Nucleic Acids Research, 2005, Vol. 33, Web Server issue W589–W591
doi:10.1093/nar/gki419

dsCheck: highly sensitive off-target search softwarefor double-stranded RNA-mediated RNA interference
Yuki Naito1, Tomoyuki Yamada3, Takahiro Matsumiya3, Kumiko Ui-Tei1,2,
Kaoru Saigo1 and Shinichi Morishita3,*
1

Received February 14, 2005; Revised and Accepted March 21, 2005

ABSTRACT
Off-target effects are one of the most serious problems in RNA interference (RNAi). Here, we present
dsCheck (http://dsCheck.RNAi.jp/), web-based online
software for estimating off-target effects caused by
the long double-stranded RNA (dsRNA) used in
RNAi studies. In the biochemical process of RNAi,the long dsRNA is cleaved by Dicer into shortinterfering RNA (siRNA) cocktails. The software simulates this process and investigates individual 19 nt
substrings of the long dsRNA. Subsequently, the software promptly enumerates a list of potential off-target
gene candidates based on the order of off-target
effects using its novel algorithm, which significantly
improves both the efficiency and the sensitivity of
the homology search. The website not only provides
a rigorous off-target search to verify previously
designed dsRNA sequences but also presents ‘offtarget minimized’ dsRNA design, which is essential
for reliable experiments in RNAi-based functional
genomics.

INTRODUCTION
RNA interference (RNAi) is now widely used to knockdown
gene expression in a sequence-specific manner, making it apowerful tool for studying gene function (1–3). The process ofRNAi is mediated by double-stranded RNA (dsRNA) that
contains a sequence homologous to the target mRNA. Long
dsRNA introduced into the cell is cleaved by the enzyme Dicer
into short-interfering RNA (siRNA) followed by incorporation

into the RNA-induced silencing complex (RISC), which is
responsible for target mRNA degradation (4).
One of the most serious problems in RNAi is ‘off-target’silencing effects (5). Off-target silencing effects are caused bysiRNA (introduced directly into cells, or produced in vivo fromlong dsRNA) that has sequence similarities with unrelated
genes. In Caenorhabditis elegans, Drosophila or plants,
RNAi experiments are usually performed using long dsRNAs.
In these cases, there is a high risk of cross-suppression or
co-suppression between closely related genes that share ahighly conserved region.
To minimize the possibility of off-target effects, it is necessary to perform an off-target search to design dsRNA or
siRNA that has limited sequence similarities with unrelated
genes. Recently, fast and sensitive off-target search software
for siRNA design has been reported (6,7), but commonly used
siRNA design servers are not useful in performing off-target
searches for long dsRNAs. DEQOR server uses BLAST to
perform off-target searches for endoribonuclease-preparedsiRNAs (8), although BLAST frequently fails to identify
off-targets (6). Therefore, we have developed a new webbased online software system, dsCheck, to provide fast andaccurate off-target searches for long dsRNA sequences. The
software ‘dices’ the input sequence into an siRNA cocktail andperforms an exhaustive scan for each siRNA to find off-target
gene candidates, simulating the biochemical process ofdsRNA-mediated RNAi in vivo. dsCheck also provides efficient design of ‘off-target minimized’ dsRNA by avoiding
regions that share a considerable number of diced siRNAs
with a specific off-target gene, and monitoring the total number of off-target hits. The software should be especially usefulfor checking whether previously designed dsRNAs have offtarget gene candidates, as well as for designing target-specificdsRNA when off-target effects are suspected.

*To whom correspondence should be addressed. Tel: +81 47 136 3984; Fax: +81 47 136 3977; Email: moris@k.u-tokyo.ac.jp
Correspondence may also be addressed to Kumiko Ui-Tei. Tel: +81 3 5841 3044; Fax: +81 3 5841 3044; Email: ktei@biochem.s.u-tokyo.ac.jp
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authorsª The Author 2005. Published by Oxford University Press. All rights reserved.
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Department of Biophysics and Biochemistry, Graduate School of Science, 2Undergraduate Program for
Bioinformatics and Systems Biology, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan and 3Department of Computational Biology, Graduate School of Frontier Sciences,
University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan

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Nucleic Acids Research, 2005, Vol. 33, Web Server issue

METHODS

conserved POU domain shown in Figure 1C, indicating ahigh risk of cross-suppression by dsRNA targeting this region.

