BMC Bioinformatics

BioMed Central

SoftwareOpen AccesssiDirect 2.0: updated software for designing functional siRNA with
reduced seed-dependent off-target effectYuki Naito*1, Jun Yoshimura2, Shinichi Morishita2 and Kumiko Ui-Tei*1
Address: 1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan and 2Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa,
Chiba 277-8562, Japan
Email: Yuki Naito* - y-naito@RNAi.jp; Jun Yoshimura - yoshimura@cb.k.u-tokyo.ac.jp; Shinichi Morishita - moris@cb.k.u-tokyo.ac.jp;
Kumiko Ui-Tei* - ktei@bi.s.u-tokyo.ac.jp* Corresponding authors

Published: 30 November 2009
BMC Bioinformatics 2009, 10:392

doi:10.1186/1471-2105-10-392

Received: 3 August 2009
Accepted: 30 November 2009This article is available from: http://www.biomedcentral.com/1471-2105/10/392© 2009 Naito et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering
RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic
applications. RNAi, requiring perfect complementarity between the siRNA guide strand and thetarget mRNA, was believed to be extremely specific. However, a recent growing body of evidence
has suggested that siRNA could down-regulate unintended genes whose transcripts possess
complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide
incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, anefficient algorithm for designing functional siRNAs with minimal off-target effect based on themechanistic features is considered of value.
Results: We present siDirect 2.0, an update of our web-based software siDirect, which provides
functional and off-target minimized siRNA design for mammalian RNAi. The previous version of
our software designed functional siRNAs by considering the relationship between siRNA sequenceand RNAi activity, and provided them along with the enumeration of potential off-target genecandidates by using a fast and sensitive homology search algorithm. In the new version, the siRNAdesign algorithm is extensively updated to eliminate off-target effects by reflecting our recent
finding that the capability of siRNA to induce off-target effect is highly correlated to thethermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which isformed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand andits target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm <21.5°C) followed by the elimination of unrelated transcripts with nearly perfect match should
minimize the off-target effects.
Conclusion: siDirect 2.0 provides functional, target-specific siRNA design with the updated
algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs
could form seed-target duplexes with Tm values below 21.5°C, and their 19-nt regions spanning
positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts,
siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq.
siDirect 2.0 is available at http://siDirect2.RNAi.jp/.

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BackgroundRNA interference (RNAi) mediated by double-stranded
RNA has become a powerful tool not only for studying
gene functions, but also for therapeutic applications [1,2].
In mammalian cells, RNAi is induced by small interfering
RNA (siRNA), a duplex of 21-nucleotide (nt) RNAs containing 2-nt 3' overhangs. The siRNAs incorporated into
cells are transferred to the RNAi effector complex called
RNA-induced silencing complex (RISC) [3,4]. RISC
assembles on one of the two strands of siRNA duplex, andis activated upon the removal of the passenger strand [59]. The activated RISC is a ribonucleoprotein complex
minimally consisting of the core protein Argonaute (Ago)and single-stranded siRNA, which acts as the guide to target complementary sequences within mRNAs [10-13].
The 5' end of the siRNA guide strand is anchored in thebinding pocket of the Mid domain of Archaeoglobus fulgidus Ago-like protein [14,15], and the 3' end is anchoredto the PAZ domain of human [16] and Drosophila [17] Agoin the RISC complex. Thus, in the siRNA guide strand, 19
nucleotides positioned at 2-20 from 5' end may be
responsible for target RNA recognition, leading to thesilencing of gene expression by cleaving target mRNA [1013]. Since RNAi is based on sequence recognition by thesiRNA, it can give rise to the silencing of other genes withsimilar sequences. This phenomenon is referred to as an
off-target effect, and the growing evidence from large-scale
knockdown experiments indicates that the off-target
silencing is induced by the base-pairing between the seedregion at positions 2-8 from the 5' end of the RISC-loaded
siRNA strand, and its complementary sequences in the 3'
UTR of the unrelated mRNAs [18-23]. Although RNAi is
now widely and routinely used as an experimental tool,
the remaining fundamental concern is whether the target
gene can be specifically silenced. Especially, accurate
knowledge of RNAi specificity is critical for therapeutictechnologies.
To avoid off-target effects, one approach may be to selectthe siRNA whose seed sequence is not complementary to
any sequences in the 3' UTR of all non-targeted genes.
However, this approach is problematic because random
7-nt sequence is predicted to appear in every 16,384 bp on
average. In fact, we analyzed the human 3' UTR databaseand it proved impossible to select such siRNAs. That is,human siRNAs with the most infrequent 7-nt seedsequence still have seed-complementarities with 17 3'
UTR sequences. Recently, we have revealed that the capability of siRNAs to induce seed-dependent off-target effectis highly correlated to the thermodynamic stability of theduplex formed between the seed region of siRNA guidestrand and its target mRNA [23]: the melting temperature(Tm) of the seed-target duplex showed strong positive correlation with the induction of seed-dependent off-target
effects. The results suggested that the Tm of 21.5°C may

