REVIEW ARTICLE
published: 04 June 2012
doi: 10.3389/fgene.2012.00101

Thermodynamic control of small RNA-mediatedgene silencing
Kumiko Ui-Tei 1,2 *, Kenji Nishi 1,2 , Tomoko Takahashi 1,2 and Tatsuya Nagasawa 1,2
1
2

Ui-Tei Lab, Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan
Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan

Edited by:Michael Rossbach, Genome Instituteof Singapore, Singapore
Reviewed by:
Tohru Yoshihisa, Nagoya University,
Japan
Timothy Bowen, Cardiff University
School of Medicine, UK*Correspondence:
Kumiko Ui-Tei , Department ofBiophysics and Biochemistry,
Genome Information Biology, The
University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan.
e-mail: ktei@bi.s.u-tokyo.ac.jp

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are key regulators of posttranscriptional gene silencing, which is referred to as RNA interference (RNAi) or RNA silencing.
In RNAi, siRNA loaded onto the RNA-induced silencing complex (RISC) downreugulates
target gene expression by cleaving mRNA whose sequence is perfectly complementary
to the siRNA guide strand. We previously showed that highly functional siRNAs possessed
the following characteristics: A or U residues at nucleotide position 1 measured from the5 terminal, four to seven A/Us in positions 1–7, and G or C residues at position 19. Thisﬁnding indicated that an RNA strand with a thermodynamically unstable 5 terminal is easily
retained in the RISC and functions as a guide strand. In addition, it is clear that unintended
genes with complementarities only in the seed region (positions 2–8) are also downregulated by off-target effects. siRNA efﬁciency is mainly determined by the Watson–Crickbase-pairing stability formed between the siRNA seed region and target mRNA. siRNAs
with a low seed-target duplex melting temperature (T m ) have little or no seed-dependent
off-target activity. Thus, important parts of the RNA silencing machinery may be regulated by nucleotide base-pairing thermodynamic stability. A mechanistic understanding ofthermodynamic control may enable an efﬁcient target gene-speciﬁc RNAi for functional
genomics and safe therapeutic applications.
Keywords: siRNA, RNAi, off-target effect, thermodynamic stability, seed region

INTRODUCTION
Small RNA molecules, including small interfering RNAs (siRNAs)and microRNAs (miRNAs), are crucial regulators of posttranscriptional gene silencing referred to as RNA interference (RNAi)or RNA silencing. RNAi is an evolutionarily conserved pathway
induced by siRNAs, 21–23-nt double-stranded RNAs (dsRNAs)
with 2-nt 3 overhangs (Figure 1). siRNAs incorporated into cells
are transferred to an RNAi effector complex called the RNAinduced silencing complex (RISC; Hutvagner and Simard, 2008;
Jinek and Doudna, 2009). The RISC assembles on one of the two
strands of the siRNA duplex and is activated upon removal of thepassenger strand (Martinez et al., 2002; Schwarz et al., 2002, 2003;
Khvorova et al., 2003; Ui-Tei et al., 2004). We and others reported
that asymmetrical features of both siRNA terminals are common
to functional siRNAs (Amarzguioui and Prydz, 2004; Reynolds
et al., 2004; Ui-Tei et al., 2004). An RNA strand with a thermodynamically unstable 5 terminal is easily retained in the RISC.
The activated RISC is a ribonucleoprotein complex that minimallyconsists of the core protein Argonaute (Ago) and a siRNA guidestrand, which recognizes mRNAs with complementary sequences(Liu et al., 2004; Meister et al., 2004; Song et al., 2004). In siRNAmediated RNAi, the Ago2 protein siRNA guide strand usuallybase-pairs with mRNA that is perfectly complementary and cleaves
them (Figure 1). However, this can also lead to silencing of othergenes with incompletely complementary sequences. This phenomenon is referred to as an off-target effect (Figure 1). The target
recognition mechanism of the off-target effect is similar to that ofwww.frontiersin.org

