Functional shRNA expression system with reduced off-target effects
Yuki Naito, Kenji Nishi, Aya Juni and Kumiko Ui-Tei
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
Vector-based short hairpin RNA (shRNA) expression
systems are used to induce sustained RNAi in cells with thehelp of plasmid constructs. Early designs of shRNAs hadstems of 19–29 nucleotides with minimal flanking RNA
sequences and were expressed using strong RNA
polymerase III (pol III) promoters, such as those that drive
the expression of endogenous U6 small nuclear RNA(snRNA) or H1 RNA [15–17]. Later, an expression systemthat mimics natural microRNA (miRNA) was constructed[18,19]. Natural miRNAs are a class of endogenous
dsRNAs that are transcribed by pol II as long primary5-capped and polyadenylated miRNAs (pri-miRNAs)[20,21]. The pri-miRNA is cropped first to release 70–90
nts of a hairpin-shaped precursor (pre-miRNA) by amember of the ribonuclease III family (RNase III), Drosha,
and its cofactor, DGCR8, in the context of the Microprocessor complex [22–24]. The pre-miRNA remains witha 2-nt 3 overhang at one end, which is critical for miRNAbiogenesis because it defines the position at one end of themolecule. This distinctive structure is recognized by the
Exportin-5-Ran-GTP heterodimer, and the pre-miRNA isshuttled to the cytoplasm [25,26]. In the cytoplasm, theDicer, an RNase III type protein, cuts the transported
pre-miRNA to generate ~22 nt miRNA duplex [27–29].
One strand of the Dicer product remains as a mature
miRNA and is assembled into an effector complex known
as the miRNA-induced silencing complex (miRISC) [4,5],
which exerts a gene-silencing effect. Thus, the pol II-driven
shRNA expression system is constructed by replacing themiRNA region with siRNA.
Recent microarray profiling studies have revealed that
“off-target” or unintended gene silencing is frequently
brought about by sequence complementarity between target
mRNA and the “seed” region of the siRNA (nucleotides
2–8 from the 5 end of the siRNA guide strand) [30–35]
through a mechanism similar to that of target silencing by
miRNAs [36–38]. Furthermore, we have shown that thecapability of siRNA to induce off-target effects is highly
correlated with the thermodynamic stability or calculated
melting temperature (Tm) for the formation of theprotein-free seed-target duplex [35]. siRNAs with lower
seed-target duplex stabilities (benchmark Tm value< 21.5 ºC) should minimize off-target effects.
In this study, we analyzed changes in microarray
expression profiles by transfecting HeLa cells with shRNA
expression constructs. Seed-dependent off-target genesilencing was induced by shRNA in a similar manner asthat by siRNA; no or little off-target effect was observed

Abstract:
The capability to induce off-target effects by small interfering
RNA (siRNA) is highly correlated with the calculated melting
temperature (Tm ) of the protein-free seed-target duplex,
indicating that its thermodynamic stability is a major factor in
determining the degree of the off-target effect. In this study,
we analyzed changes in microarray expression profiles
following transfection of short hairpin RNAs (shRNAs). In asimilar manner to siRNA, no or little off-target effects wereobserved by shRNA with a low Tm value in the seed-target
duplex, whereas a high degree of off-target effect was inducedby shRNA with a high Tm. Furthermore, shRNAs were often
cleaved by Drosha and/or Dicer at the shifted sites in theirprimary transcripts, resulting in unexpected siRNA productswith shifted sequences. Based on these results, the following
conditions should be satisfied to ensure a functional shRNA
with reduced off-target effects: the presence of A/U residues atnucleotide positions 1 and -1 from the 5 end of the guidestrand, three to six A/Us in nucleotide positions 2–7, G/C atpositions 19 and 18, and no GC stretch of more than nine
nucleotides. In addition, the Tm values of the seed regions inboth strands should be low.

1. INTRODUCTION
RNA interference (RNAi) is an evolutionarily conserved,
sequence-specific gene-silencing process triggered by thepresence of double-stranded RNA (dsRNA); it is involvedin the innate defense against viral infection andtransposable elements [1]. RNAi has also been used as a
research tool to control the expression of specific genes innumerous experimental organisms and has potential as atherapeutic strategy for several human diseases [2]. RNAisilencing is triggered by small interfering RNA (siRNA)
helices, which comprise 21 nucleotides (nts) containing
2-nt 3 overhangs in mammalian cells. siRNA incorporated
into the cells is transferred to the RNAi effector complex,
resulting in an RNA-induced silencing complex (RISC) [3].
However, the two strands of siRNA are handled differently.
The strand with the less stable duplex at its 5 end isincorporated preferentially into RISC [4–8]. The activated
RISC is a ribonucleoprotein complex consisting of at leastan Argonaute protein and a single-stranded siRNA [9–11].
In human and mouse cells, four Argonaute family proteins(human and mouse Ago1–4) have been found [12]. Amongthem, only Ago2 is endonucleolytically active [10, 11, 13].
The RISC containing the siRNA guide strand and Ago2
binds to the target RNA and silences gene expression bycleaving target mRNA by an RNase H-like active site inAgo2 [10,14].
1
978-1-4244-5095-4/09/$26.00 ©2009 IEEE

- 291 -

after introducing shRNA with a low Tm in the seed-target
duplex, whereas a high degree off-target effect was inducedby shRNA with a high Tm. Furthermore, it was also evident
that primary shRNA transcripts are often cleaved at theshifted sites. In view of these results, we proposed feasible
rules for constructing shRNA with a low off-target effect.