http://www.biomedcentral.com/1471-2105/10/392

serve as the benchmark, which discriminates the almost
off-target-free seed sequences from the off-target-positive
ones. Thus, selecting the siRNAs with low Tm of the seedtarget duplex should minimize seed-dependent off-target
silencing.
We have previously released highly effective, target-specific siRNA design software, siDirect [24], in which siRNAsequences were selected using our guidelines establishedthrough extensive experiments to clarify the relationshipbetween siRNA sequences and RNAi activities [7]. In order
to exclude potential cross-hybridization candidates, siDirect used the rigorous homology search algorithm toselect siRNA sequences that have at least three mismatches
to any other non-targeted transcripts [25]. In the updated
software, siDirect 2.0, the siRNA design algorithm has
been extensively updated to select off-target minimized
siRNAs by considering the thermodynamic stability of theseed-target duplex. By using the default parameters, atleast one functional siRNA could be designed for >94% of
the human mRNA sequences in RefSeq release 30.

Implementation
Overall flow of siRNA selection in siDirect 2.0 is illustrated in Figure 1. All possible 23-mer subsequences, corresponding to the complementary sequence of 21-nt
guide strand and 2-nt 3' overhang of the passenger strand
within the target sequence, are generated and filtered inthree selection steps described below.
Selection of highly functional siRNAs
In the first step, highly functional siRNA sequences wereselected using our algorithm [7] (Figure 1, Step 1). We
have revealed that efficient RNAi could be induced by thesiRNAs that satisfies the following three sequence conditions simultaneously: A/U at the 5' terminus of the guidestrand; G/C at the 5' terminus of the passenger strand; atleast 4 A/U residues in the 5' terminal 7 bp of the guidestrand. In addition, G/C stretch longer than 9 bp shouldbe absent [7]. The experimental validation showed that
98% of the siRNAs predicted to be functional have
reduced the target gene expression [26]. The proportion offunctional siRNA sequences selected by this algorithm is14.7% of all human 23-mer sequences generated from
RefSeq 30 (Figure 1A, see Step 1).
Reduction of seed-dependent off-target effects
We have found that the off-target effect is highly correlated with the thermodynamic stability or Tm of the seedtarget duplex, which is formed between the nucleotides
positioned at 2-8 from the 5' end of the siRNA guidestrand and its target sequence [23]. In the second step, toavoid off-target effect, Tm for the seed-target duplex was
calculated using the nearest neighbor model and the thermodynamic parameters for the formation of RNA duplex

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All 23-mer subsequences
within the target mRNA
100%

Step 1






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Selection of functional siRNAs

Satisfies functional siRNA rule
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No 75.3%
Yes 14.7%
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Reduction of seed-dependent
off-target effects

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Tm in theseed-target duplex
21.5 ºC

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Step 3Elimination of

At least 2 mismatches against
any non-targeted mRNAs

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Functional, seed-dependent
off-target reduced, and targetspecific siRNA candidates

FigureOverall 1
flow of siRNA selection in siDirect 2.0
Overall flow of siRNA selection in siDirect 2.0. The functional and target-specific siRNAs were selected by three selection steps. In Step 1, functional siRNA sequences were selected according to our algorithm [7]. In Step 2, siRNAs with Tm values below 21.5°C in the seed-target duplex were selected. In Step 3, nucleotides positioned in the 2-20 of both strands of thesiRNAs were subjected to the near-perfect match searches, and siRNAs that have at least two mismatches to any other nontargeted transcripts were selected. The percentages denote the proportions of selected ('Yes') or unselected ('No') siRNA
candidates calculated using all 23-mer subsequences (56,375,087; 100%) generated from human mRNAs in RefSeq release 30.