miRNA-mediated gene silencing (Jackson et al., 2003, 2006a; Bartel, 2004; Scacheri et al., 2004; Lewis et al., 2005; Lim et al., 2005;
Lin et al., 2005; Birmingham et al., 2006; Grimson et al., 2007). The
transcripts with sequences complementary to the seed region (i.e.,
nucleotide positions 2–8 from the 5 end of siRNA guide strand
or miRNA loaded on Ago1–4 proteins) are mainly reduced. Thisis likely because seed nucleotides are present on the Ago surface
in a quasi-helical form to serve as the entry or nucleation site
for small RNAs in the RISCs (Ma et al., 2005; Yuan et al., 2005;
Ui-Tei et al., 2008a). Thus, siRNA target recognition might be partially determined by structural features. However, the off-target
effect silencing efﬁciency is mainly determined by the thermodynamic properties of nucleotide base-pairing between the siRNA
guide strand seed region and their off-target mRNAs (Ui-Tei et al.,2008a). Understanding thermodynamic control of the siRNA offtarget effect may make it possible to avoid the off-target effect fora target gene-speciﬁc RNAi.

FUNCTIONAL siRNA SEQUENCES
RNA interference efﬁciency in mammalian cells varies considerably depending on the siRNA sequence (Holen et al., 2002;
Harborth et al., 2003). We showed that there are three siRNA
classes based on their RNAi gene silencing activity (Ui-Tei et al.,2004). Class I siRNAs, which are highly functional in mammalianRNAi, have A or U residues at nucleotide position 1, four to seven
A/Us in nucleotide positions 1–7 (AU ≥ 57%) and G/C at position
19, with the nucleotide position measured from the 5 end of theJune 2012 | Volume 3 | Article 101 | 1

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FIGURE 1 | The mechanism of RNAi and its off-target effect inmammalian cells. An RNA strand with A or U at position 1, four to seven
A/Us in positions 1–7 and G/C at position 19 (measured from the guide strand
5 end) are easily unwound from the 5 end and retained in the RISC. The
passenger strand is dissociated from the Ago1 ∼ 4-containing RISC following
unwinding, but cleaved in the Ago2-containing RISC. The guide strand

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Thermodynamic control of RNA silencing

recognizes target and off-target transcripts with complementary sequences
to seed region positions 2–8. The target transcript, which has complete
complementarity in positions 9-21, in addition to positions 2–8, is knocked
down by RNAi. Conversely, off-target transcripts are downregulated according
to the thermodynamic stability in the duplex formed between the siRNA seedregion and target mRNA.

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guide strand (Figure 1). In addition, a GC stretch of no morethan nine nucleotides occurs in class I siRNA sequences. Class III
siRNAs have opposite features with respect to the ﬁrst three conditions and cause the least RNAi-silencing effects. The remaining
siRNAs belong to class II and are a mixture of functional andnon-functional siRNAs.
We and others demonstrated that functional siRNA with an
unstable RNA strand 5 terminal in the siRNA duplex is functional as a guide strand (Amarzguioui and Prydz, 2004; Reynolds
et al., 2004; Ui-Tei et al., 2004); A or U residues at the 5 end of theguide strand are especially important. In RNAi, thermodynamic
asymmetry is not essential for target gene silencing because thepassenger strand of most double-stranded siRNAs loaded onto
RISC are cleaved by catalytic activity of the Ago2 protein anddegraded (Figure 1; Kawamata et al., 2009; Yoda et al., 2010). Thus,in this case, A/U nucleotide itself at 5 terminal might be strongly
contributed to the RNAi activity, as nucleotide monophosphates,
AMP, and UMP, bind to Ago2 with up to 30-fold higher afﬁnity
than either CMP or GMP (Frank et al., 2010). However, when thesiRNA duplex is loaded into other Ago proteins without slicer
activity, siRNAs might be unwound into a single-strand fromthe thermodynamically unstable 5 terminal as shown in miRNAmediated gene silencing (Figure 1; Matranga et al., 2005; Miyoshi
et al., 2005; Leuschner et al., 2006; Kawamata et al., 2009; Yoda
et al., 2010). As off-target gene silencing is performed using bothmechanisms for eliminating the passenger strand, siRNA thermodynamic asymmetry in addition to A/U nucleotide itself at
the 5 terminal might be involved in seed-dependent off-target
effects.

