Reduced base–base interactions between the DNA seed and RNA target are themajor determinants of a significant reduction in the off-target effect due toDNA-seed-containing siRNA
1

Kumiko Ui-Tei1, Kenji Nishi1, Yuki Naito1, Shuhei Zenno1,2, Aya Juni1 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, 1Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisadori-cho, Maebashi-shi,
Gunma 371-0816, Japan
deoxyribonucleotides with little or no loss of gene silencing
activity [9]. In contrast, replacing the 3'-proximal RNA
sequence of the guide strand with its DNA counterpart
resulted in almost complete loss of gene silencing activity.
Unlike non-modified siRNAs, DNA-modified siRNAs, inwhich the seed region consists of DNA and the remainder isRNA, exerted a limited, if any, off-target effect. Thesefindings imply that most mammalian genes can be knocked
down effectively without a substantial off-target effect bytreating cells with a special class of DNA-seed-containing
siRNAs.
In this study, we further confirmed the close relationshipbetweenthermodynamic
stabilityinseed-target
base-pairing and the level of the off-target effect byanalyzing gene silencing in mammalian cells transfectedwith DNA-seed-containing siRNAs or non-modified
siRNAs other than those belonging to class I.

Abstract:
Previously, we showed that DNA replacement of theseed-containing region of class I siRNAs significantly reduced
the off-target effect without substantial loss of target genesilencing activity (Ui-Tei et al, Nucleic Acids Res., 36,
2136-2151, 2008). Separately, we also showed that the level ofthe off-target effect due to non-modified class I siRNA isdetermined primarily by the calculated melting temperature(Tm) of the seed-target duplex, indicating that, at least in class
I-siRNA-mediated gene silencing, Tm or thermodynamic
stability of the complex between the siRNA seed and target
mRNA is the most important factor determining the degree ofoff-target effects (Ui-Tei et al., Nucleic Acids Res., 36,
7100-7109, 2008). Here, using non-modified siRNAs otherthan class I siRNAs and DNA-seed-containing siRNAs, we
examined the relationship between the thermodynamic
stability of the seed–target duplex and the degree of the
off-target effect. We found that the degree of the off-target
effects is generally determined primarily by the Tm of thecorresponding seed‒target duplex, indicating that its
thermodynamic stability is the most important generaldeterminant of the degree of the off-target effect in genesilencing.

2. EXPERIMENTAL PROCEDURE
2.1. Cell culture
Human HeLa cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Gibco BRL) at 37˚C. Media forboth cell lines were supplemented with 10 %heat-inactivated fetal bovine serum (FBS; Mitsubishi
Kagaku) and antibiotics (10 units/ml of penicillin (Meiji)
and 50 µg/ml of streptomycin (Meiji)).

1. INTRODUCTION
RNA interference (RNAi) has been used to study genefunction in a variety of organisms and it holds great
promise for therapeutic applications. However, a growing
body of evidence from microarray profiling and large-scale
knockdown experiments shows that gene-silencing due toauthentic siRNA is associated with activity that knocks
down many unintended nontarget genes (i.e. the off-target
effect) [1–5]. Most, if not all, transcripts of these
false-positive
genes
possess
virtually
completecomplementarity only to the siRNA “seed” region(nucleotide positions 2–8 from the 5'-end of the guidestrand) [3–7]. Consistent with this, the capability of class I
siRNAs [8] to induce the off-target effect was found to behighly correlated with the thermodynamic stability or
calculated melting temperature (Tm) of the duplex formed
between the guide-stand seed arm and target mRNA
(seed-target, or seed duplex) [7].
Previously, we examined the effects of systematic
deoxyribonucleotide substitutions of highly functional
siRNAs on gene silencing, and found that the 5'-proximal
seed arm of the guide strand could be replaced with cognate