as described previously [23] (Figure 1, Step 2). The formula for calculating Tm is: Tm = {(1000 × ΔH)/(A + ΔS +R ln(CT/4))} - 273.15 + 16.6 log [Na+], where ΔH (kcal/mol) is the sum of the nearest neighbor enthalpy change,
A is the helix initiation constant (-10.8), ΔS is the sum of
the nearest neighbor entropy change [27], R is the gas constant (1.987 cal/deg/mol), and CT is the total molecularconcentration of the strand (100 μM). [Na+] was fixed at
100 mM. As shown in our previous report, calculated Tmof 21.5°C may be a benchmark to discriminate almost
off-target-free seed sequences from the off-target-positive
ones [23], and thus used as the initial standard in thisstudy. Furthermore, it has been revealed that RNAi silencing is occasionally induced by the passenger strands offunctional siRNAs [23], and that the passenger strands
also take part in the seed-dependent off-target gene silencing [18,28]. Thus, siRNAs whose seed-target Tm is below
21.5°C for both guide and passenger strands were selectedin this study. In consequence, 3.0% of all human 23-mersequences remained available (Figure 1A, see Step 2). Cal-

culated Tm value for each siRNA is shown in the siDirect
2.0 output page (Figure 2A).
Elimination of near-perfect matched genes
Several studies have indicated that the effect of single-basemismatches between the siRNA guide strand and the target mRNA varies, according to the positions of the mismatch and/or the sequence of siRNA [21,29]. However, as
shown in our previous report, it is obvious that even when
the Tm value of the seed-target duplex is sufficiently low,
the target gene silencing can still take place if the non-seedregion is completely complementary [23]. Therefore, inthe third step, siRNAs that have near-perfect matches toany other non-targeted transcripts were eliminated. In
siDirect 2.0, off-target searches are performed for 19-mersequences at positions 2-20 of both strands of the siRNA
duplex (Figure 1B, Step 3), because these 19 nucleotides
are thought to be involved in target mRNA recognition.
Since widely-used BLAST tends to overlook near-perfectmatch candidates frequently, we used our fast and sensi-

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A

http://www.biomedcentral.com/1471-2105/10/392

B

Effective siRNA candidates
targetpositionfunctional
siRNA
selection:
Ui-TeiRNA oligo sequences
21nt guide (5 3 )21nt passenger (5 3 )target sequence
21nt target + 2nt overhangseed - duplex
stabilty (Tm);
guidepassenger

specificity check:minimum number ofmismatches against
any off- targets;
guide

TTGTCAACATGACCAACAAGTGTACUUGUUGGUCAUGUUGACAAGUCAACAUGACCAACAAGUGU

U

16.7 °C

20.5 °C

2 [detail] 3 [detail]

AGUUGUAGCUCAUGGAAAGAG
CUUUCCAUGAGCUACAACUUG

U

19.0 °C

20.1 °C

3 [detail] 2 [detail]152- 174 TGCTTGGATTCCTACAAAGAAGCUUCUUUGUAGGAAUCCAAGCA
CUUGGAUUCCUACAAAGAAGC

U

19.2 °C

20.1 °C

2 [detail] 3 [detail]302- 324 CCGCATTGACCATCTATGAGATGUCUCAUAGAUGGUCAAUGCGGGCAUUGACCAUCUAUGAGAUG

U

17.8 °C

20.5 °C

4 [detail] 3 [detail]326- 348 TCCAGAACATCTTTGCTATTTTCAAAUAGCAAAGAUGUUCUGGACAGAACAUCUUUGCUAUUUUC

U

19.7 °C

19.2 °C

2 [detail] 2 [detail]331- 353 AACATCTTTGCTATTTTCAGACAUCUGAAAAUAGCAAAGAUGUUCAUCUUUGCUAUUUUCAGACA

U

12.2 °C

12.0 °C

2 [detail] 2 [detail]338- 360 TTGCTATTTTCAGACAAGATTCAAAUCUUGUCUGAAAAUAGCAAGCUAUUUUCAGACAAGAUUCA

U

19.2 °C- 4.3 °C

3 [detail] 2 [detail]372- 394 CTGGAATGAGACTATTGTTGAGAUCAACAAUAGUCUCAUUCCAGGGAAUGAGACUAUUGUUGAGA

U

12.1 °C

20.4 °C

2 [detail] 3 [detail]409- 431 GTCTATCATCAGATAAACCATCTAUGGUUUAUCUGAUGAUAGAC
CUAUCAUCAGAUAAACCAUCU

U

20.0 °C

17.4 °C

3 [detail] 3 [detail]581- 603 TGGAAATCCTAAGGAACTTTTACAAAAGUUCCUUAGGAUUUCCAGAAAUCCUAAGGAACUUUUAC

U

13.3 °C

18.7 °C

2 [detail] 2 [detail]587- 609 TCCTAAGGAACTTTTACTTCATT

68- 9019 + GCTTGGATTCCTACAAAGA exon(2)||||||||||||||||||| M28622 | Hs#S1166 Human interferon beta- 1 (IFN- beta- 1) mRNA, complete cds.
GCTTGGATTCCTACAAAGA NM_002176.2 | Homo sapiens interferon, beta 1, fibroblast (IFNB1), mRNA