SEED-DEPENDENT OFF-TARGET EFFECT EFFICIENCY VARIES
DEPENDING ON SEED SEQUENCE
Accumulated evidence from large-scale knockdown experiments(Jackson et al., 2003, 2006a; Scacheri et al., 2004; Lin et al.,2005; Birmingham et al., 2006) suggests that siRNA can generate

FIGURE 2 | Schematic presentation of downregulation of transcripts
with seed-complementary sequences. In the left panel, transcripts
possessing 3 UTR complementarity to a given 7-nt-long guide strand
sequence were divided into 15 groups based on the position of thecomplementary sequence in the siRNA guide strand. Transcripts labeledwith “1” and “7” at both ends possess complementarity to nucleotides

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Thermodynamic control of RNA silencing

off-target effects through a mechanism similar to that of miRNAtarget silencing (Lewis et al., 2005; Lim et al., 2005; Grimson
et al., 2007). The 3 UTRs of off-target transcripts or miRNAtargets are complementary to the guide strand seed region (i.e.,
nucleotide positions 2–8; Figure 2; Lim et al., 2005; Lin et al.,2005; Birmingham et al., 2006; Jackson et al., 2006a). We determined the relationship between class I siRNA seed sequences andoff-target effect using the expression reporter plasmid, psiCHECK,
which encodes the Renilla luciferase gene. Three tandem repeatsof seed-matched target sequences (Figure 3C) complementary to
the entire seed-containing region (positions 1–8), but not to theremaining non-seed region (positions 9–21), were introduced into
the region corresponding to the 3 UTR of the luciferase mRNA togenerate psiCHECK-sm and used to determine the efﬁciency ofthe seed-dependent unintended off-target effect (see Figure 4A;
Ui-Tei et al., 2008a). Although all siRNAs examined exhibited highactivity for intended gene silencing at 50 nM, the off-target genesilencing calculated using psiCHECK-sm was much less effective
and more susceptible to changes in siRNA concentration (UiTei et al., 2008a). These ﬁndings indicated that variations in the
efﬁciency of unintended off-target gene silencing were due toa difference in the interactions between the guide strand RNA
entrapped in the RISC and mRNA.

SEED-DEPENDENT OFF-TARGET EFFECT EFFICIENCY VARIES
DEPENDING ON SEED REGION GC CONTENTClass I siRNA seed region GC content used in our previous study(Ui-Tei et al., 2008a) ranged from 0 to 57%. To further determine
the relationship between seed region GC content exceeding 57%
and off-targeting efﬁciency of the corresponding siRNA, six functional class II siRNAs with high GC content in the seed region
were arbitrarily selected (Figure 3A), and their capability to exert
off-target effects was examined using luciferase reporter assays(Figure 4; Ui-Tei et al., 2009). Note that two of the six class II siRNAs (siLuc-1063 and siLuc-1430) possessed a 100% GC content

1–7 of the siRNA guide strand and vice versa. The horizontal arrow
indicates a transcript group with seed complementarity. In the right panel,changes in gene expression levels are shown by log 2 of fold change ratio
to mock transfection. Note that the groups of transcripts labeled with 2–8
are the most sensitive to the off-target effects, suggesting that guidestrand nucleotides 2–8 serve as a “seed.”