2.2.
siRNA
and
DNA-seed-containing
siRNA
preparation
Passenger- and guide-strand RNA or DNA-substituted
RNA oligonucleotides (Table 1) were chemicallysynthesized (Proligo), mixed in a 1:1 fashion in 10 mM
NaCl and 20 mM Tris–HCl (pH 7.5), and annealed byincubating at 95ºC for 15 min, 37ºC for 30 min, and 25ºC
for 30 min. Annealed products were examined using 3 %agarose gel electrophoresis in TBE buffer, which canseparate 21-bp long double-stranded siRNA from 21-nt
long single-stranded RNA. Almost all RNA was recovered
as dsRNA.
2.3. Construction of psiCHECK derivatives and theassay of seed-dependent off-target activity
The plasmids psiCHECK-cm included 3 copies ofcompletely matched (cm) target sequences while1
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psiCHECK-sm included 3 copies of seed-matched (sm)target sequences (see Table 2 and Fig. 2A). Chemicallysynthesized oligodeoxynucleotides (75 bp in length)
including three copies of the same 23bp sm or cm target
sequences with cohesive XhoI/EcoRI ends were inserted
into the psiCHECK-1 (Promega) XhoI/EcoRI site, which issituated in the region encoding the 3' UTR of Renilla luc
mRNA. The cm sequence is completely matched that of theguide strand of non-modified or DNA-modified siRNAs,and is expressed as a part of the target mRNA in transfected
cells. In contrast, the sm sequence in the target mRNA
consists of two parts. Its 3'-terminal third is complementary
in sequence to the 8bp-long 5' proximal region of the guidestrand of siRNA or DNA-seed-containing siRNA, while theremaining two-thirds, totally non-homologous. One
milliliter of HeLa cell suspension (1 x 105 cells/ml) was
inoculated in a 1.5-cm well 24 hours prior to transfection.
Cells were transfected simultaneously with one ofpsiCHECK derivatives (10 ng each), pGL3-Control(Promega, 0.5 µg) or phLuc-Control (0.5 µg) [7], andsiRNA or DNA-seed-containing siRNA. Lipofectamine
2000 reagent (Invitrogen) was used for transfection. Cells
were harvested and luc activity was measured using theDual-Luciferase Reporter Assay System (Promega). The
reduction of Renilla luc activity by the addition of siRNAagainst the firefly luc gene is normalized with firefly luc
activity (internal control). pGL3-Control, encoding thefirefly luc, served as a control for Renilla luc silencing
assay for the siRNAs (siVIM-270, -596, -812, and -1128,
siOct-797, and -821, siGRK4-189, and -934) along withcognate DNA-seed-containing siRNAs. The gene silencing
assay for siRNAs (siLuc-36, -49, -309, -774, -1048, -1063,-1120, -1430 and 2-153) and cognate DNA-seed-containing
siRNAs was carried out using as a control phLuc-Control
coding for a modified firefly luc gene. The siGY441 is an
siRNA for knock-down of GFP and serves as an siRNA
control.

seed-GC content exceeding 57 %Three classes of siRNAs exist based on their RNAi
gene-silencing activity [8]. Class I siRNAs, which arehighly functional in mammalian RNAi, have A or U
residues at nucleotide position 1, three to six A/Us in
nucleotide positions 2–7 and G/C at position 19, with thenucleotide position measured from the 5'-end of the guidestrand, as shown in Fig. 1A. In addition, no GC stretch ofmore than nine nucleotides occurs in class I siRNAsequences. Class III siRNAs have the opposite featureswith respect to the first three conditions and cause little orno mammalian gene silencing. The remaining siRNAs
belong to class II and are a mixture of highly orintermediately functional siRNAs and nonfunctional
siRNAs.
Previously[7],
wedeterminedthatthermodynamic stability and Watson–Crick base-pairing inthe seed–target duplex [the duplex formed between theguide-strand seed (positions 2–8) and target mRNA] are themajor determinants of the efficiency of the off-target
effects due to class I siRNAs, for which the seed-region GC
content ranges from 0% to 57%.
To determine the relationship between thethermodynamic stability of seed–target duplexes with GC
content exceeding 57% and the off-targeting efficiency ofthe corresponding siRNA, six functional class II siRNAs
with high seed-GC contents were chosen arbitrarily, andtheir capability to exert off-target effects was examined(Fig. 2M–R). Note that two of the six class II siRNAs
examined here (siLuc-1430 and siLuc-1063) possess a
100%-GC seed region (Table 1). To compare the degreesof the off-target effect and target gene silencing, 17 pairs ofcompletely matched (cm) and seed-matched (sm) target
sequences corresponding to the above-mentioned six class