passenger

131- 153 CTCTTTCCATGAGCTACAACTTG

Similar Sequences

17 + GCTTGGATTCCTACAAAGA unaligned - notalign(1)
X|||||||||X|||||||| BX648613 | Hs#S16818038 Homo sapiens mRNA; cDNA DKFZp686N06224 (from clone DKFZp686N06224)

ACTTGGATTCATACAAAGAUGAAGUAAAAGUUCCUUAGGACUAAGGAACUUUUACUUCAUU

U

14.6 °C

19.9 °C

2 [detail] 2 [detail]591- 613 AAGGAACTTTTACTTCATTAACAUUAAUGAAGUAAAAGUUCCUUGGAACUUUUACUUCAUUAACA

U

8.9 °C

13.3 °C

2 [detail] 2 [detail]595- 617 AACTTTTACTTCATTAACAGACTUCUGUUAAUGAAGUAAAAGUUCUUUUACUUCAUUAACAGACU

U

11.8 °C

4.9 °C2 [detail] 3 [detail]601- 623 TACTTCATTAACAGACTTACAGGUGUAAGUCUGUUAAUGAAGUACUUCAUUAACAGACUUACAGG

U

21.4 °C

8.9 °C

3 [detail] 2 [detail]720- 742 TGGCTAATGTACTGCATATGAAAUCAUAUGCAGUACAUUAGCCAGCUAAUGUACUGCAUAUGAAA

U

21.1 °C

11.6 °C

3 [detail] 2 [detail]721- 743 GGCTAATGTACTGCATATGAAAGUUCAUAUGCAGUACAUUAGCC
CUAAUGUACUGCAUAUGAAAG

U

14.9 °C

8.5 °C

3 [detail] 2 [detail]744- 766 GACACTAGAAGATTTTGAAATTT

AUUUCAAAAUCUUCUAGUGUCCACUAGAAGAUUUUGAAAUUU

U

7.7 °C18.9 °C

2 [detail] 2 [detail]746- 768 CACTAGAAGATTTTGAAATTTTTAAAUUUCAAAAUCUUCUAGUG
CUAGAAGAUUUUGAAAUUUUU

U

7.4 °C

20.2 °C

2 [detail] 2 [detail]749- 771 TAGAAGATTTTGAAATTTTTATTUAAAAAUUUCAAAAUCUUCUAGAAGAUUUUGAAAUUUUUAUU

U- 12.0 °C

5.3 °C2 [detail] 2 [detail]16 + GCTTGGATTCCTACAAAGA exon(2)XX|||X||||||||||||| M30640 | Hs#S2104 Human endothelial leukocyte adhesion molecule I (ELAM1) mRNA, complete cds.
CTTTGCATTCCTACAAAGA NM_000450.1 | Homo sapiens selectin E (endothelial adhesion molecule 1) (SELE), mRNA
16 + GCTTGGATTCCTACAAAGA exon(2)
X||X|X||||||||||||| BX648671 | Hs#S16817980 Homo sapiens mRNA; cDNA DKFZp686B0247 (from clone DKFZp686B0247)
ACTGGAATTCCTACAAAGA NM_182523.1 | Homo sapiens hypothetical protein MGC61571 (MGC61571), mRNA
16 + GCTTGGATTCCTACAAAGA exon(1)
X||||X|||||||X||||| AY010114 | Hs#S3438475 Homo sapiens unknown mRNA sequencetcttgcattcctataaaga
16 + GCTTGGATTCCTACAAAGA exon(1)
X|||||X||||X||||||| NM_001001786.1 | Homo sapiens BRCC2 mRNA (BRCC2), mRNA