Off-target search strategies for long dsRNAThe key idea of the program follows the biochemical processof dsRNA-mediated RNAi shown in Figure 1A. The input
dsRNA sequence is diced into 19 nt substrings of an siRNA
cocktail, and an exhaustive off-target search is performed for
all individual siRNAs using the siDirect engine, which makes
it possible to enumerate the complete set of off-targets in areasonable amount of time (7). In dsCheck, the in silico dicing
size is set to 19, as a complete match at the 19 nt doublestranded region of an siRNA is sufficient for the target mRNAdegradation. For example, an input 500 bp dsRNA sequence isprocessed into 482 substrings each 19 nt in length, which are
subjected to the off-target search individually. In the next step,
all the hits with a complete match (i.e. 19/19 matches), one
mismatch (18/19 matches) or two mismatches (17/19 matches)
are counted individually for every off-target gene candidate
and sorted in descending lexicographic order for the output.
Figure 1B shows a typical output for a 1497 bp query
sequence of the Drosophila POU domain protein, pdm2
(NM_078834, coding region). The result shows significant
hits against pdm2 (two splicing variants: NM_078834 and
NM_165017), and two unrelated genes, nub (NM_057311)
and vvl (NM_079224). These proteins share the highly

To design off-target minimized dsRNA sequences, one
approach would be to suppose that the off-target effects arecaused by a considerable number of collaborative hits by dicedsiRNAs on the same gene, and to select a region that minimizes the maximum number of collaborative off-target hits,
which are defined as complete or partial matches of multiple
19 nt substrings against the same off-target gene. According
to this criterion, dsCheck starts by selecting a region thatminimizes the maximum number of ‘complete match’ collaborative off-target hits. If multiple regions are optimal, it also
examines the maximum number of ‘partial match’ collaborative off-target hits to select the best one. If the complete
match, collaborative hits on a sequence exceed 80% of thetotal number of diced 19 nt substrings, dsCheck regards
the sequence as the intended target gene.
Some dsRNA sequences include 19 nt substrings that may
react with a large number of off-target genes, which differsfrom the collaborative silencing effects acting on a single offtarget gene. An additional criterion is necessary to evaluate thesilencing effect of one siRNA sequence on many off-targets,

B

Target mRNA

dsRNA

dsCheck Results

Cleavage by Dicer
0 mis : Total hits with a complete match (19/19 matches).

siRNAs

1 mis : Total hits with one mismatch (18/19 matches).
2 mis : Total hits with two mismatches (17/19 matches).

off-target effects

0 mis 1 mis 2 mis Description:

Unrelated mRNAs

nub (NM_057311)

C

2500

vvl (NM_079224)

1500
2000

1000
1

Query (pdm2 ; NM_078834)