2.4. Calculation of melting temperature
Melting temperature (Tm) was calculated based on thenearest-neighbor model [10] and the thermodynamic values
for RNA-RNA [11] and RNA-DNA [12]. The formula for
calculating Tm is as follows. Tm ={(1000 x ΔH)/(A + ΔS
+ R ln (Ct/4))}-273.15 + 16.6log[Na+]. ΔH (Kcal/mol),sum of nearest neighbor enthalpy change. A, helix initiation
constant (-10.8). ΔS, sum of nearest neighbor entropychange. R, gas constant (1.987 cal/deg/mol). Ct, total
molecular concentration of strand (100 µM). [Na+] was
fixed at 100 mM. Standard Gibbs free energy change (ΔG)
and dissociation constant (Kd) were calculated as described
previously [7].

Fig. 1 Structures of siRNA (A) and DNA-seed-containing
siRNA (B). The nucleotide position is counted from the 5'-endof the guide strand. The seed region (positions 2–8) isunderlined. In B, the DNA and RNA nucleotides are blue andred, respectively. Note that the 5' one-third of the guide strand
containing the seed region and its complementary region in thepassenger strand with the 3'-overhang are replaced with DNA.

3. RESULTS AND DISCUSSION
3.1 Variation in the efficiency of off-target genesilencing due to functional class II siRNAs with a2- 299 -

II siRNAs along with 11 arbitrarily chosenclass I siRNAs (Table 1) were introduced
into an expression reporter plasmid,
psiCHECK (Fig. 2A), and the change in luc
activity in transfected HeLa cells was
examined as a function of the siRNAconcentration (Fig. 2B-R). psiCHECK
encodes the Renilla luc gene. Three tandem
repeats of cm or sm target sequences wereintroduced into the region corresponding to
the 3'-UTR of the luc mRNA to generate
psiCHECK-cm
orpsiCHECK-sm,
respectively
(Fig.
2A).
The
21-nuleotide-longcm
targets
werecompletely complementary to the siRNA
guide strands (Table 2) and were used todetermine the efficiency of intended genesilencing, basically RNAi (Fig. 2). The sm
sequence was completely comlementary to
the entire seed-containing region (positions
1–8), but not to the remaining non-seedregion, positions 9–21 (Table 2), and wasused to determine the efficiency of theseed-dependent unintended off-target effect(Fig. 2). Under our experimental conditions,the amount of luc mRNA produced withincells was estimated to be about 300 copies
per ng of total RNA [7], a value
Fig. 2 Dose-dependent gene silencing effects ofauthentic siRNA and DNA-seed-containing
siRNA for completely matched (cm) andseed-matched (sm) targets. Both class I siRNAs
and functional class II siRNAs are included. The
gene-silencing effects were examined using
HeLa cells transfected with psiCHECK-cm or-sm plasmids containing various cm and sm
targets. The relative luc activity in transfected
HeLa cells was determined using the dual
luciferase assay. (A) The structures of thepsiCHECK-cm and -sm plasmids and themechanism of gene silencing with authentic
non-modified
siRNAs(left)
andDNA-seed-containing siRNAs (right). Three
tandem repeats of cm or sm target sequenceswere introduced into the region corresponding to
the 3'-UTR of the luc mRNA. In B–R, theeffects of transfection of non-modified siRNAs(left pair) and DNA-seed-containing siRNAs(right pair) on cm and sm-targets are shown.
(B–L) class I siRNAs, (M–R) class II siRNAs,(B) siLuc-309, (C) VIM-812, (D) GRK4-934,(E) Oct-821, (F) Luc-774, (G) VIM-1128, (H)Luc2-153, (I) VIM-596, (J) Oct-797, (K)VIM-270, (L) Luc-36, (M) Luc-1120, (N)Luc-1430, (O) Luc-1063, (P) Luc-1048, (Q)Luc-49, (R) GRK4-189. The sequences of thesiRNA and DNA-seed-containing siRNAs, andsm and cm target sequences are shown in Tables
1 and 2.