67-mer oligonucleotides into BamHI/HindIII sites in thepSUPER.retro.Puro vector (OligoEngine) containing an H1
promoter; this construct was named pSUPER.H1 (Fig. 1B).
From the inserted oligonucleotides, the transcript was
encoded in the following order: (i) a 21-nt siRNA guidestrand; (ii) a human miR-23a loop, and (iii) a 19-nt
passenger strand of the identical siRNA lacking a 3overhang. The inserted synthetic oligonucleotide sequenceswere as follows:
pSUPER.H1-VIM269, 5-GATCCCGCCATCAACACCG
AGTTCAAGGCTTCCTGTCACCTTGAACTCGGTGTT
GATGGCTTTTTTA-3 and 5-AGCTTAAAAAAGCCATCAACACCGAGTTCAAGGTGACAGGAAGCCTTGAACTCGGTGTTGATGGCGG-3;and pSUPER.H1-VIM812, 5-GATCCCGTACGTCAGCAATATGAAAGTGCTTCCTGTCACACTTTCATATTGCTGACGTACTTTTTTA-3 and 5-AGCTTAAAAAAGTACGTCAGCAATATGAAAGTGTGACAGGAAGCACTTTCATATTGCTGACGTACGG-3.
The pol II-driven shRNA expression vector was aderivative of pENTR.U6 (Invitrogen), and was named
pENTR.CMV (Fig. 1C). It contains the cytomegalovirus(CMV) immediate-early enhancer/promoter and the
genome region from which human miR-187 is transcribed.
The CMV promoter and the miR-187 expression region
were PCR-amplified from pSH-CMV using primers (5-CC
GCTCGAGTCAATATTGGCCATTAGC-3 and 5-CTA
GCTAGCTAGTTCATTAATGCAGTCGAGTTC-3) anddigested with XhoI/NheI. The digested product was ligatedinto pENTR.U6 digested with SalI/XbaI. pSH-CMV was aderivative of pTRE2 (Clontech). The Tet response element(TRE) in pTRE2 was replaced with the CMV promoter ofpCI-neo (Promega) and the genomic sequence with themiR-187 expression site was inserted into pTRE2. The
CMV promoter was PCR-amplified with primers (5-TTAA
TTGGCGCGCCTCAATATTGGCCATTAGC-3 and
5-AACTGCAGAACCAATGCATGATCTGACGGTTCACTAAACG-3) and digested with AscI/NsiI. The productwas inserted into pTRE2 previously treated with the same
restriction enzymes. The human miR-187 expression region(271 bp) was PCR-amplified from HeLa genomic DNA
using primers (5-CGCGGATCCATCGGGATGCACAGC
AAGT-3 and 5-GCTCTAGACCCACCAGAGCCTGGAC
TTTC-3) and digested with BamH1/XbaI and ligated into
pTRE2 previously digested with the same restriction
enzymes. From the annealed oligonucleotides inserted into
DraIII/PshAI sites in the miR-187 expression region, thetranscript was encoded in the following order: (i) the5-flanking region of miR-187, (ii) a 21-nt siRNA guidestrand, (iii) a human miR-30 loop, (iv) a 19-nt passenger
strand of the identical siRNA lacking a 3 overhang, and (v)a 3-flanking region of miR-187. The inserted oligonucleotide sequences were as follows:
pENTR.CMV-VIM269, 5-GTGACCCTCCAGCCAUCAACACCGAGUUCAAGTGTGAAGCCACAGATGGCTTGAACTCGGTGTTGATGGCTTGAGGGACGC-3 and2. EXPERIMENTAL PROCEDURE
2.1. Cell culture
Human HeLa cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Gibco-BRL) at 37°C. Mediawas supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Mitsubishi Kagaku) and antibiotics (10units/ml penicillin [Meiji] and 50 g/ml streptomycin[Meiji]).
2.2. Preparation of siRNA and construction of theshRNA expression plasmid
RNA oligonucleotides were purchased from Proligo(Sigma-Aldrich) and siRNA was prepared as described
previously (Fig. 1A) [8]. The siRNA sequences were as
follows:
siVIM269, 5-GCCAUCAACACCGAGUUCAAG-3 and5-UGAACUCGGUGUUGAUGGCGU-3;siVIM270, 5-CCAUCAACACCGAGUUCAAGA-3 and5-UUGAACUCGGUGUUGAUGGCG-3;and siVIM812, 5-GUACGUCAGCAAUAUGAAAGU-3
and 5-UUUCAUAUUGCUGACGUACGU-3.
The pol III-driven shRNA expression vectors wereconstructed by inserting annealed synthetic oligonucleotides consisting of two strands composed of single-stranded

A

B

C

siRNA:

siRNA:

Passenger strand

siRNA:
Guide strand

shRNA:shRNA:UU

Dicer
G or A
↓

Dicer
TTTTT
7mGppp

H1

DroshaAAAAA

5´ LTRPuro

pSUPER.H1
3´ LTR

CMV

β-globin poly A

attL1
pUC ori

Amp

attL2

T1
T2

pENTR.CMV

pUC ori

Kan

Fig. 1 The structures of siRNA and shRNA expression
constructs. (A) siRNA. Blue line: passenger strand. Red line:
guide strand. (B) pSUPER.H1 vector. A primary transcript driven
by the H1 promoter preferentially starts at G or A and terminates
at the poly(T) tract. shRNA is cleaved by Dicer (brown triangle)
to generate mature siRNA. (C) pENTR.CMV vector. The
5'-capped and polyadenylated transcript is driven by the CMV
promoter. The transcript is first cleaved by Drosha (green
triangle) and subsequently by Dicer (brown triangle) to generatemature siRNA.

