Ui-Tei, Gene Technol 2012, 1:1
http://dx.doi.org/10.4172/gnt.1000102

Gene Technology
Letter to the Editor

Open AccesssdRNA: siRNA with a DNA Seed for an Efficient and Target-gene Specific
RNA Interference
Kumiko Ui-Tei*
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Abstract
RNA interference (RNAi), a process through which small interfering RNAs (siRNAs) induce sequence-specific post-transcriptional
gene silencing, is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. To
achieve accurate target gene function and successful therapeutic applications, it is necessary to select an efficient and target genespecific siRNA with minimal off-target effects. We found that the ability to induce off-target effects on unintended genes is strongly
correlated to the thermodynamic stability of the duplex formed between the seed region (positions 2-8 from the 5' end of the siRNA guidestrand) and target mRNA. Consistent with this property, we found that DNA-RNA chimeric siRNA (chiRNA) with deoxyribonucleotides
in the 5' proximal eight nucleotides of the guide strand and the complementary nucleotides in the passenger strand exerted virtually no
off-target effect due to low stability of the DNA-RNA duplex in seed-target base-pairing. However, the corresponding RNAi activities forprimary target genes were also decreased to one-tenth on average by the DNA substitutions. Here, we report that siRNAs with sevendeoxyribonucleotides exclusively in the seed region (sdRNA) may exhibit efficient target-specificity, but off-target effect-reduced RNAi
activity.

Keywords: DNA modification; Off-target effect; Seed region; Smallinterfering RNA; siRNA; RNA interference; RNAiIntroduction
RNA interference (RNAi) is an evolutionarily conserved pathway
induced by small interfering RNAs (siRNAs), which are 19 bp in length,
double-stranded RNAs (dsRNAs) with two-nucleotide 3' overhangs[1]. siRNAs incorporated into cells are transferred to an RNAi effector
complex called an RNA-induced silencing complex (RISC) [2,3]. RISCs
are assembled on one of the two strands of the siRNA duplex and are
activated upon removal of the passenger strand [4-6]. Others reported
that asymmetric features at both siRNA terminals are common tofunctional siRNAs [6-8]. An RNA strand with an unstable 5' terminus
is easily retained in a RISC. An activated RISC is a ribonucleoprotein
complex consisting minimally of a core protein, Argonaute (Ago) andan siRNA guide strand [9-11]. The siRNA guide strand retained in an
RISC recognizes mRNAs with perfectly complementary sequences andthe Ago2 protein cleaves them.
The RNAi effect of a siRNA has been assumed to be extremely
specific. However, accumulated evidence has revealed that siRNAs may
downregulate many unintended genes with partial complementarities
mainly in the seed region (nucleotides 2-8 from the 5' end of the siRNA
guide strand) [12-16]. This phenomenon is referred to as an off-target
effect. The recognition mechanism of an off-target effect is known tobe similar to that of microRNA-mediated gene silencing [13-15,1721]; seed nucleotides are present on the Ago surface in a quasi-helicalform to serve as the entry or nucleation site for small RNAs in the RISC
[12,22,23]. To achieve accurate target gene function and successful
therapeutic applications, it may be critical to select a target genespecific siRNAs with minimal off-target effects.
We found that ability to induce an off-target effect is strongly
correlated to the thermodynamic stability of the duplex formed between
the seed region of siRNA guide strand and target mRNA (seed-target
duplex) (Figure 1a) [12]. Furthermore, we have developed a DNARNA chimeric siRNA (chiRNA) with deoxyribonucleotides in the 5'
proximal eight nucleotides of the guide strand and the complementary
nucleotides. The chiRNA showed virtually no off-target effects,
probably because the stability of the seed-target duplex was reducedby the DNA substitutions in the siRNA seed region (Figure 1b) [16].
For target-specific RNAi with reduced off-target effects, we propose an
improved chiRNA, referred to as an sdRNA, in which seven nucleotides
in the seed region is exclusively replaced with DNA without including a
nucleotide at the 5' terminus (Figure 1c).
Gene Technol
ISSN: GNT, an open access journal

Materials and Methods
siRNA
RNAi efficiency in mammalian cells varies considerably dependingon the siRNA sequence. We have shown that highly functional siRNAs
for mammalian RNAi have A or U residues at nucleotide position 1
measured from the 5' end of the guide strand, four to seven A/Us at
nucleotide positions 1-7 (AU ≥ 57%) and a G/C at position 19 (Figure1) [6]. In addition, a GC stretch of no more than nine nucleotides iscontained in siRNA sequences. All of the double-stranded small RNAs
used in this study shown in S1 simultaneously satisfied these conditions.