3
- 300 -

one-hundredth that of β-actin mRNA. The luc
activities
measuredusing
different
psiCHECK-sm and -cm constructs targeted by
the corresponding siRNAs were roughly
proportional to the levels of mRNA [7],indicating that at least under these conditions
most, if not all, of the luc activity reduction due
not only to RNAi, but also to the off-target
effect, is attributable to siRNA-dependent luc
mRNA degradation.
As with the 11 class I siRNAs with 0–57 %GC content in the seed region, all six functionalclass II siRNAs examined here, for which theseed-region GC content ranged from 57% to100%, showed high activity of the intended
gene silencing at 50 nM (Fig. 2M–R). Even at
0.5 nM, most, if not all, of the class II siRNAs
examined here reduced the activity of the lucgene with the cm target to less than 20 %. In
contrast to class I siRNAs, which have little or
no off-target effect, the class II siRNAs were
frequently associated with a considerable levelof off-target gene silencing (Fig. 2).
The Tm and standard free energy change(ΔG) for the formation of the seed–target
duplexaregoodmeasuresofthethermodynamic stability of the protein-free
seed–target duplex. In a previous experiment[7], using class I siRNAs, we verified the close
linear relationship between ΔG and Tm, apositive strong correlation between sm-luc
activity and ΔG, and a negative strong
correlation between Tm and sm-luc activity.
Note that the relative sm-luc activity iscompromised by the off-target effect. By
replacing class I siRNAs with a mixture ofclass I and II siRNAs, the range of ΔG
expanded from between –13 and –7 to between–19 and –7 kcal/mol (Fig. 3A,B,D), while therange of Tm expanded from between –8 and28ºC to –8 and 55ºC (Fig. 3A,C,D). However,as shown in Fig. 3A–C, the linear relationships
among these parameters were almost invariant.
The value of ΔG may be converted into thedissociation constant (Kd) using the formula
ΔG = –RTln(1/ Kd). The Kd values of the 17
siRNAs were calculated and are listed in Fig.
3D. The highest Kd was more than 108 times
greater than the lowest one. Therefore, it may
follow that in both functional class I and II
siRNA-mediated gene silencing, the degree ofoff-target effects is governed primarily by thethermodynamic stability of the seed-target
duplex, which is 7 base pairs long.

4- 301 -

Table 1 Sequences of the guide strand and passenger strand of siRNAs
and the corresponding DNA-seed-containing siRNAs.
Authentic siRNAClass I
siLuc-309
siVIM-812
siGRK4-934
siOct-821
siLuc-774
siVIM-1128
siLuc2-153
siVIM-596
siOct-797
siVIM-270
siLuc-36
Class II

CCGCGAACGACAUUUAUAAUG
GUACGUCAGCAAUAUGAAAGUCUUGAAGCCUGAGAAUAUUCUCGCCAGAAGGGCAAAAGAUCAGAUUUCGAGUCGUCUUAAUGU
CUCGUCACCUUCGUGAAUACC
CGUACGCGGAAUACUUCGAUUGAAAACACCCUGCAAUCUUUC
GUUCGAGUAUGGUUCUGUAAC
CCAUCAACACCGAGUUCAAGACAUUCUAUCCGCUGGAAGAUGUUAUAAAUGUCGUUCGCGGGCUUUCAUAUUGCUGACGUACGUAAUAUUCUCAGGCUUCAAGUCAUCUUUUGCCCUUCUGGCGCC
AUUAAGACGACUCGAAAUCCAUAUUCACGAAGGUGACGAGCCUCGAAGUAUUCCGCGUACGUGAAGAUUGCAGGGUGUUUUCGGUACAGAACCAUACUCGAACCAUUGAACUCGGUGUUGAUGGCGUCUUCCAGCGGAUAGAAUGGC