2- 292 -

5-GCGTCCCTCAAGCCAUCAACACCGAGUUCAAGC
CATCTGTGGCTTCACACTTGAACTCGGTGTTGATG
GCTGGAGGGTCACACT-3;and pENTR.CMV-VIM812, 5-GTGACCCTCCAGUACGUCAGCAAUAUGAAAGUTGTGAAGCCACAGATGGACTTTCATATTGCTGACGTACTTGAGGGACGC-3 and
5-GCGTCCCTCAAGUACGUCAGCAAUAUGAAAGU
CCATCTGTGGCTTCACAACTTTCATATTGCTGACG
TACTGGAGGGTCACACT-3.

A

0.2

0.4

0.6

0.8

1052
3476
4386
3867
2858
4274
3332
3336
996
933
1084
1276
906
4140
5261
4516

0.0

Cumulative fraction

1.0

6-mer

siVIM2702.3. Microarray analysis
HeLa cells (1  105 cells/ml) were inoculated 24 h prior totransfection into individual wells of a culture plate. Cells
were transfected with 50 nM siRNA, 500 ng pSUPER.H1,
or 500 ng pENTR.CMV. Total RNA (3 g) was purified
using the RNeasy Kit (Qiagen) 24 h after transfection, andhybridized to the Human Genome U133 Plus 2.0 GeneChip(Affymetrix) containing about 47,400 human transcripts
according to the manufacturer’s protocol. RNA frommock-transfected cells treated with transfection reagent in
the absence of siRNA were used as the siRNA transfection
control. The plasmids pSUPER.H1 and pENTR.CMV, bothwithout the shRNA transcription unit, were used as shRNA
controls. The transcript expression value was calculatedusing Microarray Suite 5.0 (MAS5) [39] with quantile
normalization [40]. Transcripts with hybridization signals
strong enough to be called present (P) were used in thisstudy. To identify downregulated transcripts on the array,the cumulative distribution and mean fold changes of eachtranscript groups with the site versus those with nocanonical site were compared.

−2.0

−1.5

−1.0

5′ C C A U C A A C A C C G A G U U C A A G A 3′
: : : : : : : : : : : : : : : : : : :3′ G C G G U A G U U G U G G C U C A A G U U 5′765432
5′ C G C C A U C A A C A C C G A G U U C A A G A 3′

−0.5

0.0

0.5

1.0

0.05

0.10

0.5

1.0

0.05

0.10

0.5

1.0

0.05

0.10

Log2 Fold Change

mRNA

−0.20

−0.15

B

−0.10

−0.05

0.00

Mean Log2 Fold Change

0.2

0.4

0.6

0.8

295
1176
1053
790
1099
993
899
274
217
404
276
244
192
1734
1890

0.0

Cumulative fraction

1.0

7-mer

siVIM270

−2.0

−1.5

−1.0

5′ C C A U C A A C A C C G A G U U C A A G A 3′
: : : : : : : : : : : : : : : : : : :3′ G C G G U A G U U G U G G C U C A A G U U 5′8765432
5′ C G C C A U C A A C A C C G A G U U C A A G A 3′

−0.5

0.0

Log2 Fold ChangemRNA

2.4. Motif analysis of 3 UTR and CDS
The probe sequences, which were taken from theannotation table provided on the Affymetrix website(http://www.affymetrix.com/), were mapped to the RefSeq
human mRNA sequences (release 24) to identify the target
transcripts. We found 67,220 annotations for humantranscripts that corresponded to 54,675 of the probe sets. In
microarray analyses using siVIM269, siVIM270, andsiVIM812, the number of transcripts identified as P by

−0.20

−0.15

C

−0.05

0.00

0.4

0.6

0.8

74
190
269
292
179
207
36
83
90
99
48
74
55
695

0.0

0.2

Cumulative fraction

1.0

8-mer

Fig. 2 Microarray transcript profiles following transfection ofsiVIM270. Transcripts possessing the complementarity to a
6-mer (A), 7-mer (B), and 8-mer (C) guide strand sequences of
siVIM270 were divided into 16, 15, and 14 groups,
respectively, based on their positions in the siRNA. (upper
panel) Change in gene expression is shown by log2-foldchange ratio to mock transfection. The cumulative distributionof transcript groups is colored as shown in each figure. The
number of transcripts belonging to each group is presented at
the side of each colored line. (lower panel) Blue character:passenger strand sequence. Red character: guide strand
sequence. Black character: target mRNA sequence. Histograms of mean fold changes (log2) of each transcript groups
are represented on the right.

−0.10

Mean Log2 Fold Change

siVIM270

−2.0

−1.5

5′ C C A U C A A C A C C G A G U U C A A G A 3′
: : : : : : : : : : : : : : : : : : :3′ G C G G U A G U U G U G G C U C A A G U U 5′98765432
5′ C G C C A U C A A C A C C G A G U U C A A G A 3′

−1.0

−0.5

0.0

Log2 Fold ChangemRNA−0.248

−0.20

−0.15

−0.10

−0.05

0.00

Mean Log2 Fold Change

3- 293 -

MAS5 was estimated at 19,856. Of these, 16,783, which
represented RefSeq entries (25%) with the 3 UTR, wereconsidered here. In the experiments using pSUPER.H1VIM269 and pSUPER.H1-VIM812, 17,858 of 21,543
transcripts defined as P were analyzed. In the experiments
using pENTR.CMV-VIM269 and pENTR.CMV-VIM812,
17,435 of 20,957 transcripts were analyzed. Seven nucleotide sequences matching the seed region (nucleotides 2–8)
of both strands of siRNA were assigned as extracted 3
UTR sequences.

sequences inserted into two types of shRNA expression
vectors, pol III-driven pSUPER.H1 and pol II-driven
pENTR.CMV, respectively. The shRNA expression
plasmids were transfected into HeLa cells at 0.5 g/welland analyzed after 24 h. Both pSUPER.H1-VIM269 andpENTR.CMV-VIM269 plasmids induced high levels ofoff-target effects (Fig. 3B,C), and decreased the expression
level of vimentin to 46.0 and 32.7 %. However, trace
off-target effects were observed with pSUPER.H1-VIM812
and pENTR.CMV-VIM812 (Fig. 3E,F), although vimentin
expression was clearly reduced to 54.0 and 47.7 % by these
constructs, respectively. These results indicate thatseed-dependent off-target effects are induced in a similarmanner as that induced by siRNA; the degree of off-target
effect is primarily governed by the thermodynamic stability
or Tm value of the seed-target duplex.