Cell culture and silencing assay
Human HeLa cells were cultured and subjected to gene silencing
as described previously [6,12]. Briefly, a 1 ml suspension of human
HeLa cells (1×105 cells/ml) was inoculated in a 1.5 cm well 24 h prior
to transfection. The cells were transfected with pGL3 or pGL2 control(1 µg; Promega), both of which encode firefly luciferase (luc), and theRenilla luc-encoding plasmid psiCHECK-completely matched (CM) or
seed matched (SM) (0.1 µg; Promega) with siRNA, chiRNA, sdRNA,or sd/chiRNA. Lipofectamine 2000 regent (Invitrogen) was used fortransfection. Each of siRNA, chiRNA, sdRNA, or sd/chiRNA (0.5, 5,
50, 500, 5,000, and 50,000 pM) was diluted in 50 µl of Opti-MEMI
without fetal bovine serum (FBS) and mixed with 50 µl Opti-MEMI
containing 1µl of Lipofectamine 2000 for 30 min in room temperature.
After the culture medium was changed with FBS-free medium, 100µl of siRNA, chiRNA, sdRNA, or sd/chiRNA-Lipofectamine 2000
complex were added to each well. After 4 h incubation, the medium
was replaced with fresh medium with FBS. The cells were harvested*Corresponding author: Kumiko Ui-Tei, Department of Biophysics andBiochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, Tel: +81 3 5841 3043; Fax: +81 3 5841 3043;
E-mail: ktei@bi.s.u-tokyo.ac.jp
Received October 27, 2012; Accepted November 20, 2012; Published November
26, 2012
Citation: Ui-Tei K (2012) sdRNA: siRNA with a DNA Seed for an Efficient andTarget-gene Specific RNA Interference. Gene Technol 1:102. doi:10.4172/
gnt.1000102
Copyright: © 2012 Ui-Tei K. This is an open-access article distributed under theterms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author andsource are credited.

Volume 1 • Issue 1 • 1000102

Citation: Ui-Tei K (2012) sdRNA: siRNA with a DNA Seed for an Efficient and Target-gene Specific RNA Interference. Gene Technol 1:102.
doi:10.4172/gnt.1000102
Page 2 of 6

(a)

(b)
chiRNA-mediatedsilencingsiRNA-mediatedsilencing
siRNA
3'

19

87

G or C1-3'-5'

AU richA or U

3'

5'

19

sdRNA

passenger strand
87

G or C1

AU rich-3'-5'
A or U

3'

19

5'

87

1

AU rich-3'-5'
A or Uguide strand

AAAAA

-5'
Seed region

RISC

passenger strand

G or C

guide strand

guide strand

3'sdRNA-mediatedsilencing

chiRNA RISC

RISC

passenger strand

5'

(c)

AAAAA
-5'
Seed region

3'

Strong base-pairing

AAAAA
-5'
Seed region

3'

Weak base-pairing

Weak base-pairing

Off-target mRNA

Off-target mRNA

Off-target mRNA

Translational repression

Translational repression

Translational repression

AAAAA

-5'
Seed region

3'

AAAAA

AAAAA

-5'
Seed region

3'-5'
Seed region

3'

Strong off-target effectWeak off-target effectWeak off-target effectOn-target mRNA

On-target mRNA

On-target mRNA

Cleavage
3'

Strong RNAi

Cleavage

Cleavage
AAAAA
-5'

AAAAA
-5'

3'

Weak RNAi

AAAAA
-5'

3'

Strong RNAi

Figure 1: Comparison of the RNAi activities and off-target effects of (a) siRNA, (b) chiRNA and (c) sdRNA. The highly functional double-stranded small RNAs
used in this study contained A or U residues at nucleotide position 1, four to seven A/Us at nucleotide positions 1-7, G/C at position 19, and a GC stretch of no
more than nine nucleotides is not contained. The small double-stranded RNA unwound into a single-stranded RNA in the RISC. The guide strand retained in the
RISC recognized on- and off-target mRNAs with complementary sequences to the seed region at positions 2-8 from the 5' end of the guide strand. The on-target
mRNAs had perfectly complementary sequences. However, off-target effects were observed for mRNAs with seed-complementary sequences. The siRNA showeda strong off-target effect and strong RNAi activity. The chiRNA showed a weak off-target effect and weak RNAi activity. The sdRNA was ideal showing a weak offtarget effect and strong RNAi activity.