TCTTGGGTTCCAACAAAGA
16 + GCTTGGATTCCTACAAAGA exon(1)
X||||||||X|||X||||| NM_024638.2 | Homo sapiens queuine tRNA- ribosyltransferase domain containing 1 (QTRTD1), mRNA

tcttggattgctataaaga
16 + GCTTGGATTCCTACAAAGA exon(7)X||||||||||X||||X|| AL833606 | Hs#S4621785 Homo sapiens mRNA; cDNA DKFZp686L2267 (from clone DKFZp686L2267)
ACTTGGATTCCAACAAGGA NM_003872.2 | Homo sapiens neuropilin 2 (NRP2), transcript variant 2, mRNA
NM_018534.3 | Homo sapiens neuropilin 2 (NRP2), transcript variant 4, mRNA
NM_201264.1 | Homo sapiens neuropilin 2 (NRP2), transcript variant 6, mRNA
NM_201266.1 | Homo sapiens neuropilin 2 (NRP2), transcript variant 1, mRNA
NM_201267.1 | Homo sapiens neuropilin 2 (NRP2), transcript variant 5, mRNA
NM_201279.1 | Homo sapiens neuropilin 2 (NRP2), transcript variant 3, mRNA
16 + GCTTGGATTCCTACAAAGA exon- exon junction(1)|X|||XX|||||||||||| XM_035572.9 | PREDICTED: Homo sapiens chromosome 4 open reading frame 9 (C4orf9), mRNA

GTTTGCTTTCCTACAAAGA
16 + GCTTGGATTCCTACAAAGA exon(1)|X|||||X|||||||||X| BF895230 | Hs#S3158510 CM2- MT0158 - 301100- 572- e12 MT0158 Homo sapiens cDNA, mRNA sequence

GATTGGACTCCTACAAATA
16 + GCTTGGATTCCTACAAAGA exon(1)|X|||||X|||||||||X| XM_373814.2 | PREDICTED: Homo sapiens hypothetical LOC388572 (LOC388572), mRNA

GATTGGACTCCTACAAATA

Graphical view of effective siRNA candidates
10
20
30
40
50
60
70
80
90
100
ACAT T CT AACT GCAACCT T T CGAAGCCT T T GCT CT GGCACAACAGGT AGT AGGCGACACT GT T CGT GT T GT CAACAT GACCAACAAGT GT CT CCT CCAAA 1- 100
68- 90 U
110
120
130
140
150
160
170
180
190
200
T T GCT CT CCT GT T GT GCT T CT CCACT ACAGCT CT T T CCAT GAGCT ACAACT T GCT T GGAT T CCT ACAAAGAAGCAGCAAT T T T CAGT GT CAGAAGCT CCT 101- 200
131- 153 U
152- 174 U
210
220
230
240
250
260
270
280
290
300
GT GGCAAT T GAAT GGGAGGCT T GAAT ACT GCCT CAAGGACAGGAT GAACT T T GACAT CCCT GAGGAGAT T AAGCAGCT GCAGCAGT T CCAGAAGGAGGAC 201- 300
310
320
330
340
350
360
370
380
390
400
GCCGCAT T GACCAT CT AT GAGAT GCT CCAGAACAT CT T T GCT AT T T T CAGACAAGAT T CAT CT AGCACT GGCT GGAAT GAGACT AT T GT T GAGAACCT CC 301- 400
302- 324 U
326- 348 U

16 + GCTTGGATTCCTACAAAGA exon(1)|X|||||X|||||||||X| AK125099 | Hs#S16887605 Homo sapiens cDNA FLJ43109 fis, clone CTONG2025516, moderately similar to
GATTGGACTCCTACAAATA Homo sapiens general transcription factor II, i (GTF2I)
16 + GCTTGGATTCCTACAAAGA exon(1)|X|||||X|||||||||X| AL135733 | Hs#S1712524 DKFZp434H0831_r1 434 (synonym: htes3) Homo sapiens cDNA clone
GATTGGACTCCTACAAATA DKFZp434H0831 5', mRNA sequence
16 + GCTTGGATTCCTACAAAGA exon(2)|X|||||||||||XX|||| AB094093 | Hs#S15632068 Homo sapiens DLNB14 mRNA, complete cds
GTTTGGATTCCTATGAAGA NM_198489.1 | Homo sapiens similar to DLNB14 (DLNB14), mRNA
16 + GCTTGGATTCCTACAAAGA exon(2)||XXX|||||||||||||| AF091263 | Hs#S1368572 Homo sapiens RNA binding motif protein 5 (RBM5) mRNA, complete cds
GCGCCGATTCCTACAAAGA NM_005778.1 | Homo sapiens RNA binding motif protein 5 (RBM5), mRNA
16 + GCTTGGATTCCTACAAAGA exon(2)||XX|||X||||||||||| BC023552 | Hs#S5517855 Homo sapiens stratifin, mRNA (cDNA clone MGC:19713 IMAGE:3534328),
GCGAGGACTCCTACAAAGA complete cds
NM_006142.3 | Homo sapiens stratifin (SFN), mRNA
16 + GCTTGGATTCCTACAAAGA unaligned - notalign(1)|||X|X|||||||||||X| BM665772 | Hs#S4081636 UI- E- CK1-afn- h- 18- 0- UI.s1 UI- E- CK1 Homo sapiens cDNA clone UI- E- CK1gctagaattccTACaaaaa afn- h- 18- 0- UI 3', mRNA sequence