3. RESULTS AND DISCUSSION
3.1 Thermodynamic stability of the seed-target duplex
determines shRNA off-target effects
To confirm the notion that off-target gene silencing inducedby shRNA is also determined primarily by seed-target
duplex stability as it is by siRNA, genome-wide expression
profiling was performed using three siRNAs and fourshRNA expression plasmids, all of which target the humanvimentin gene. Two siRNAs, siVIM269 and siVIM270,
which have seed sequences with Tm values in 100 mM
NaCl of 27.4°C and 26.5°C, respectively, were predicted as
good off-target inducers. The seed duplex generated by
siVIM812 had a Tm value of 8.8°C and was considered apoor off-target inducer. HeLa cells were transfected witheach of 50 nM siRNA and their corresponding shRNA
expression constructs, and the changes in the expression
level of transcripts were analyzed after 24 h. siVIM269 andsiVIM270 decreased the expression level of target vimentin
gene to 26.8 and 15.8 %, respectively, and siVIM812
decreased vimentin expression to 15.2 %. The expression
levels of the transcripts with seed complementary
sequences (positions 2–8 of the siRNA guide strand) in 3
UTRs were most significantly reduced by siVIM269 (Fig.
3A) and siVIM270 (Fig. 2A). To evaluate the off-target
effects of every 6-, 7-, and 8-mer sequence in the siRNA
guide strand, we analyzed differences in transcriptional
expression levels with complementarities with siVIM270
sequence (Fig. 2). Although the transcripts with 6-mercomplementarities at positions 2–7 and 3–8 were similarly
reduced (Fig. 2A), those with 7-mer complementarities at
positions 2–8 were prominently reduced (Fig. 2B). Highly
reduced transcripts contained complementarities at
positions 2–9 in 8-mer complementary transcripts whereas
the neighboring 8-mers also reduced considerably (Fig. 2C).
These results confirm that seed-dependent off-target effects
are highly correlated with the 7-mer sequence positioned at
2–8 in the seed region. The transfection with siVIM812
exhibited no or little off-target effect (Fig. 3D). To evaluate
the off-target effects induced by shRNA expression
plasmids, we used two siRNA (siVIM269 and siVIM812)

3.2 Shift in cleavage site by Dicer and Drosha
The primary transcript of pol III-driven shRNA is cleaved
by Dicer to generate an siRNA duplex (Fig. 1B), and pol
II-driven shRNA is processed by Drosha before Dicer
cleavage (Fig. 1C). We investigated the displacement of
the cleavage site by Dicer or Drosha. Dicer cleaves the site
close to the loop as shown in Figures 1B and C. Because
both of our pol III- and pol II-driven shRNA expression
plasmids had only 1 nt of clearance to the root of the loop
region from the 3 end of the passenger strand, the cleaved
site could be shifted only up to 1 nt. siVIM269 significantly
reduced the expression of transcripts with seed
complementarities between the siRNA sequence at
positions 2–8 from the 5 end of the guide strand and 3
UTRs (Fig. 3A). However, the transcripts withcomplementarities in the 7-mer at positions 1–7 were
mostly reduced by the transfection of pSUPER.H1VIM269 and pENTR.CMV-VIM269 (Fig. 3B,C). This
indicates that, as anticipated, the processed site by Dicer
was shifted 1 nt towards the loop. Drosha cleaves out theflanking region of the primary transcript of shRNA driven
by the pol II promoter (Fig. 1C). The passenger strand of
siVIM812 showed a seed-dependent off-target effect (Fig.
3G). The transcripts with complementarity in positions 2–8
were also significantly reduced by the addition ofsiVIM812. The passenger strand of shVIM812 derived
from pENTR.CMV-VIM812 decreased transcripts withcomplementarities in positions 3–9 as well as 2–8 (Fig. 3I),
indicating that a part of the sites processed by Drosha in
shVIM812 was shifted 1 nt away from the flanking region.
These results clearly demonstrate that the sites processedby Dicer and Drosha fluctuate, although the shift incleavage site may differ according to the inserted shRNA
sequence.

4- 294 -

D

siVIM269

5′ G C C A U C A A C A C C G A G U U C A A G 3′ Passenger strand: : : : : : : : : : : : : : : : : : :3′ U G C G G U A G U U G U G G C U C A A G U 5′
Guide strand
8765432
5′ A C G C C A U C A A C A C C G A G U U C A A G 3′

G

siVIM812

5′ G U A C G U C A G C A A U A U G A A A G U 3′ Passenger strand: : : : : : : : : : : : : : : : : : :3′ U G C A U G C A G U C G U U A U A C U U U 5′
Guide strand
8765432
5′ A C G U A C G U C A G C A A U A U G A A A G U 3′

mRNA

mRNA
297
313
1225
1078
782
1104
1007
952
290
236
447
289
262
211
1785

mRNA
2325
2787
3221
2921
2617
1727
1730
2375
2300
2382
1439
255
352
252
124

25
65
137
202
290
312
1436
1732
1555
1817
1540
1335
2330
2768
3225

−0.10

−0.05

0.00

0.05

0.10

−0.15

−0.10

Mean Log2 Fold Change

E

pSUPER.H1-VIM269U
CU C
5′ G C C A U C A A C A C C G A G U U C A A G G
C: : : : : : : : : : : : : : : : : : : : : :U
3′ U G C G G U A G U U G U G G C U C A A G U U C C A
CUG
8765432
5′ A C G C C A U C A A C A C C G A G U U C A A G 3′

−0.15

−0.05

0.00

0.05

0.10

H

−0.15

..