24 h after transfection and their luc activity was measured using a
Dual-Luciferase Reporter Assay System (Promega). The siRNAs
used in this study were chemically synthesized (Sigma) (see S1).
siGY-441, an siRNA for GFP knockdown, was used as a control.
IC 50 was determined from the results shown in figures 2 and 3 byusing the following formula:
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ISSN: GNT, an open access journal

IC50 = 10∧ log ( A / B ) × ( 50 − C ) / ( D − C ) + log ( B ) 

where in A: of the two points on each Figure which bracket 50% inhibition,
the higher concentration of siRNA, B: of the two points on each Figure whichbracket 50% inhibition, the lower concentration of siRNA, C: inhibitory activity(%) at the concentration B, D: inhibitory activity (%) at the concentration A.
Volume 1 • Issue 1 • 1000102

Citation: Ui-Tei K (2012) sdRNA: siRNA with a DNA Seed for an Efficient and Target-gene Specific RNA Interference. Gene Technol 1:102.
doi:10.4172/gnt.1000102
Page 3 of 6

(a)

siRNA
3'

passenger strand

5'

sdRNA

chiRNA-3'-5'

guide strand

mRNA
5'

3'

5'

passenger strand

-3'
-5'

guide strand

3'

sd/chiRNA

passenger strand

5'

-3'
-5'

guide strand

-3'5'

-3'5'

passenger strand

5'

-3'
-5'

guide strand-3'5'

guide strand

guide strand

3'

-3'

guide strand

guide strand

20
0
0

0

0
00

120

60
40
20
0
5

0.

5

60
40
20
0
5

5

50

0

0
50

0

5

0
00

siVIM270 (pM)

Relative luc activity (%)

80

50
0

Relative luc activity (%)

IC50 = 16.2 pM

0.

50

0

0

0
50

120

0
00

60
40
20
0
5

5

50

0

0

0
50

Relative luc activity (%)

0
5

5

50

00

50

120

00

60
40
20
0
5

5

50

0

0
50

0
00

5

chiVIM270 (pM)

120

0

0
00

60
40
20
0
5

5

50

0

0
50

0

20
0
5

5

0.

50

00

50

0

00

50

120

IC50 = 149.3 pM

100
80
60
40
20
0
5

0.

5

50

00

50

0

00

50

sd/chiFL2-153 (pM)

80

0.

00

40

5

IC50 = 27.8 pM

100

50

60

sd/chiFL2-153 (pM)

80

0.

0

80

0
50

IC50 = 129.8 pM

100

00

50

IC50 = 19.6 pM

100

0

20

120

50

40

0.

50

50

00

0

5

5

0.

50

60

sdFL3-36 (pM)

80

0.

0

80

5

IC50 = 114.6 pM

100

50

IC50 = 15.9 pM

100chiFL3-36 (pM)(e) VIM270
100

5

0

sdFL2-153 (pM)80

siFL3-36 (pM)

120

00

0
50

IC50 = 336.1 pM

100

20

0

40

0
50

00

50

40

50

60

50

50

60

00

0
50

sdVIM270 (pM)120

IC50 = 43.4 pM

100
80
60
40
20
0
5

0.

5

50

50
0

80

5

5

80

Relative luc activity (%)

5

Relative luc activity (%)

IC50 = 105.6 pM

100

5

0

120

chiFL2-153 (pM)

Relative luc activity (%)

120

0.

20
0.

IC50 = 12.8 pM

100

sd/chiCLTC2416 (pM)

Relative luc activity (%)

00

0
50

40

Relative luc activity (%)

00

50

60

siFL2-153 (pM)0Relative luc activity (%)(d) FL3-36

50

00

50

120

sdCLTC2416 (pM)

80

0

5

50

5

0

0

50

Relative luc activity (%)

20
0.

5

5

0.