331- 353 U
338- 360 U
372- 394 U
410
420
430
440
450
460
470
480
490
500
T GGCT AAT GT CT AT CAT CAGAT AAACCAT CT GAAGACAGT CCT GGAAGAAAAACT GGAGAAAGAAGAT T T CACCAGGGGAAAACT CAT GAGCAGT CT GCA 401- 500
409- 431 U
510
520
530
540
550
560
570
580
590
600
CCT GAAAAGAT AT T AT GGGAGGAT T CT GCAT T ACCT GAAGGCCAAGGAGT ACAGT CACT GT GCCT GGACCAT AGT CAGAGT GGAAAT CCT AAGGAACT T T 501- 600
581- 603 U
587- 609 U
591- 613 U
595- 617 U
610
620
630
640
650
660
670
680
690
700
T ACT T CAT T AACAGACT T ACAGGT T ACCT CCGAAACT GAAGAT CT CCT AGCCT GT GCCT CT GGGACT GGACAAT T GCT T CAAGCAT T CT T CAACCAGCAG 601- 700

16 + GCTTGGATTCCTACAAAGA exon(1)|||||X||||||X|||X|| CR749825 | Hs#S21591852 Homo sapiens mRNA; cDNA DKFZp686E19106 (from clone DKFZp686E19106)

gcttgaattccttcaagga
16 + GCTTGGATTCCTACAAAGA exon(1)|||||||XX||X||||||| NM_003803.2 | Homo sapiens myomesin 1 (skelemin) 185kDa (MYOM1), mRNA

GCTTGGAGGCCAACAAAGA
16 + GCTTGGATTCCTACAAAGA exon(1)|||||||XX||X||||||| NM_002196.2 | Homo sapiens insulinoma-associated 1 (INSM1), mRNA

GCTTGGAGACCCACAAAGA
16 + GCTTGGATTCCTACAAAGA exon(2)|||||||X|X|||||X||| BX647741 | Hs#S16819013 Homo sapiens mRNA; cDNA DKFZp686C0786 (from clone DKFZp686C0786)
GCTTGGACTTCTACAGAGA NM_198283.1 | Homo sapiens EGF- like- domain, multiple 11 (EGFL11), mRNA
16 + GCTTGGATTCCTACAAAGA exon(2)|||||||||X||X|||X|| BC007372 | Hs#S3603344 Homo sapiens tripartite motif- containing 52, mRNA (cDNA clone MGC:16175
GCTTGGATTACTTCAAGGA IMAGE:3636274), complete cds
NM_032765.2 | Homo sapiens tripartite motif- containing 52 (TRIM52), mRNA

601- 623 U
710
720
730
740
750
760
770
780
790
800
AT GCT GT T T AAGT GACT GAT GGCT AAT GT ACT GCAT AT GAAAGGACACT AGAAGAT T T T GAAAT T T T T AT T AAAT T AT GAGT T AT T T T T AT T T AT T T AAA 701- 800
720- 742 U

16 + GCTTGGATTCCTACAAAGA exon(1)|||||||||X||||X||X| BC039511 | Hs#S6158647 Homo sapiens, clone IMAGE:5579213, mRNA

GCTTGGATTTCTACCAATA

721- 743 U
744- 766 U
746- 768 U
749- 771 U

16 + GCTTGGATTCCTACAAAGA exon(2)|||||||||X|||||XX|| AK023166 | Hs#S2651220 Homo sapiens cDNA FLJ13104 fis, clone NT2RP3002343
GCTTGGATTGCTACATGGA NM_012461.1 | Homo sapiens TERF1 (TRF1)- interacting nuclear factor 2 (TINF2), mRNA