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FIGURE 3 | Structures and sequences of siRNAs and chiRNAs usedin this study and their seed-matched target sequences. (A) The
structures of 11 human class I siRNAs and six class II siRNAs.
(B) The structures of 11 human class I chiRNAs and six class II

in the seed region (Figure 3A). In contrast to class I siRNAs, whichhave little or no off-target effects, class II siRNAs were frequently
associated with a considerable level of off-target gene silencing on
the seed-matched targets (Figure 4). This apparent difference inthe off-target effect may be due to differences in the GC content
in the seed region between functional class I and II siRNAs.

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chiRNAs. (C) The seed-matched target sequences used forpsiCHECK-sm constructs shown in Figures 4 and 7. The red box
indicates the seed region, and blue indicates the DNA-substituted
regions within the chiRNA.

SEED-DEPENDENT OFF-TARGET EFFECT IS DETERMINED BY
THERMODYNAMIC STABILITY IN THE DUPLEX FORMED
BETWEEN THE siRNA GUIDE STRAND SEED REGION AND
TARGET mRNA
The results shown above indicated that siRNAs with high GC content in the seed sequence have strong seed-dependent off-target

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FIGURE 4 | Concentration-dependent gene silencing effects ofauthentic siRNA of seed-matched targets. Both class I siRNAs andfunctional class II siRNAs were included. The gene silencing effects were
examined using HeLa cells transfected with psiCHECK-sm plasmids
containing various seed-matched targets. The relative luciferase (luc)activity in transfected HeLa cells was determined using a dual luciferase
assay. (A) Authentic, non-modiﬁed siRNA psiCHECK-sm plasmid
structures and gene silencing mechanism. Three tandem repeats of

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Thermodynamic control of RNA silencing

seed-matched target sequences were introduced into the region
corresponding to the 3 UTR of the luciferase mRNA. In (B–R), the effects
of non-modiﬁed siRNA transfection on seed-matched 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) GRK4-189, (N)Luc-1120, (O) Luc-49, (P) Luc-1048, (Q) Luc-1063, (R) Luc-1430. siRNAsequences and structures are shown in Figure 3A.

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A

D

B

E

C

F

FIGURE 5 | The close relationship between the efficiency ofseed-dependent off-target gene silencing and seed-target duplex
thermodynamic stability. Both class I siRNAs and functional class II
siRNAs (A–C) and class I chiRNAs and functional class II chiRNAs (D–F)
were analyzed. Solid red circles and open red circles represent the class I
and II siRNA data, respectively. Solid blue circles and open blue circles
represent the class I and II chiRNA data, respectively. (A,D) The
calculated T m of the seed-target duplex decreased with increasing
standard free energy (ΔG) for seed-target duplex formation (correlation
coefﬁcient: −0.98 and −0.91, respectively). (B,E) Luciferase activity

Frontiers in Genetics | Non-Coding RNA(seed-dependent off-target gene silencing at a 50 nM siRNAconcentration) was positively correlated with ΔG (correlation coefﬁcient:
0.72 and 0.71, respectively). (C,F) The correlation between theseed-dependent gene silencing activity (luciferase activity) and thecalculated T m of the seed-target duplex. Luciferase activity based onseed-dependent gene silencing with 50 nM siRNA was obtained from
Figures 4 and 7, respectively. Seed-target duplex ΔG and T m were
calculated using the nearest neighbor method. The relative luciferase
activity and calculated T m were correlated with each other and had a
coefﬁcient of −0.76 and −0.79, respectively.

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Table 1 | Relative luciferase activities and T ms, ΔGs, and K ds at seed regions of class I and II siRNAs.
Luciferase activity (% at 50 nM)

Seed region GC number

T m 2–8 (˚C)

ΔG 2–8 (kcal/mol)

K d (M)