UGAA
pENTR.CMV-VIM269
GGCAC
GCCAUCAACACCGAGUUCAAGU: : : : : : : : : : : : : : : : : : : : : :C
3′ A G U G C G G U A G U U G U G G C U C A A G U U C G
AGG8765432..
CU
5′ A C G C C A U C A A C A C C G A G U U C A A G 3′ A G A

mRNA

.

−0.10

−0.05

0.00

0.05

0.10

−0.15

F

C
A
C
C
G

C

−0.05

0.00

0.05

0.10

AG AU

−0.15

−0.10

−0.05

0.00

0.05

0.10

I

pENTR.CMV-VIM812

GG 3′
GACUUUCAUAUUGCUGACGUACGU G A: : : : : : : : : : : : : : : : : : : : : :UUGAAAGUAUAACGACUGCAUG C
AGC
8765432AAGU
5′ACUUUCAUAUUGCUGACGUACGU
5′

mRNA

3′

2420
2907
3369
3046
2749
1820
1817
2491
2444
2486
1525
279
376
259
134

29
74
142
209
311
326
1492
1836
1626
1882
1647
1415
2442
2905
3373

−0.10

−0.15

Mean Log2 Fold Change

mRNA
306
319
1243
1106
814
1147
1042
975
294
228
435
288
274
220
1841

0.10

2500
2945
3448
3121
2815
1859
1849
2536
2489
2566
1566
276
387
266
135

UGAA
pENTR.CMV-VIM812
GGCAC
GUACGUCAGCAAUAUGAAAGUU: : : : : : : : : : : : : : : : : : : : : :C
3′ A G U G C A U G C A G U C G U U A U A C U U U C A GAGG8765432..
CU
5′ A C G U A C G U C A G C A A U A U G A A A G U 3′ A G A
5′

0.05

pSUPER.H1-VIM812

Mean Log2 Fold Change

C

0.00

mRNA

Mean Log2 Fold Change

C

−0.05

GU CA
C A C U U U C A U A U U G C U G A C G U A C G U 3′
U
: : : : : : : : : : : : : : : : : : : : : :C
G U G A A A G U A U A A C G A C U G C A U G 5′
C UUC8765432
5′ A C U U U C A U A U U G C U G A C G U A C G U 3′

28
83
151
217
327
355
1521
1867
1641
1925
1662
1454
2499
2963
3454

−0.10

−0.10

Mean Log2 Fold Change

mRNA

−0.15

.

0.10

pSUPER.H1-VIM812

308
331
1264
1144
833
1171
1062
991
306
242
451
296
290
227
1880

..

0.05

U
CU C
5′ G U A C G U C A G C A A U A U G A A A G U GC: : : : : : : : : : : : : : : : : : : : : :U
3′ U G C A U G C A G U C G U U A U A C U U U C A C A
CUG
8765432
5′ A C G U A C G U C A G C A A U A U G A A A G U 3′

mRNA

5′

0.00

.

B

−0.05

Mean Log2 Fold Change

..

−0.15

siVIM812

5′ U U U C A U A U U G C U G A C G U A C G U 3′ Guide strand: : : : : : : : : : : : : : : : : : :3′ U G A A A G U A U A A C G A C U G C A U G 5′
Passenger strand
8765432
5′ A C U U U C A U A U U G C U G A C G U A C G U 3′

..

A

−0.10

Mean Log2 Fold Change

−0.05

0.00

0.05

Mean Log2 Fold Change

0.10

−0.15

−0.10

−0.05

0.00

0.05

0.10

Mean Log2 Fold Change

Fig. 3 Seed-dependent off-target effects after transfection of siRNA and shRNA. Changes in the expression levels of the transcripts
possessing the 7 nt complementarity with the siRNA guide strand are shown as mean log2-fold change. siRNA and shRNA structuresare shown in the upper left. Blue character: passenger strand sequence. Red character: guide strand sequence. Black character: target
mRNA sequence. The number of transcripts belonging to each group is presented at the side of each colored line. Histograms of themean fold changes (log2) of each transcript group are represented on the right. The off-target effect of the passenger strand is also
analyzed in G–I.

Furthermore, no or little off-target effect was observedin the transcripts with seed complementarity with thepassenger strand of shVIM812 derived from pSUPER.H1VIM812 (Fig. 3H). The 5 end of the primary transcriptfrom pSUPER.H1-VIM812 corresponded to the transcription start site and the 3 end corresponded to the terminationsite. Therefore, the results suggest that the start and/or
termination site are not rigid and ambiguous.

guide strand and the corresponding sites in the passenger
strand (Fig. 4D). In the pol II system, the cleavage sites ofboth Drosha and Dicer were often shifted 1 nt (Fig. 4E).
Furthermore, shRNA as well as siRNA (Fig. 4F) with low
Tm (< 21.5°C) at seed regions are preferred to reduce
off-target effect.
Considering the sequence rules for the design offunctional siRNA, the thermodynamic stability of theseed-target duplex, and the shift in Dicer and Drosha
cleavage sites, a functional shRNA with reduced off-target
effects should satisfy the following conditions: the presenceof A/U residues at nucleotide positions 1 and -1 from the 5
end of the guide strand, three to six A/Us in nucleotide
positions 2–7, G/C at positions 19 and 18, and no stretch ofmore than nine GC base pairs. In addition, the Tm values of
7 nt at positions 2–8 of the guide strand and 11–17 of thepassenger strand should be low (Fig. 4G,H). Furthermore,particularly in the pSUPER.H1 system, a G residue shouldbe the first nucleotide because the RNA pol III promoter
prefers to initiate transcription with a purine. Stretches of 4 T’s or A’s in the siRNA sequence should also be
avoided due to the addition of a poly(T) tract as atermination signal for RNA pol III (Fig. 4G).