5

IC50 = 38.0 pM

100

0

40

120

50

60

Relative luc activity (%)

80

50

Relative luc activity (%)

IC50 = 12.6 pM

1000chiCLTC2416 (pM)(c) FL2-153
120

0

0
00

Relative luc activity (%)

siCLTC2416 (pM)

00

50

20

0

5

50

40

50

5

60

0

5

0.

80

50

0

0
00

50

00

50

0

0

50

5

20

50

5

0.

0

0

40

IC50 = 11.2 pM

100

0

20

60

120

50

40

80

Relative luc activity (%)

60

IC60 = 19.0 pM

100

0

80

120

50

IC60 = 9.4 pM

100

Relative luc activity (%)

120

50

Relative luc activity (%)(b) CLTC2416

00

50

0

00

50

sd/chiVIM270 (pM)

Figure 2: RNAi activity assay using reporter plasmids containing the CM target sequence. (a) Structures of the siRNAs, chiRNAs, sdRNAs, and sd/chiRNAs andtheir base-pairing patterns with completely matched targets; RNAi activities of the siRNAs, chiRNAs, sdRNAs, and sd/chiRNAs of (b) CLTC2416; (c) FL2-153; (d)FL3-36 and (e) VIM270.

Plasmid construction
All plasmids constructed were derivatives of psiCHECK-1 (Promega).
Chemically synthesized oligodeoxynucleotides, including one copy of the
23-bp CM target sequences (S2) or three copies of the SM target sequences(S3) with cohesive XhoI/EcoRI ends, were inserted into the psiCHECK-1(Promega) XhoI/EcoRI site, which is situated in the region encoding the 3'
UTR of Renilla luc mRNA; they are referred to as psiCHECK-CM and SM,
respectively. The inserted CM or SM targets were expressed as part of the 3'
UTR of Renilla luc mRNA in the transfected cells. The CM target completely
Gene Technol
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matched the siRNA guide strand (Figure 2a), whereas the SM target consistedof two parts: the eight 3' terminal nucleotides of the target were complementary
in sequence to the 5' end (nucleotide position 1) and the seed (positions 2-8)of the corresponding siRNA guide strand, while the remaining thirteen
nucleotides were totally non-homologous to the guide strand (Figure 3a).

Results and Discussion
Designing sdRNA
We developed an improved method using siRNAs with DNA in
Volume 1 • Issue 1 • 1000102

Citation: Ui-Tei K (2012) sdRNA: siRNA with a DNA Seed for an Efficient and Target-gene Specific RNA Interference. Gene Technol 1:102.
doi:10.4172/gnt.1000102
Page 4 of 6

chiRNA

siRNA

(a)
3'

5'

passenger strand-5'

guide strand

mRNA
5'

-3'

5'

3'

sdRNA

passenger strand
guide strand

-3'
-5'

5'

3'

sd/chiRNA

passenger strand-5'

guide strand

passenger strand

5'

guide strand

-5'

-3'

-3'

guide strand

guide strand

guide strand

guide strand

3'-3' 5'-3' 5'-3' 5'-3'40
20

sdVIM270 (pM)

0
00

50

0

0
00

50
0
00

50

00

50

50

80
60
40
20

00

0

0

00

50
00
0

50
0
50
00

5

0

IC50 = 3636.3 pM

50
0

20

100

5

40

120

50

60

0

0

0.
5

80

00

20

sd/chiFL3-36 (pM)

Relative luc activity (%)

IC50 = 2663.8 pM

50

50

40

sdFL3-36 (pM)

5

50

0.
60

50

0
00

80

50

0

00

50

50

50

5

0

IC50 = ∞

100

5

20

120

50

40

100

0

0

5

60

00

20

0.

80

120

50

40

sd/chiFL2-153 (pM)

Relative luc activity (%)

IC50 = ∞100

0.

Relative luc activity (%)

120

50

60

5

0
00

80

50

00

0
50

50

5

50

5

0

IC50 = 4864.3 pM

100

50

20

120

5

40

5

sd/chiCLTC2416 (pM)

Relative luc activity (%)

60

50

0

0.
5

0
00

60

50

chiVIM270 (pM)

80

50

0

80

0.

50
00
0

50
0
50
00

0

00

50

IC50 = 3045.0 pM

100

IC50 = ∞100

sdFL2-153 (pM)

Relative luc activity (%)

20

50

5

50

0.
5
120

0.