siLuc-1120
siLuc-1430
siLuc-1063
siLuc-1048
siLuc-49
siGRK4-189
Control

GGAUCUGGAUACCGGGAAAACGGUGAACUUCCCGCCGCCGUUGGAUGAUAAACCGGGCGCGGUGAUUACACCCGAGGGGGAUGAGGAAGAUGGAACCGCUGGAGAGACGUCUCUUCAGGCAGUUCUUUUCCCGGUAUCCAGAUCCACCGGCGGCGGGAAGUUCACCGG
CGCGCCCGGUUUAUCAUCCCC
AUCCCCCUCGGGUGUAAUCAG
UCCAGCGGUUCCAUCUUCCAGAACUGCCUGAAGAGACGUCUU

siGY441

GCCACAACGUCUAUAUCAUGGAUGAUAUAGACGUUGUGGCUG

DNA-seedcontaining siRNAClass I
chiLuc-309
CCGCGAACGACatttataatg
chiVIM-812
GUACGUCAGCAatatgaaagt
chiGRK4-934
CUUGAAGCCUGagaatattct
chiOct-821
CGCCAGAAGGGcaaaagatca
chiLuc-774
GAUUUCGAGUCgtcttaatgt
chiVIM-1128
CUCGUCACCUUcgtgaatacc
chiLuc2-153
CGUACGCGGAAtacttcgatt
chiVIM-596
GAAAACACCCUgcaatctttc
chiOct-797
GUUCGAGUAUGgttctgtaac
chiVIM-270
CCAUCAACACCgagttcaaga
chiLuc-36
CAUUCUAUCCGctggaagatg
Class IIttataaatGUCGUUCGCGGGC
tttcatatUGCUGACGUACGU
aatattctCAGGCUUCAAGUC
atcttttgCCCUUCUGGCGCC
attaagacGACUCGAAAUCCA
tattcacgAAGGUGACGAGCC
tcgaagtaUUCCGCGUACGUG
aagattgcAGGGUGUUUUCGG
tacagaacCAUACUCGAACCA
ttgaactcGGUGUUGAUGGCG
tcttccagCGGAUAGAAUGGC

chiLuc-1120
chiLuc-1430
chiLuc-1063
chiLuc-1048
chiLuc-49
chiGRK4-189
Control

GGAUCUGGAUAccgggaaaac
GGUGAACUUCCcgccgccgtt
GGAUGAUAAACcgggcgcggt
GAUUACACCCGagggggatga
GGAAGAUGGAAccgctggaga
GACGUCUCUUCaggcagttct

tttcccggUAUCCAGAUCCAC
cggcggcgGGAAGUUCACCGG
cgcgcccgGUUUAUCAUCCCC
atccccctCGGGUGUAAUCAG
tccagcggUUCCAUCUUCCAG
aactgcctGAAGAGACGUCUU

chiGRK4-189

GACGUCUCUUCaggcagttct

aactgcctGAAGAGACGUCUU

Lower case, deoxyribonucleotide. Upper case, ribonucleotide. Red and blue indicate the A or U (T)residue and the G or C residue in the seed region, respectively.

3.2
EffectsofDNA
replacement in the siRNAseedregionontheseed-dependent off-target
effectOur previous experimentshowedthat
DNAreplacementof
theseed-containing
sequence(DNA-seed-containing
siRNA; Fig. 1B) in class I
siRNAs almost completely
eliminates off-target effects[7] (also see Fig. 2B–L). In
contrast, as shown in Fig.
2M–R, functional class II
siRNAs frequently could noteffectively eliminate theoff-target effects by DNA
replacement in the seedregion.
This
apparent
difference in the off-target
effect may be due to the great
difference in the seed-GC
content between functionalclass I and II siRNAs.
To clarify this point, weTable 2 Sequences of the cm- and sm-targets.
sm-Target