3.3 Design of shRNA expression system with reduced
off-target effects
We reported previously that highly functional siRNA in
mammalian RNAi simultaneously satisfies the following
four sequence conditions (Fig. 4A): A or U residues at
position 1 from the 5 end of the guide strand, three to six
A/Us in positions 2–7, G/C at position 19; and no GC
stretch of more than nine nucleotides present. Therefore,
oligonucleotides inserted into shRNA expression vectors totranscribe shRNAs must at least satisfy these sequence
conditions (Fig. 4B and C).
In this study, we found that the transcription start site
might not be strictly defined, and that the Dicer cleavage
site could be shifted at the position between -1 and -2 of the5- 295 -

A

B

Passenger strand

G/C

C

G/C

A/U

5′ C C A U C A A C A C C G A G U U C A A G A 3′
: : : : : : : : : : : : : : : : : : :3′ G C G G U A G U U G U G G C U C A A G U U 5′

A/U

U
CU C
5′ C C A U C A A C A C C G A G U U C A A G A GC: : : : : : : : : : : : : : : : : : : : : :U
3′ U U G G U A G U U G U G G C U C A A G U U C U C A
CUG

Guide strand AU richTranscription startU
CU C
5′ C C A U C A A C A C C G A G U U C A A G A GC: : : : : : : : : : : : : : : : : : : : : :U
3′ U U G G U A G U U G U G G C U C A A G U U C U C A
CUGTranscription termination

F

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

GA/U

5′ C C A U C A A C A C C G A G U U C A A G A 3′
: : : : : : : : : : : : : : : : : : :3′ G C G G U A G U U G U G G C U C A A G U U 5′

AU richno long consecutive G/Cs

Drosha cleavage site
(pENTR.CMV-VIM812)

Dicer cleavage site
(pENTR.CMV-VIM269). . 5′
.
C

UGAA
G
G
CA
C
CCAUCAACACCGAGUUCAAGAU: : : : : : : : : : : : : : : : : : : : : :C
G U UGGU AGU UGUGGC U C A AGU U C UG
A
A
G
G
..
U A G AC3′nucleotide postion
A/U

U
CU C
5′ C C A U C A A C A C C G A G U U C A A G A GC: : : : : : : : : : : : : : : : : : : : : :U
3′ U U G G U A G U U G U G G C U C A A G U U C U C A
CUG

U

UA GA

G
C
C
A
C

H

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

no long consecutive G/Cs

A/U

UGAA

E
Dicer cleavage site
(pSUPER.H1-VIM269)

AU richG/CGCACCAUCAACACCGAGUUCAAGAU: : : : : : : : : : : : : : : : : : : : : :G U UGGU AGU UGUGGC U C A AGU U C UG
Gno long consecutive G/Cs

D

low TmGA..
3′

AU richno long consecutive G/Cs

G/C

. . 5′.
C

low Tm

AU richno long consecutive G/Cs
no AAAA or UUUU

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

G
..
3′

G/C

A/U

G
CA
CCAUCAACACCGAGUUCAAGAU: : : : : : : : : : : : : : : : : : : : : :A G U UGGU AGU UGUGGC U C A AGU U C UGG

low Tm

AU richno long consecutive G/Cs

UGAA

UA GA

G
C
C
A
C

Fig. 4 The functional and reduced off-target effectof siRNA and shRNA sequences. Functional
siRNA (A) and shRNAs derived from pSUPER.H1(B) and pENTR.CMV (C). The potential cleavage
sites by Drosha and/or Dicer in shRNAs derived
from pSUPER.H1 (D) and pENTR.CMV (E). The
functional and reduced off-target effect of siRNA(F), shRNAs derived from pSUPER.H1 (G), andpENTR.CMV (H). Blue character: passenger
strand sequence. Red character: guide strand
sequence. Green triangles indicate Drosha cleavage
sites (small triangle: original cleavage site; large
triangle: shifted cleavage site). Brown triangles
indicate Dicer cleavage sites (small triangle:
original cleavage site; large triangle: shiftedcleavage site). Blue triangles indicate transcription
start or termination sites. Yellow box indicates theregion with low thermodynamic stability (Tm).
Nucleotide positions are shown in the upper
regions in F, G, and H. Position 1 indicates the 5
end of the guide strand.