Relative luc activity (%)

0
00

40

120

sdCLTC2416 (pM)

50
0
00

60

Relative luc activity (%)

Relative luc activity (%)

0

00

0

20

50

0

00

50

50

80

50

50
0
50
00
50
00
0

0

IC50 = 7968.7 pM

100

5

20

120

5

40

5

50

0

0.

60

50

40

chiFL3-36 (pM)

Relative luc activity (%)

IC50 = 46.0 pM

80

0.
5

00

20

siFL3-36 (pM)

Relative luc activity (%)5040(e) VIM270

siVIM270 (pM)

50

0.
60

5

0
00

80

50

00

0
50

50

5

50

5

0

IC50 = ∞

100

5

20

120

0.

40

Relative luc activity (%)

60

0.

Relative luc activity (%)

80

100

60

chiFL2-153 (pM)

IC50 = ∞

120

0

0

siFL2-153 (pM)

100

80

50

20

(d) FL3-36
120

0

40

00

0

60

50

00

0
50

50

5

50

5

0

80

5

20

IC50 = ∞

100

50

40

120

5

60

Relative luc activity (%)

80

0.

Relative luc activity (%)

IC50 = 1261.5 pM

100

IC50 = ∞100

chiCLTC2416 (pM)(c) FL2-153
120

120

5

siCLTC2416 (pM)

00

0

00

0

20

50

0

00

50

50

50

5

0

40

50

20

60

50

40

80

5

60

IC50 = ∞

100

50

80

120

0.
5

IC50 = 137.5 pM

100

Relative luc activity (%)

120

0.
5

Relative luc activity (%)(b) CLTC2416

sd/chiVIM270 (pM)

Figure 3: Off-target effect activity assay using a reporter plasmid containing SM target sequence. (a) Structures of the siRNAs, chiRNAs, sdRNAs, and sd/chiRNAs
and their base-pairing patterns with seed-matched targets; Off-target effect activities of the siRNAs, chiRNAs, sdRNAs, and sd/chiRNAs of (b) CLTC2416; (c) FL2153; (d) FL3-36 and (e) VIM270.

the seed region (sdRNA), in which ribonucleotides were replaced with
DNA at positions 2-8 from the 5' end of the siRNA guide strand but aribonucleotide at the 5' terminal end was not replaced with DNA. The
complementary ribonucleotides of the guide strand seed region in thepassenger strand were also replaced with DNA (Figure 1c). The sdRNA
was expected to have strong target-specific RNAi activity and weak offtarget effect activity, because it had a DNA seed sequence that could
form a DNA-RNA seed-target duplex with low stability compared tothat formed by an RNA-RNA duplex; the other positions (at 1 and 9-21)
were ribonucleotides. In particular, A or U ribonucleotide residues at
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the 5' end of the guide strand might be important because the A/U
nucleotide itself at the 5' terminal was shown to contribute strongly toRNAi activity. This observation might be explained by the fact that thenucleotide monophosphates AMP and UMP bind to Ago2 with up to
30-fold higher affinity than that observed with either CMP or GMP [24].

Comparison of the on-target RNAi activities of siRNAs,
chiRNAs, sdRNAs, and sd/chiRNAs
To compare the RNAi activities of siRNAs, chiRNAs, and sdRNAs
Volume 1 • Issue 1 • 1000102

Citation: Ui-Tei K (2012) sdRNA: siRNA with a DNA Seed for an Efficient and Target-gene Specific RNA Interference. Gene Technol 1:102.
doi:10.4172/gnt.1000102
Page 5 of 6(a) RNAi activity
400

9000

siRNA
chiRNAsdRNA
sd / chiRNA

350
300

7000

250
200
150
100

6000
5000
4000
3000
2000

50
0

siRNA
chiRNAsdRNA
sd / chiRNA

8000

IC50 (nM)

IC50 (nM)(b) Off-target effect activity

1000↑
10 VIM270 100↑↑↑FL2-153 FL3-36

1

1000

CLTC2416

0
10

↑ 100
1000
↑
↑
VIM270
CLTC2416 FL2-153

IC50 of siRNA (nM)

10000

Comparison of the off-target effect activities of the siRNAs,
chiRNAs, sdRNAs, and sd/chiRNAs