Figure 2 from siDirect 2.0 webserver
Screenshots
Screenshots from siDirect 2.0 webserver. (A) A typical output of siDirect 2.0: siRNAs targeting human interferon β-1(NM_002176) are designed. (B) By clicking the individual siRNA in (A), a detailed list of off-target gene candidates with nearperfect matches is displayed separately for each siRNA strand. The alignment between each off-target sequence and the siRNAsequence clearly visualizes the positions of mismatches.

tive algorithm [25]. In addition, all of the near-perfectmatch hits are precomputed for all the functional humansiRNAs to accelerate the computational performance.
Precomputed results are stored in the memory engine of
MySQL relational database management system. This
makes it possible to return the list of siRNA candidates
within a few seconds (Figure 2A). The output page
includes the minimum number of mismatches against
any near-perfect match candidates for each siRNA (Figure2A). By clicking the individual siRNA in Figure 2A, adetailed list of candidate genes will appear (Figure 2B). By
default, siRNA sequences that have at least two mismatches to any other non-targeted transcripts are selected.

Results and Discussion
We performed a genome-wide design of siRNAs forhuman mRNAs in RefSeq release 30 with the following
parameters: 1) satisfying our functional siRNA design
algorithm [7,24], 2) Tm values at the seed-target duplex ofboth the guide and the passenger strands below 21.5°C,
and 3) no off-target hits with less than two mismatches.
The degree of off-target effects is shown to be correlatedwith the thermodynamic stability or the calculated Tm
value of the seed-target duplex [23]. The initial boundary
Tm value was set to 21.5°C to discriminate the off-targetfree sequences from the off-target-positive ones, according

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to our previous report [23]. Among the entire siRNAsequence population that have at least two mismatches toany other non-targeted transcripts, the siRNA sequenceswith seed-target Tm below 21.5°C account for 2.1% of
about 56 million 23-mer fragments found in humanmRNAs (Figure 3A), and one or more siRNA can be
designed for 94.7% of all human mRNAs (Figure 3B).
However, the strong correlation between the calculated
Tm and the off-target gene silencing activity indicates thatthe seed-dependent off-target effect is definitively reduced
when the siRNA with lower Tm of seed-target duplex areselected. The population of siRNAs among all human 23mer sequences with the Tm in the seed-target duplex ofless than 15°C and 10°C is 0.7% and 0.3%, respectively(Figure 2A), and the fraction of human mRNAs which canbe targeted by more than one siRNA within such criteria
decreases to 85.1% and 72.7%, respectively. (Figure 3B).

designed for only 47.0% and 18.5%, respectively (Figure
3B). Furthermore, the percentage of human mRNAs drops
severely to 0.15% if the near-perfect match hits with lessthan four mismatches are filtered. Thus, siDirect 2.0 filters
siRNAs with less than two mismatches by default to avoid
severe reduction in the number of siRNA candidates.

It is also desirable to select siRNA that contains as manymismatches as possible to any non-targeted mRNAs. In
addition to the Tm value of below 21.5°C, siRNAsequences with at least two mismatches to any other nontargeted transcripts are selectable for 94.7% of humanmRNAs (Figure 3B). However, if the siRNAs having nearperfect match hits with less than three mismatches, with
their Tm of seed sequences below 21.5°C, are selected,
one or more siRNA can be designed for only 77.2% of thehuman mRNAs (Figure 3B). When siRNAs with seed Tm
below 15°C and 10°C were selected, siRNAs can be

Although most existing web servers for designing siRNA
incorporate BLAST [30] to avoid off-target effects [31-38],
several sites including WI siRNA Selection Server [34],siDRM [39], DSIR [40] and Dharmacon siDESIGN Center
consider seed-dependent off-target effects. Current version of WI siRNA Selection Server and siDRM enumerates
the transcripts with full homology to the seed region, andDSIR and Dharmacon siDESIGN Center calculate seed frequencies for each siRNA candidate. Therefore, we analyzed the relationship between the calculated Tm and the
distribution of each seed sequence in human 3' UTRs. Cal-We were unable to design functional, off-target minimized siRNAs for 5.3% of the RefSeq mRNAs using thedefault parameters. Typical examples of these mRNAs are
the histone clusters (NM_003523, etc.) and ribosomal
proteins (NM_002952, etc.), which are known to form
multigene families. When designing siRNAs targetingsuch genes, users can manually investigate the detailed listof off-target gene candidates (Figure 2B) and select thesiRNA that does not have off-target hits to unrelated transcripts.