[5] Schwarz,D.S., Hutvagner,G., Du,T., Xu,A., Aronin,N. andZamore,P.D. (2003) Asymmetry in the assembly of the RNAi
enzyme complex. Cell, 115, 199-208.
[6] Matranga,C., Tomari,Y., Shin,C., Bartel,D.P. and Zamore, P.D.
(2005) Passenger-strand cleavage facilitates assembly of siRNA
into Ago2-containing RNAi enzyme complexes. Cell, 123,
607-620.
[7] Rand,T.A., Petersen,S., Du,F. and Wang,Z. (2005) Argonaute2
cleaves the anti-guide strand of siRNA during RISC activation.
Cell, 123, 621-629.
[8]
Ui-Tei,K.,
Naito,Y.,
Takahashi,F.,
Haraguchi,T.,
Ohki-Hamazaki,H., Juni, A., Ueda,R. and Saigo,K. (2004)
Guidelines for the selection of highly effective siRNA sequences
for mammalian and chick RNA interference. Nucleic Acids Res.,32, 936-948.
[9] Elbashir,S.M., Lendeckel,W. and Tuschl, T. (2001) Functional
anatomy of siRNAs for mediating efficient RNAi in Drosophilamelanogaster embryo lysate. Genes Dev., 15, 188-200.
[10]
Liu,J.,
Carmell,M.A.,
Rivas,F.V.,
Marsden,C.G.,
Thomson,J.M.,, Song,J.J., Hammond,S.M., Joshua-Tor,L. and
Hannon,G.J. (2004) Argonaute2 is the catalytic engine of
mammalian RNAi. Science, 305, 1437-1441.
[11] Meister,G., Landthaler,M., Patkaniowska,A., Dorsett,Y.,
Teng,G. and Tuschul, T. (2004) Human argonaute2 mediates
RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell, 15,
185-197.
[12] Doi,N., Zennno,S., Ueda,R., Ohki-Hamazaki,H., Ui-Tei,K.
and Saigo, K. (2003) Short-interfering-RNA-mediated genesilencing in mammalian cells requires Dicer and eIF2C
translational initiation factors. Curr. Biol., 13, 41-46.
[13] Rivas,F.V., Tolia,N.H., Song,J.J., Aragon,J.P., Liu,J.,
Hannon,G.J. and Joshua-Tor, L. (2005) Purified argonaute2 andan siRNA form recombinant human RISC. Nat Struct. Mol. Biol.,12, 340-349.
[14] Song,J-J., Smith,S.K., Hannon,G.J. and Joshua-Tor,L. (2004)
Crystal structure of argonaute and its implications for RISC slicer
activity. Science, 304, 1434-1437.

4. CONCLUSION
In summary, we found that shRNA induces off-target
effects depending on the thermodynamic stability or Tm of
the seed-target duplex, in a similar manner as does siRNA.
However, the processing of shRNA by Drosha or Dicer was
not rigid but rather fluctuated slightly. Based on theseresults, a functional shRNA with reduced off-target effects
should satisfy the following conditions: the presence of
A/U residues at nucleotide positions 1 and -1 from the 5
end of the guide strand, three to six A/Us in nucleotide
positions 2–7, G/C at positions 19 and 18, and no GC
stretch of more than nine nucleotides. In addition, the Tm
values of the seed region in both strands should be low.
ACKNOWLEGEMENTS
We are grateful to T. Haraguchi for providing pSH-CMV
plasmid. This work was supported in part by grants from
the Ministry of Education, Culture, Sports, Science andTechnology of Japan to K.U-T.
REFERENCES[1] McManus,M.T. and Sharp,P.A. (2002) Gene silencing in
mammals by small interfering RNAs. Nat. Rev. Genet., 3,
737-747.
[2] Dykxhoorn,D.M. and Lieberman,J. (2006) Knocking down
disease with siRNAs. Cell, 126, 231-235.
[3] Hammond,S.M., Bernstein,E., Beach,D. and Hannon,G.J.
(2000) An RNA-directed nuclease mediates post-transcriptional
gene silencing in Drosophila cells. Nature, 404, 293-296.
[4] Khvorova,A., Reynolds,A. and Jayasena,S.D. (2003)
Functional siRNAs and miRNAs exhibit strand bias. Cell, 115,
209-216.

6- 296 -

[15] Brummeikamp,T.R., Bernards,R. and Agami,R. (2002) A
system for stable expression of short interfering RNAs in
mammalian cells. Science, 296, 550-553.
[16] Paddison,P.J., Caudy,A.A., Bernstein,E., Hannon,G.J. andConklin,D.S. (2002) Short hairpin RNAs (shRNAs) induce
sequence-specific silencing in mammalian cells. Genes Dev., 16,
948-958.
[17] Xia,H., Mao,Q., Pauison,H.L. and Davidson,B.L. (2002)
siRNA-mediated gene silencing in vitro and in vivo. Nat.
Biotechnol., 20, 1006-1010.
[18] Zeng,Y., Wagner,E.J. and Cullen,B.R. (2005) Both naturaland designed microRNAs can inhibit the expression of cognate
mRNAs when expressed in human cells. Mol. Cell, 9, 1327-1333.
[19] Zeng,Y., Cai,X. and Cullen,B.R. (2005) Used of RNApolymerase II to transcribe artificial microRNAs. Methods
Enzymol., 392, 371-380.
[20] Lee,Y., Kim,M., Han,J., Yeom,K.H., Lee,S., Baek,S.H. and
Kim,V.N. (2004) MicroRNA genes are transcribed by RNA
polymerase II. EMBO J., 23, 4051-4060.
[21] Cai,X., Hagedorn,C.H. and Cullen,B.R. (2004) Human
microRNAs are processed from capped, polyadenylatedtranscripts that can also function as mRNAs. RNA, 10, 1957-1966.
[22] Gregory,R.K., Yan,K.P., Amuthan,G., Chendrimada,T.,
Doratotaj,B., Coock,N. and Shiekhattar,R. (2004) The
microprocessor complex mediates the genesis of microRNAs.
Nature, 432, 235-240.
[23] Han,J., Lee,Y., Yeom,K.H., Kim,Y.K., Jin,H. and Kim,V.N.
(2004) The Drosha-DGCR8 complex in primary microRNA
processing. Genes Dev., 18, 3016-3027.
[24] Han,J., Lee,Y., Yeom,K-H., Nam,J-W., Heo,I., Rhee,J-K.,
Sohn,S.Y., Cho,Y., Zhang,B-T. and Kim,V.N. (2006) Molecularbasis for the recognition of primary microRNAs by the
Drosha-DGCR8 complex. Cell, 125, 887-901.
[25] Yi,R., Qin,Y., Macara,I.G. and Cullen,B.R. (2003)
Exportin-5 mediates the nuclear export of pre-microRNAs andshort hairpin RNAs. Genes Dev., 17, 3011-3016.
[26] Lund,E., Guttinger,S., Calado,A., Dahlberg,J.E. and Kutay,U.
(2004) Nuclear export of microRNA precursors. Science, 303,
95-98.
[27] Grishok,A., Paquinelli,A.E., Conte,D., Li,N., Parrish,S., Ha,I.,
Baillie,D.L., Fire,A., Ruvkun,G. and Mello,C.C. (2001) Genes
and mechanisms related to RNA interference regulate expressionof the small temporal RNAs that control C.elegans developmental
timing. Cell, 106, 23-34.
[28] Hutvagner,G., McLachlan,J., Pasquinelli,A.E., Balint,E.,
Tuschl,T.a nd Zamore, P.K. (2001) A cellular function for the
RNA-interference enzyme Dicer in the maturation of the let-7
small temporal RNA. Science, 293, 834-838.
[29] Ketting,R.F., Fischer,S.E., Bernstein,E., Sijen,T., Hannon,G.J.
and Plasterk,R.H. (2001) Dicer functions in RNA interference andin synthesis of small RNA involved in developmental timing in C.
elegans. Genes Dev., 15, 2654-2659.
[30] Jackson,A.L., Bartz,S.R., Schelter,J., Kobayashi,S.V.,
Burchard,J., Mao,M., Li,B., Cavet,G. and Linsley,P.S. (2003)