1497

1479

4

4

NM_078834.2| Drosophila melanogaster CG12287-PA (pdm2) mRNA, complete cds

1337

11

23

NM_165017.1| Drosophila melanogaster CG12287-PB (pdm2) mRNA, complete cds

128

45

53

NM_057311.4| Drosophila melanogaster CG6246-PA (nub) mRNA, complete cds

20

28

72

NM_079224.3| Drosophila melanogaster CG10037-PA (vvl) mRNA, complete cds

3

15

128

NM_168571.1| Drosophila melanogaster CG32133-PA (CG32133) mRNA, complete cds

2

7

36

NM_142597.2| Drosophila melanogaster CG12254-PA (Arc92) mRNA, complete cds

2

4

48

NM_176557.1| Drosophila melanogaster CG33106-PB (mask) mRNA, complete cds

2

4

48

NM_176556.1| Drosophila melanogaster CG33106-PA (mask) mRNA, complete cds

2

4

15

NM_132116.1| Drosophila melanogaster CG14441-PA (CG14441) mRNA, complete cds

2

4

13

NM_135957.2| Drosophila melanogaster CG31738-PB (CG31738) mRNA, complete cds

2

4

7

NM_131968.2| Drosophila melanogaster CG32772-PA (CG32772) mRNA, complete cds

2

4

5

NM_165168.1| Drosophila melanogaster CG31738-PA (CG31738) mRNA, complete cds

2

3

16

NM_133124.1| Drosophila melanogaster CG7502-PA (CG7502) mRNA, complete cds

2

3

10

NM_079524.3| Drosophila melanogaster CG1030-PA (Scr) mRNA, complete cds

2

3

10

NM_206443.1| Drosophila melanogaster CG1030-PB (Scr) mRNA, complete cds

2

3

10

NM_206442.1| Drosophila melanogaster CG1030-PC (Scr) mRNA, complete cds

2

3

7

NM_057525.2| Drosophila melanogaster CG7937-PA (C15) mRNA, complete cds

2

3

4

NM_134555.1| Drosophila melanogaster CG15454-PA (CG15454) mRNA, complete cds

2

3

2

NM_057268.3| Drosophila melanogaster CG6944-PA (Lam) mRNA, complete cds

1

18

33

NM_132246.2| Drosophila melanogaster CG10555-PA (CG10555) mRNA, complete cds

1

17

109

NM_167335.1| Drosophila melanogaster CG4013-PC (Smr) mRNA, complete cds

1

17

109

NM_167334.1| Drosophila melanogaster CG4013-PB (Smr) mRNA, complete cds

1

17

109

NM_080536.2| Drosophila melanogaster CG4013-PA (Smr) mRNA, complete cds

1

10

65

NM_078592.2| Drosophila melanogaster CG11172-PA (NFAT) mRNA, complete cds

1

7

4

NM_132281.1| Drosophila melanogaster CG12660-PA (CG12660) mRNA, complete cds

1

6

48

NM_143409.1| Drosophila melanogaster CG11873-PA (CG11873) mRNA, complete cds

1

6

45

NM_206357.1| Drosophila melanogaster CG33261-PD (Trl) mRNA, complete cds

Figure 1. (A) Biochemical process of dsRNA-mediated RNAi. Off-target effects are caused by ‘diced’ siRNAs that have sequence similarities with unrelated genes.
(B) The output for the 1497 bp query sequence of the Drosophila pdm2 gene (NM_078834, coding region). Significant hits against two off-target genes, nub(NM_057311) and vvl (NM_079224) were detected. (C) pdm, nub and vvl share a highly conserved POU domain. Each dot represents a position with 17/19 or more
matches.

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A

Designing off-target minimized dsRNA sequencesNucleic Acids Research, 2005, Vol. 33, Web Server issue

A

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matches
19/19>18/19>17/19

100

50

0

B

matches
19/19>18/19>17/19

1000
0
10
00
1
10

1

1497

Figure 2. Designing ‘off-target minimized’ dsRNA for the Drosophila pdm2 gene (NM_078834, coding region). (A) The maximum number of ‘collaborative
off-target hits’ by 82 adjacent 19 nt substrings in 100 bp dsRNAs. The arrowhead indicates the recommended region for designing an off-target minimized dsRNA of
100 bp in length. (B) Total number of off-target hits. The 19 nt substrings in the shaded area may react with a large number of off-target genes.

although the effect may not be as serious as the collaborative
silencing effect, as the concentration of single siRNA is low indiced siRNA cocktails. One reasonable measure would be thetotal number of off-target hits for each 19 nt substring ofdesigned dsRNA. To attract attention to this risk, dsCheck
displays a warning if the total number of off-target hits
exceeds a specified threshold.
Figure 2 illustrates how dsCheck designs target-specificdsRNA for the Drosophila pdm2 gene (NM_078834, coding
region). Given that the length of dsRNA is 100 bp, dsCheck
returns the positions 424–523 for the target-specific region thatsuccessfully avoids the collaborative silencing effects on the
major off-target genes nub (NM_057311) and vvl (NM_079224).
Efficacy of each diced siRNAIn mammalian RNAi, the efficacy of each siRNA varies widely
depending on its sequence; hence, several groups have reported
guidelines for the selection of siRNAs (9–12). However, inDrosophila cells, it is reported that most, if not all, siRNAsequences may act as effective silencers (9). Incorporation ofsiRNA efficacy prediction may run the risk of underestimating
off-target effects in non-mammalian RNAi. Therefore, allsiRNA sequences are treated equally in dsCheck.
Database maintenance
Currently, off-target searches can be performed against the
Drosophila, C.elegans, Arabidopsis and Oryza sativa
mRNA sequences stored in the NCBI RefSeq database (13).
Since off-target searches demand a substantial number ofmRNA sequences that are likely to cover the entire set oftranscripts, we plan to incorporate additional species whenample cDNA collections are available.
ACKNOWLEDGEMENTS
This work was supported in part by the Special Coordination
Fund for Promoting Science and Technology to K.S., theLeading Project for Biosimulation to S.M. and Grants-inAid for Scientific Research to K.U.-T., K.S. and S.M. from

the Ministry of Education, Culture, Sports, Science andTechnology of Japan. Funding to pay the Open Access publication charges for this article was provided by the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
Conflict of interest statement. None declared.
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