bp DNA guide brought about a complete
loss of cleavage at site 4 (c). Target mRNA cleavage at sites
1 and 4 was eliminated almost completely by the 9–10-bp
DNA arm (d, e). The 11-bp DNA arm significantly-344-

suppressed target cleavage even at the major cleavage site(f).
Considering the conformation of target mRNA/guidestrand with 5' proximal DNA, mRNA cleavage at the minor
sites is unlikely to be attributable to the Ago RNase H
activity. Double-stranded DNA surrounding the nucleosome
core protein surface is cleaved at an interval of ten
nucleotides by non-specific DNase [29]. Therefore, we
consider that mRNA cleavage at minor sites is due to attackof a duplex consisting of mRNA and a guide strand with the
DNA arm bound to the surface of Ago by non-specificcellular RNase and that this RNase activity is prevented by
the presence of DNA in the vicinity of a cleavable site.
3.4 Seed positioning in DNA-modified siRNA containing
RISC
In the authentic RNAi, the seed arm in RISC is believed tobe correctly positioned via the interaction between the 5'
phosphate of the guide strand and the Mid domain pocket [5,8, 9]. Therefore, this was examined in DNA-modified
siRNA-dependent gene silencing using siRNA with a longer
DNA arm (LDNA-siRNA; Fig. 4, 2-4). For example,
LDNA19-siRNA is an active modified siRNA with 8-bp
GS-DNA associated with an 11-bp DNA extension (19-bp in
total length). As shown in Fig. 4 (1–4), the extension of the
DNA arm did not affect gene silencing activity. If the 5' endof the guide strand is a determinant of seed positioning of
RISC, DNA extension should change the mRNA target
sequence. To examine this possibility, an LDNA19-typesiRNA was constructed containing DNA extension ofdifferent sequences. The results indicated that 8 bp of DNA
immediately adjacent to the RNA is capable of acting as aseed for mRNA recognition, but no seed activity was foundto be associated with the DNA extension, indicating that, incontrast to the case of nonmodified siRNA, the 3' but not 5'
GS end may be responsible for positioning of the seed in
DNA-modified siRNA-containing RISC. Consistent with
this, changes in the RNA sequence in the 3' end of guidestrand significantly affected target silencing (Fig. 4, 6).
4. CONCLUDING REMARKS
We found that class I siRNAs with DNA seed arm arecapable of inducing highly effective gene silencing without
any substantial off-target effect in mammalian cells. The
presence of the DNA arm in the GS terminal half eliminates
minor cleavage of mRNA possibly due to cellular nonspecific RNase. The molecular mechanism of DNAmodified siRNA-dependent gene silencing is similar to thatof nonmodified siRNA-dependent gene silencing. However,in DNA-modified siRNA-dependent gene silencing, the 5'
end of the guide strand may not be involved in correct
positioning of the seed sequence in RISC.
ACKNOWLEDGEMENTSWe thank N. Fukunishi for technical assistance. This work waspartially supported by Special Coordination Fund for promoting
Science and Technology to K.S., and grants from the Ministry of
Education, Culture, Sports, Science and Technology of Japan to
K.U.-T.

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