Expression profiling reveals off-target gene regulation by RNAi.
Nat. Biotech., 21, 635-637.
[31] Scacheri,
P.C., Rozenblatt-Rosen,O., Caplen,N.J.,
Wolfsberg,T.G.,
Umayam,L.,
Lee,J.C.,
Hughes,C.V.,
Shanmugam,K.S., Bhattacharjee,A., Meyerson,M. and Collins,F.S.
(2004) Short interfering RNAs can induce unexpected anddivergent changes in the levels of untargeted proteins inmammalian cells. Proc. Natl. Acad. Sci., USA., 101, 1892-1897.
[32] Lin,X.,
Ruan,X.,
Anderson,M.G,
McDowell,J.A.,
Kroeger,P.E., Resik,S.W. and Shen,Y. (2005) siRNA-mediatedoff-target gene silencing triggered by a 7 nt complementation.
Nucleic Acids Res., 33, 4527-4525.
[33] Jackson,A.L., Burchard,J., Schelter,J., Chau,B.N., Cleary,M.,
Lim,L. and Linsley,P.S. (2006) Widespread siRNA “off-target”
transcript silencing mediated by seed region sequence
complementarity. RNA, 12, 1179-1187.
[34]
Birmingham,A.,
Anderson,E.M.,
Reynolds,A.,
Ilsley-Tyree,D.,
Leake,D.,
Fedorov,Y.,
Baskerville,S.,
Maksimova,E., Robinson,K., Karpilow,J., Marshall,W.S. andKhvorova,A. (2006) 3 UTR seed matches, but not overall identity,
are associated with RNAi off-targets. Nat. Methods, 3, 199-204.
[35] Ui-Tei,K., Naito,Y., Nishi,K., Juni,A. and Saigo,K. (2008)
Thermodynamic stability and Watson-Crick base pairing in theseed duplex are major determinants of the efficiency of thesiRNA-based off-target effect. Nucleic Acids Res., 36, 7100-7109.
[36] Grimson, A., Farh,K.K., Johnston,W.K., Garrett-Engele,P.,
Lim,L.P. and Bartel,D.P. (2007) MicroRNA targeting specificity
in mammals: determinants beyond seed pairing. Mol. Cell, 27,
91-105.
[37] Lim, L.P., Lau,N.C., Garrett-Engele,P., Grimson,A.,
Schelter,J.M., Castle,J., Bartel,D.P., Linsley,P.S. and Johnson,J.M.
(2005) Microarray analysis shows that some microRNAs
downregulate large numbers of target mRNAs. Nature, 433,
769-773.
[38] Lewis,B.P., Burge,C.B. and Bartel,D.P. (2005) Conserved
seed pairing, often flanked by adenosines, indicates that thousandsof human genes are microRNA targets. Cell, 120, 15-20.
[39] Hubbell,E., Liu,W.M. and Mei,R. (2002) Robust estimatorsfor expression analysis. Bioinformatics, 18, 1585-1592.
[40] Bolstad,B.M., Irizarry,R.A., Gautier,L. and Wu,Z. (2005)
Preprocessing
high-density
oligonucleotide
arrays.
In
Gentleman,R., Carey,V.J., Huber,W., Irizarry, R.A. and Dudoit,S.
(eds.), Bioinformatics and Computational Biology Solutions Using
R and Bioconductor. Springer, New York, USA, pp. 13-32.
[41] Panjkovich, A. and Melo, F. (2005) Comparison of different
melting temperature calculation methods for short DNA
sequences. Bioinformatics, 21, 711-722.
[42] Freier, A. M., Keirzek, R., Jaeger, J. A., Sugimoto, N.,
Caruthers, M. H., Neilson, T. and Turner, D. H. (1986) Improved
free-energy parameters for predictions of RNA duplex stability.
Proc. Natl. Acad. Sci. USA, 83, 9373-9377.
[43] Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A.,
Nakamuta, H., Ohmichi, T., Yoneyama, M. and Sasaki, M. (1995)
Thermodynamic parameters to predict stability of RNA/DNA
hybrid duplexes. Biochemistry, 34, 11211-11216.



7- 297 -