CLASS I
siLuc-309

76

0

−8.1

−7.2

5.3 × 10−6

siVIM-812

95

1

8.8

−9.3

1.5 × 10−7

siGRK4-934

102

1

6.7

−9.6

9.2 × 10−8

siOct-821

64

2

12.2

−10.3

2.8 × 10−8

siLuc-774

75

2

14.6

−10

4.7 × 10−8

siVIM-1128

79

3

21.2

−12.8

4.1 × 10−10

siLuc2-153

52

3

21.0

−11.7

2.6 × 10−9

siVIM-596

49

3

26.4

−11.6

3.1 × 10−9

siOct-797

29

3

25.7

−12.4

8.1 × 10−10

siVIM-270

25

3

26.2

−12.2

1.1 × 10−9

siLuc-36

49

4

28.4

−13

3.0 × 10−10

siGRK4-189

9

4

40.1

−15.5

4.3 × 10−12

siLuc-1120

44

5

42.3

−16.7

5.7 × 10−13

siLuc-49

6

6

46.3

−17.6

1.3 × 10−13

siLuc-1048

30

6

49.7

−17.8

8.9 × 10−14

siLuc-1063

18

7

54.5

−18.6

2.3 × 10−14

siLuc-1430

20

7

54.5

−18.6

2.3 × 10−14

CLASS II

FIGURE 6 | Correlation between seed-dependent gene silencing
activity of siRNA and chiRNA and calculated T m of the protein-free
seed duplex. Gene silencing activity was measured using relative
luciferase activity in HeLa cells transfected with psiCHECK-sm andcognate siRNAs or chiRNAs at a 50 nM concentration, as shown in
Figures 4 and 7. T m of the protein-free seed region (positions 2–8) was
determined using the nearest neighbor method. (A,B) All possible 7-nt
seed sequences (47 = 16,384) were ordered as a function of GC content

effects. Thus, one possible efﬁciency regulator of the seeddependent off-target effect might be the thermodynamic stabilityof the nucleotide duplex. The melting temperature (T m ) and standard free energy change (ΔG) of the seed–target duplex formation
are good measures of the thermodynamic stability of the proteinfree seed–target duplex. In a previous experiment using class I

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and T m values of their double-stranded counterparts with RNA (A) andDNA (B). Note that because of its deﬁnition, class I siRNA or chiRNAcannot possess more than four GCs in the seed region. Open red (A) or
blue (B) circles represent combinations of target and siRNA resulting inless than 50% relative luciferase activity. Solid red (A) or blue (B) circles
represent combinations of target and siRNA with little or no off-target
effect (luciferase activity >50%). The horizontal line at 21–25˚C (A) or
28–41˚C (B) may correspond to 50% luciferase activity reduction.

siRNAs (Ui-Tei et al., 2008a), we veriﬁed a close linear relationshipbetween ΔG and T m in seed region positions 2–8; a strong positive correlation between luciferase activity and ΔG (r = 0.69), anda strong negative correlation between T m and luciferase activity(r = −0.72) was found. By replacing class I siRNAs with a mixtureof class I and II siRNAs, the ΔG range expanded from −13 andJune 2012 | Volume 3 | Article 101 | 7

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Table 2 | Thermodynamic siRNA modification.
Chemical modification

Nucleotide position

Modified base-pairing stability

LNA

The 5 end of the passenger

Increase the stability at 5 end of the passenger

Elmén et al.

strandstrand(2005)

Four residues on both ends of

Increase the stability at 3 end of the guide strand

4 -Thioribonucleoside

Functional modification

Reference

Hoshika

the passenger strand and 3

Enhancement of

end of the guide strand

selective RISC loading of

2-Thiouracil

The 3 end of the guide strand

Increase the stability at 3 end of the guide strand

Dihydrouracil

The 3 end of the passenger

Decrease the stability at 3 end of the passenger

strandstrandPosition 2 of the guide strand

The conformational alteration of RISC by theand positions 1 + 2 of theguide strand modiﬁcation reduces the rate ofpassenger strand

RISC formation to dissociate off-target transcripts

Reduction of thewith weaker binding to the guide strands

seed-dependent

Positions 1–8 of the guide

Decrease the stability in the seed region of the

off-target effectstrand and positions 12–21 ofguide strand

the guide strand

et al. (2007)
Sipa et al.
(2007)