cm-Target

Luc-309-sm

ACUAACCCCUAAUAUUUAUAAUG

Luc-309-cm

GCCCGCGAACGACAUUUAUAAUG

VIM-812-sm

CUAGGUGAAACUUAUAUGAAAGU

VIM-812-cm

ACGUACGUCAGCAAUAUGAAAGU

GRK4-934-sm AGGCAAGCGGCGAAGAAUAUUCU

GRK4-934-cm GACUUGAAGCCUGAGAAUAUUCU

Oct-821-sm

CACUGAGCGGUUUCAAAAGAUCA

Oct-821-cm

GGCGCCAGAAGGGCAAAAGAUCA

Luc-774-sm

UAGGAUGCCAUCGGUCUUAAUGU

Luc-774-cm

UGGAUUUCGAGUCGUCUUAAUGU

VIM-1128-sm

GAGGCAAAUGACGCGUGAAUACC

VIM-1128-cm

GGCUCGUCACCUUCGUGAAUACC

Luc2-153-sm

AAGCGGUGCCAAGUACUUCGAAA

Luc2-153-cm

CACGUACGCGGAAUACUUCGAAA

VIM-596-sm

CGUUACUCUCGGAGCAAUCUUUC

VIM-596-cm

CCGAAAACACCCUGCAAUCUUUC

Oct-797-sm

GCUGAAGCUAGUAGUUCUGUAAC

Oct-797-cm

UGGUUCGAGUAUGGUUCUGUAAC

VIM-270-sm

AAGGAAGGGAGAGGAGUUCAAGA

VIM-270-cm

CGCCAUCAACACCGAGUUCAAGA

Luc-36-sm

CUUUGGAUGAAACCUGGAAGAUG

Luc-36-cm

GCCAUUCUAUCCGCUGGAAGAUG

Luc-1120-sm

CCUACAGCGGCUGCCGGGAAAAC

Luc-1120-cm

GUGGAUCUGGAUACCGGGAAAAC

Luc-1430-sm

CACAGAGGACUAACGCCGCCGUU

Luc-1430-cm

CCGGUGAACUUCCCGCCGCCGUU

Luc-1063-sm

CUCUGCGAGGUCACGGGCGCGGU

Luc-1063-cm

GGGGAUGAUAAACCGGGCGCGGU

Luc-1048-sm

UGUGGGAGUGCAAAGGGGGAUGA

Luc-1048-cm

CUGAUUACACCCGAGGGGGAUGA

Luc-49-sm

ACACACUAGGACCCCGCUGGAGA

Luc-49-cm

CUGGAAGAUGGAACCGCUGGAGA

GRK4-189-sm AAUUUUUGUAAGAAGGCAGUAA

3.2 Effects of

GRK4-189-cm AAGACGUCUCUUCAGGCAGUUCU

Fig. 3 The close relationship between the efficiency of seed-dependent off-target genesilencing and the thermodynamic stability of the seed-target duplex. Both class I siRNAs
and functional class II siRNAs were used for the analysis. Solid red circles and open red
circles show the class I and II siRNA data, respectively. (A) The calculated Tm of theseed-target duplex decreases with increases in the standard free-energy (ΔG) for seed
duplex formation (correlation coefficient; –0.98). (B) The sm-luc activity, which is theseed-dependent off-target gene silencing at a 50nM siRNA concentration, was positively
correlated with ΔG (correlation coefficient; 0.72). (C) The correlation between theseed-dependent gene silencing activity (sm-luc activity) and the calculated Tm of theseed-target duplex. A set of sm-luc activities based on the seed-dependent gene silencing
at a 50nM siRNA concentration was obtained from Fig. 2. The Tm of the seed-target
duplex was calculated using the nearest neighbor method. The relative sm-luc activity
and calculated Tm were correlated with each other and had a coefficient of –0.76. (D)List of the seed-target duplex dissociation constants.

5- 302 -

Table 3 DNA-replacement-dependent changes in theTm, ΔG and dissosiation constants.
GC
content

ΔTm(℃)

Δ(kcal/mol)