IC50 of siRNA (nM)

Weak

Weak

chiRNA
3'

5'

5'
3'

3'

3'

5'

5'

sd/chiRNA
sdRNA
siRNA

chiRNA
-3'
-5'

3'

5'

5'
3'
-3'
-5'
3'

-3'
-5'
-3'
-5'

3'

strong
mRNA

-3'

5'

guide strand

5'5'sd/chiRNA

sdRNA

siRNA

strong
mRNA

-3'
-5'

-3'
-5'

-3'
-5'

-3'
-5'

-3'

5'

guide strand

Figure 4: RNAi activities and off-target effect activities of the siRNAs, chiRNAs,
sdRNAs, and sd/chiRNAs. Comparison of the (a) RNAi activities and (b) offtarget effect activities of the siRNAs, chiRNAs, sdRNAs, and sd/chiRNAs.
Upper panels: The horizontal axis indicates the IC50 of each siRNA. Verticalaxis indicates (a) IC50 of the RNAi activities and (b) off-target effect activities ofsiRNAs, chiRNAs, sdRNAs, or sd/chiRNAs with the same sequences as thesiRNAs shown on the horizontal axis. Lower panel: schematic representationof the (a) RNAi activities and (b) off-target effect activities of the siRNAs,
chiRNAs, sdRNAs, and sd/chiRNAs.

based on the same sequences (Figure 2a), four different sequencescomplementary to human clathrin heavy chain (CLTC2416), luc(FL2-153 and FL3-36), and human vimentin (VIM270) were used. As
detailed in the Experimental Section, we constructed psiCHECK-CM
reporters that contained Renilla luc with the complementary sequenceof each siRNA introduced into its 3' UTR. The psiCHECK-CM plasmids
were transfected into human HeLa cells together with pGL3-Control(encoding firefly luc) along with small double-stranded RNA. One
day after transfection, Renilla luc activity was measured with reference
to the firefly luc activity level in the transfected cells, and the relative
luc activities were calculated. Each of the highly functional siRNAs
reduced the relative luc activity (RNAi activity) in a dose-dependent
manner at siRNA concentrations of 0.5 pM to 50 nM (Figure 2b-e). The
calculated IC50 values of the nonmodified siRNAs varied from 9.4-105.6
pM depending on the sequence, indicating that siRNAs used in thisstudy are very active as reported by others [25,26]. The IC50 values of
the chiRNAs were from 19.0-336.1 pM, a decrease of 14-49% comparedto the IC50 values of the siRNAs. The IC50 values of the sdRNAs werefrom 11.2-129.8 pM, about 58-83% of those of the siRNAs. As shownin figure 4a, siRNAs showed the strongest RNAi activities in all of foursiRNAs used in this study, while chiRNAs showed the lowest activities.
Because sdRNAs with a deoxyribonucleotide in the seed region showed
equivalent RNAi activity with the siRNAs, the low RNAi activity of theGene Technol
ISSN: GNT, an open access journalchiRNAs might not be due to the deoxyribonucleotide substitutions inthe seed region. To investigate whether a deoxyribonucleotide at the 5'
terminal end of the guide strand and three deoxyribonucleotides at the
3' end of the passenger strand were involved in the reduction in RNAi
activity, we examined the effect of an sd/chiRNA containing an sdRNA
guide strand and chiRNA passenger strand. These sd/chiRNAs also
showed equivalent RNAi activities with the siRNAs; their IC50 values
were 37-73% of the siRNA values (Figure 2b-e), suggesting that the 5'
end ribonucleotide in the guide strand played an important role in theRNAi activity. These results show good agreement with the previously
reported result that AMP and UMP bind to Ago2 with up to 30-foldhigher affinity than either CMP or GMP [24].