2 -O-methyl

2 -Deoxy (DNA)Jacksonet al.
(2006b)Ui-Tei et al.
(2008b)the passenger strand

Table 3 | Relative luciferase activities and T ms, ΔGs, and K ds at seed regions of class I and II chiRNAs.
Luciferase activity (% at 50 nM)

Seed region GC number

T m 2–8 (˚C)

ΔG 2–8 (kcal/mol)

K d (M)

chiLuc-309

103

0

−12.2

−4.9

2.6 × 10−4

chiVIM-812

94

1

−0.5

−5.5

9.3 × 10−5

chiGRK4-934

95

1

0.2

−5.6

7.3 × 10−5
6.2 × 10−6

CLASS I

chiOct-821

95

2

4.7

−7.1

chiLuc-774

65

2

−4.8

−6.8

1.0 × 10−5

chiVIM-1128

79

3

8.8

−7.9

1.6 × 10−6

chiLuc2-153

91

3

5.4

−7.8

1.9 × 10−6

chiVIM-596

63

3

8.8

−7.8

1.9 × 10−6

chiOct-797

84

3

1.3

−7.8

1.9 × 10−6

chiVIM-270

78

3

3.1

−7.6

2.7 × 10−6

chiLuc-36

68

4

19

−8.0

1.4 × 10−6

CLASS II
chiGRK4-189

12

4

28.1

−9.6

9.2 × 10−8

chiLuc-1120

69

5

31.1

−9.8

6.5 × 10−8

chiLuc-49

31

6

30.4

−11.0

8.6 × 10−9

chiLuc-1048

16

6

45.2

−9.7

7.7 × 10−8

chiLuc-1063

58

7

41.4

−12.0

1.6 × 10−9

chiLuc-1430

53

7

35

−12.0

1.6 × 10−9

−7 to between −19 and −7 kcal/mol (Figures 5A,B), while the T mrange expanded from −8 and 28˚C to −8 and 55˚C (Figures 5A,C).
Correlation coefﬁcients between luciferase activity and ΔG or T m
were 0.72 or −0.76, respectively, indicating a close relationshipbetween the seed-dependent off-target effect and the seed duplex
ΔG and T m . The linear relationships among these parameters
were almost invariant (Figure 5A). The dissociation constant (K d )of the 17 siRNAs calculated using the formula ΔG = −RTln(1/K d )indicated that the highest K d was more than 108 times greaterthan the lowest one (Table 1). Therefore, it may follow that inboth functional class I and II siRNA-mediated gene silencing, theFrontiers in Genetics | Non-Coding RNA

degree of off-target effects is governed primarily by the thermodynamic stability of the seed-target duplex formed between theseed region of the siRNA guide strand and its mRNA counterpart. In Figure 6A, all possible 7-nt seed sequences (47 = 16,384)
were ordered as a function of GC content and T m values of
their double-stranded counterparts, and the siRNAs were plottedagainst the absence or presence of off-target effects. The data suggest that T m values between 21 and 25˚C serve as a T m boundary,which may discriminate off-target-free seed sequences from offtarget-positive ones. Approximately 22% of 7-nt sequences had
T m values under 21˚C, indicating that limited seed sequences

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FIGURE 7 | Concentration-dependent gene silencing effects of
DNA-seed-containing chiRNA for seed-matched targets. Both class I
chiRNAs and functional class II chiRNAs were included. The gene silencing
effects were examined using HeLa cells transfected with psiCHECK-sm
plasmids containing various seed-matched targets. The relative luciferase (luc)activity in transfected HeLa cells was determined using a dual luciferase
assay. (A) chiRNA psiCHECK-sm plasmid structures and gene silencing
mechanism. Three tandem repeats of seed-matched target sequences werewww.frontiersin.org