Kd-hybrid /Kd-RNAsiLuc-309

0

-4.1

2.3

siGRK4-934

1

-6.5

4.0

siVIM-812

1

-9.3

3.8

siOct-821

2

-7.5

3.2

siLuc-774

2

-19.4

3.2

siLuc2-153

3

-15.6

3.9

siOct-797

3

-24.4

4.6

siVIM-270

3

-23.1

4.6

siVIM-596

3

-17.6

3.8

siLuc-36

3

-9.4

5.0

siVIM-1128

4

-12.4

4.9

2.0E-02
1.2E-03
1.6E-03
4.5E-03
4.7E-03
1.4E-03
4.3E-04
4.1E-04
1.6E-03
2.1E-04
2.6E-04

siGRK4-189

5

-12.0

5.9

siLuc-1120

7

-11.2

6.9

siLuc-49

7

-15.9

6.6

siLuc-1048

5

-4.5

8.1

siLuc-1063

6

-13.1

6.6

siLuc-1430

4

-19.5

6.6

4.7E-05
8.8E-06
1.5E-05
1.2E-06
1.4E-05
1.4E-05-13

5

2.E-03siRNAClass IClass II

Mean

Tm = [ Tm value between the seed sequence of the DNA-seedcontaining siRNA and target mRNA (DNA-RNA hybrid)] - [Tm value
between the siRNA seed sequence and target mRNA (RNAduplex). Kd, dissociation constant. Kd-hybrid/Kd-RNA = (Kd for
DNA-RNA hybrid)/(Kd of the RNA duplex).

examined the relationship between the relative sm-luc
activity, which is compromised by the off-target effect, andthe Tm of the DNA-RNA seed duplex in 11 class I and sixclass II siRNAs (Fig. 4). We found that the linear
relationship between Tm and the relative sm-luc activity(off-target effect) is essentially invariant irrespective of thepresence or absence of DNA replacement in theseed-containing region. However, DNA replacement
reduced both the seed-duplex Tm and sm-luc activity
considerably. In all the class I DNA-seed-containing
siRNAs, the Tm was reduced to less than 20ºC, while therelative sm-luc activity at 50 nM exceeded 60%, theminimum relative luc activity necessary for a practical
off-target effect. In contrast, in three of six cases treatedwith functional class II siRNA, the relative sm-luc activity
at 50 nM was 30 % or less, although the seed-Tm was
reduced, which shows a strong negative correlation to with
the Δ G for seed-target duplex formation (Fig. 4A).
Therefore, it appears that the reduced off-target effect inDNA-seed-containing siRNA–dependent gene silencing is
generally attributable to a reduction in the thermodynamic
stability of the DNA–RNA hybrid in the seed–target duplex.
According to Fig. 4A and B and Table 3, DNA
replacement throughout the guide-strand seed region isFig. 4 The close relationship between the efficiency ofDNA-seed-dependent off-target gene silencing and thethermodynamic stability of the seed-target duplex. Solid and open
red circles show the data obatined by transfecting class I and II
nonmodified siRNAs, respectively. Solid and open blue circles
show the data obtained by transfecting class I and II
DNA-seed-containing siRNAs, respectively. (A) A strong negative
correlation was observed between the calculated Tm and ΔG witha correlation coefficient of –0.89. (B) The correlation between the
sm-luc activity and Tm. The abscissa indicates the calculated Tm
for the seed-target duplex. The ordinate is the relative sm-luc
activity at a 50 nM siRNA concentration. Arrows indicate shifts
induced on DNA replacement of the siRNA-seed sequence. The
correlation coefficient between the luc activity and Tm of theRNA-seed and RNA target duplex was –0.76, while that between
the sm-luc activity and Tm of the duplex between DNA-seed andtarget mRNA was –0.78.

6- 303 -

roughly equivalent to a 13ºC reduction in Tm, a 5 kcal/mol
increment in ΔG, and about two to three G/C→A/Uchanges in the seed duplex (Table 3).

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4. CONCLUSION
Insummary,DNA
substitution
inthesiRNA-seed-containing sequence significantly reduced theseed-dependent off-target effect without changing the Tm/off-target strength linear relationship. These results
suggest that as with authentic siRNA-mediated genesilencing, the thermodynamic stability or Tm of theseed-target duplex is the most important determinant of the
off-target
effectingenesilencingdue
toDNA-seed-containing siRNA.
ACKNOWLEGEMENTS
This work was supported in part by grants from the
Ministry of Education, Culture, Sports, Science andTechnology of Japan to K.U-T.
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