To compare the off-target effect activities of siRNAs, chiRNAs,and sdRNAs (Figure 3a), four different sequences, CLTC2416, FL2153, FL3-36, and VIM270, were used. Off-target effect activities were
measured using the psiCHECK-SM reporter system. These highly
functional siRNAs, siCLTC2416, siFL2-153, siFL-36, and siVIM270,
showed off-target effects in a dose-dependent manner until the siRNAconcentration reached 50 nM (Figure 3b-e). The calculated IC50 valuesof the off-target effects in the nonmodified siRNAs, siCLTC2416, siFL2153, and siVIM270, varied from 46.0-1261.5 pM, and that of siFL336 was not determined. However, the off-target effects of the chiRNAs
were considerably reduced as shown in our previous report [16]; theiroff-target effects were reduced to undetectable levels, and the IC50 valueof chiVIM270 was decreased to 0.6% (7968.7 pM) of the IC50 value ofsiVIM270. Previously, we showed that ability to induce an off-target
effect is correlated to the thermodynamic stability of seed-target duplex
of siRNA [12]. The melting temperature value of RNA-RNA duplex atseed positions 2-8 (Tm2-8) of siCLTC2416 is 33.2ºC, which decreases
to 12.7ºC when DNA-RNA duplex is formed by chiCLTC2416,
indicating the stability of seed-target duplex is strong for siCLTC2416
but weak for chiCLTC2416 consistent with the results shown in figure
3b. Similarly, Tm2-8 values of RNA-RNA duplexes formed between
siFL2-153, siFL3-36, and siVIM270 and each target mRNAs were21.0-26.2ºC, and the values of DNA-RNA duplexes formed between
chiFL2-153, chiFL3-36, and chiVIM270 and each target mRNAs
decreased to 3.1-18.9ºC (Figure 3c-e) [12]. Since the Tm2-8 values inthe duplexes formed between sdRNAs and target mRNAs are sameas those of DNA-RNA duplexes formed between chiRNAs and target
mRNAs, it was unexpectedly that the sdRNAs showed strong off-target
effects compared to those of chiRNAs. The IC50 values of sdFL2-153
and sdVIM270 were decreased to 41% (3045.0 pM) and 1.7% of theIC50 value of each siRNA, respectively. As shown in figure 4b, the threesiRNAs showed the strongest off-target effect activities, while thechiRNAs showed the weakest off-target effect activities; the sdRNAs
showed intermediate levels of off-target effect activity. The decreases
in off-target effect activities of the sdRNAs compared to those of thesiRNAs might be caused by the DNA substitutions in the seed region,
suggesting that decreased stabilities in the seed-target duplexes reduced
the off-target effects. However, the off-target effects of the sdRNAs
increased compared to those of the chiRNAs, suggesting that the 5' end
ribonucleotide of the guide strand and three ribonucleotides at the 3'
end of the passenger strand contributed to the increase in off-target
effects. Furthermore, the sd/chiRNAs showed weak or similar levels ofoff-target activities compared to those found with sdRNAs (Figure 4b).
This result suggests that the 5' end RNA of the guide strand contributed
strongly to the off-target effect, while the three ribonucleotides at the 3'
end of the passenger strand contributed weakly to this effect.
Volume 1 • Issue 1 • 1000102

Citation: Ui-Tei K (2012) sdRNA: siRNA with a DNA Seed for an Efficient and Target-gene Specific RNA Interference. Gene Technol 1:102.
doi:10.4172/gnt.1000102
Page 6 of 6

Conclusions
Based on our previous reports, we expected that an sdRNA that
included a DNA seed would be an appropriate tool for inducing targetspecific RNAi with reduced off-target effects. Our results confirm thatthe sdRNA showed weak off-target effect activity compared to siRNAs
and strong RNAi activity compared to chiRNAs. A 5' end ribonucleotide
in the siRNA guide strand contributed strongly to the off-target effects,
whereas the three ribonucleotides at the 3 end of the siRNA passenger
strand contributed weakly to the off-target effects. Our results indicatethat sdRNAs might be a powerful tool for developing ideal RNAi
experiments and therapeutic applications.

Supplementary Information Files
S1. Supplementary Information 1. RNA sequences.
S2. Supplementary Information 2. Target sequences in psiCHECKCM.
S3. Supplementary Information 3. Target sequences in psiCHECKSM.
Acknowledgements
I am grateful to Dr Kaoru Saigo and Dr Yukikazu Natori for their valuablecomments and discussions throughout this study, and Kiyoko Shinohara for
preparing the figures. I acknowledge the funding sources: the Ministry of
Education, Culture, Sports, Science and Technology of Japan (MEXT) [grant
numbers 21115004, 21310123, 23651200], the Cell Innovation Project (MEXT),and Core Research Project for Private University, matching fund subsidy. The
author declares no competing interests.

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