Thermodynamic control of RNA silencing

introduced into the region corresponding to the 3 UTR of the luciferase
mRNA. In (B–R), the effects of chiRNAs transfection on seed-matched targets
are shown. (B–L) class I chiRNAs, (M–R) class II chiRNAs, (B) chiLuc-309, (C)chiVIM-812, (D) chiGRK4-934, (E) chiOct-821, (F) chiLuc-774, (G) chiVIM-1128,(H) chiLuc2-153, (I) chiVIM-596, (J) chiOct-797, (K) chiVIM-270, (L) chiLuc-36,(M) chiGRK4-189, (N) chiLuc-1120, (O) chiLuc-49, (P) chiLuc-1048, (Q)chiLuc-1063, (R) chiLuc-1430. chiRNA sequences and structures are shown in
Figure 3B.

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are available for selecting siRNAs with reduced off-target
effects.

THERMODYNAMIC CONTROL OF RNA STRAND
INCORPORATION INTO THE RISC BY CHEMICALMODIFICATIONSRNA strand incorporation into the RISC is determined by siRNA
duplex thermodynamics. The RNA strand with lowest binding stability in the 5 end of the guide strand is preferentially incorporated
into the RISC. Thus, rational chemical modiﬁcations can be used
to improve selective guide strand loading into the RISC (Table 2).
High-afﬁnity modiﬁcations [e.g., locked nucleic acid (LNA)] at
the 5 end of the passenger strand increase selective loading of
the guide strand (Elmén et al., 2005). In addition, base modiﬁcations of a high-afﬁnity 2-thiouracil base at the 3 end of the guidestrand and a low-afﬁnity dihydrouracil base at the 3 end of thepassenger strand can be used to the same effect (Sipa et al., 2007).
Furthermore, a moderately active siRNA duplex is signiﬁcantly
improved by modifying the high-afﬁnity 4 -thioribonucleoside(Hoshika et al., 2007). Similarly, other modiﬁcations, such as
high-afﬁnity 5-methyluracil and 5-methylcytosine modiﬁcations(Terrazas and Kool, 2009), or low-afﬁnity 2,4-diﬂuorotoluene and5-nitroindole modiﬁcations (Addepalli et al., 2010), may also
control the efﬁciency of RISC loading.
ELIMINATION OF SEED-DEPENDENT OFF-TARGET EFFECT BY
CHEMICAL MODIFICATIONSThe seed-dependent off-target effect is also eliminated by chemical
modiﬁcations (Table 2). 2 -O-methyl modiﬁcation of the guidestrand seed region alters the RISC conformation and reduces
seed-dependent off-target effects by dissociating off-target transcripts with weak binding to the guide strand (Jackson et al.,2006b). Low-afﬁnity dihydrouracil base, 2,4-diﬂuorotoluene, or
5-nitroindole modiﬁcations in the seed region may also reduce
the seed-dependent off-target effects. Furthermore, we revealed
that 2 -deoxy modiﬁcation (DNA replacement) of nucleotides 1–
8 in the guide strand and 12–21 in the passenger strand (DNA:RNA
chimeric siRNA, chiRNA; Figure 3B) reduces thermodynamic stability in the seed-target duplex, and almost completely eliminates
off-target effects with little or no loss of target gene silencing activity (Ui-Tei et al., 2008b). 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 of thepassenger strand. As shown in Figure 7, most functional class
II siRNAs could not effectively eliminate the off-target effects by
DNA replacement in the seed region (Ui-Tei et al., 2009). We
examined the relationship between the relative luciferase activity and the ΔG or T m of the DNA:RNA seed duplex in 11
class I and six class II chiRNAs (Figure 5D–F). We veriﬁed a
close linear relationship between ΔG and T m in the seed region(Figure 5D), a strong positive correlation between luciferase activity and ΔG (r = 0.71) and a strong negative correlation between
T m and luciferase activity (r = −0.79), irrespective of the presenceor absence of DNA replacement in the seed region (Figure 5E,F).
However, DNA replacement increased ΔG and reduced both theseed-target duplex T m and luciferase activity considerably. T m
was reduced to less than 20˚C in all the class I chiRNAs, while

Frontiers in Genetics | Non-Coding RNA

FIGURE 8 | Microarray profiles of transcripts downregulated bytransfection of siRNA with high or low stability in the seed-target
duplex. (A) siRNA with high seed T m is capable of forming stable seed
duplexes with the 3 UTR counterpart of target mRNA. (B) siRNA with low
seed T m is capable of forming unstable seed duplexes with the 3 UTR
counterpart of target mRNA. Microarray-based expression proﬁles were
examined 24 h after transfection. Gene expression changes are shown bylog 2 of fold change ratio (ordinate) relative to mock transfection. The
abscissa represents transcript signal intensity (log 10). Blue and gray dots
represent transcripts complementary to the seed and those with no seed
complementarity, respectively. Target genes are colored red and indicatedby arrows. Their expression levels were similarly reduced. The number ofgenes examined is shown on the upper right edge of each panel.

the relative luciferase activity at 50 nM exceeded 60%, the minimum relative luciferase activity necessary for a practical off-target
effect. In contrast, the relative luciferase activity at 50 nM was
30% or less in three of six cases treated with functional class
II chiRNAs, even though the seed-target T m was reduced; thisdemonstrates a strong negative correlation with ΔG for seedtarget duplex formation (Figure 5D). Therefore, it appears thatthe reduced off-target effect in chiRNA-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 Tables 1 and 3, DNA replacement throughout the guide

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Ui-Tei et al.

Thermodynamic control of RNA silencing

strand seed region is roughly equivalent to a 13˚C reduction
in T m , a 5 kcal/mol increment in ΔG, and about two to three
G/C → A/U changes in the seed duplex. In Figure 6B, T m valuesof all possible 7-nt DNA:RNA hybrids are ordered and plottedagainst chiRNAs with or without off-target silencing activity. For
DNA:RNA hybrids, 28–41˚C might be a boundary line that discriminates off-target-free from off-target-positive seed sequences.
However, this boundary had higher T m values, as compared tothose of RNA duplexes shown in Figure 6A. This might be partially due to different parameters used in calculating T m valuesof RNA duplexes and DNA:RNA hybrids. The proportion of 7-nt
DNA:RNA hybrids with T m values under 28˚C was about 88%,
indicating that most 7-nt sequences are available for off-target
effect-reduced RNA silencing by replacing RNA with DNA in theseed region.

GENOME-WIDE ANALYSIS REVEALED THAT siRNA WITHLOW STABILITY IN THE SEED-TARGET DUPLEX IS CAPABLE
OF INDUCING TARGET GENE-SPECIFIC SILENCING
The hypothesis that off-target gene silencing is determined primarily by seed-target duplex stability was apparent in genome-wide
expression proﬁling using class I siRNA with high or low seed T mvalue (Figure 8). The reporter assay described above predicted thatsiRNA with high seed T m value would be good inducer, while thatwith low seed T m value would be poor inducer of the off-target
effect.
As anticipated, both siRNAs effectively reduced the amount ofcompletely matched target mRNA to less than 20% as a result of
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Conﬂict of Interest Statement: The
authors declare that the research was
conducted in the absence of any commercial or ﬁnancial relationships that
could be construed as a potential conﬂict of interest.
Received: 24 April 2012; paper pending published: 11 May 2012; accepted:
18 May 2012; published online: 04 June
2012.
Citation: Ui-Tei K, Nishi K, Takahashi
T and Nagasawa T (2012) Thermodynamic control of small RNA-mediatedgene silencing. Front. Gene. 3:101. doi:
10.3389/fgene.2012.00101
This article was submitted to Frontiers in
Non-Coding RNA, a specialty of Frontiersin Genetics.
Copyright © 2012 Ui-Tei, Nishi, Takahashi and Nagasawa. This is an openaccess article distributed under the termsof the Creative Commons Attribution
Non Commercial License, which permits non-commercial use, distribution,and reproduction in other forums, provided the original authors and source arecredited.

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