Selectable siRNAs (%)

3.0
2.5

Human mRNAs for which at least
one siRNA is selectable (%)

3.5

0 mismatch
1 mismatch
2 mismatches
3 mismatches
4 mismatches

2.0
1.5
1.0
0.5

100

25651
25000

80

20000

60

15000

40

10000
0 mismatch
1 mismatch
2 mismatches
3 mismatches
4 mismatches

20

0

0.0

< 10 ºC< 15 ºC< 21.5 ºC

Tm in the seed-target duplex

5000

Number of human mRNAs for which
at least one siRNA is selectableB

A0< 10 ºC< 15 ºC< 21.5 ºC

Tm in the seed-target duplex

FigureThe
proportion3of selectable siRNAs and mRNAs according to Tm values in the seed-target duplexes
The proportion of selectable siRNAs and mRNAs according to Tm values in the seed-target duplexes. (A) The
percentage of selectable siRNA candidates for human mRNAs according to the Tm values in the seed-target duplexes. The
total number of siRNA (56,375,087) is set to 100%. (B) The percentage of human mRNAs harboring at least one target
sequence of an siRNA whose Tm value of the seed-target duplex is below the indicated value. 100% indicates 25,651 mRNAs.
Off-target hits with 0-4 mismatches between nucleotides at positions in the 2-20 of both siRNA strands and human mRNAs
were represented as separate lines.

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culated Tm of the seed-target duplexes of all possible 7-nt
seed sequences (47 = 16,384) ranged from -12°C to 60°C,
and of these, 4488 (27.4%) 7-mers had the Tm below
21.5°C (Figure 4A). The number of 3' UTRs bearing atleast one target site of any 7-nt sequence was broadly distributed from 17 to 10,882 (Figure 4B), excluding thesequence AAAAAAA, which is found in almost all 3' UTRs
with poly(A) tails. When the siRNAs were classified into
eight groups according to their Tm of the seed-target
duplex, as shown in Figure 4C, siRNAs whose seed-target
duplexes had higher Tm, ranging from 20°C to 60°C,
were less frequent and similarly distributed. On the other
hand, the seed sequences with lower Tm were frequently
found in human 3' UTRs (Figure 4C).

A

We have extensively updated siDirect 2.0 based on our
experimental knowledge, and provided a promising website for reducing siRNA off-target silencing. The website
selects: 1) functional siRNAs that satisfy our guideline [7],2) siRNAs with reduced seed-dependent off-target effects
by considering the thermodynamic stability of the seedtarget duplex, 3) siRNAs that do not hit any non-targeted
genes with near-perfect matches. When the candidate
functional siRNAs could form seed-target duplexes withTm values below 21.5°C, and their 19-nt region spanning
positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0
can design at least one qualified siRNA for >94% of
human mRNA sequences in RefSeq. This website should
provide a wide scope of applications in RNAi studies.

B
Number of 3´ UTRs containing
each of 7-nt sequence

60
50
Calculated Tm (ºC)

Conclusion

40
30
21.5

20
10
0
-10
-20

1

4488

All possible 7-nt sequences (47 = 16384)
sorted by calculated Tm

12000
10000
8000
6000
4000
2000
0
1

16384

All possible 7-nt sequences (47 = 16384)
sorted by appearance in human 3´ UTRs

16384

C
60

Calculated Tm (ºC)

50
40
30
20
10

0

AAAAAAA

-10
-20
0

5000

10000

15000

20000

0

Average

20000

Number of 3´ UTRs containing
each of 7-nt sequence

Figure 4 Tm values and appearances of 7-nt seed sequences
Calculated
Calculated Tm values and appearances of 7-nt seed sequences. (A) Calculated Tm values of the duplex formed by all
possible 7-nt sequences. The dotted line indicates that the number of 7-nt sequences with duplex Tm below 21.5°C is 4,488(27.4%). (B) Appearance of 7-nt seed sequences in human 3' UTRs. The numbers of 3' UTR sequences containing at least one
given 7-nt sequence are shown. (C) Relationship between the appearance of each 7-nt sequence in the 3' UTRs containing atleast one 7-nt sequence and its calculated Tm. Histograms in the right panel show the appearance of each 7-nt sequence inhuman 3' UTR, divided into 10°C Tm intervals. The seed sequence whose duplex has lower Tm (colored blue) is more frequently observed in the 3' UTRs as compared to those with higher Tm (colored orange).

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BMC Bioinformatics 2009, 10:392

Availability and requirements

http://www.biomedcentral.com/1471-2105/10/392

16.

Project name: siDirect
17.

Project home page: http://siDirect2.RNAi.jp/
18.

Operating system(s): Platform independent
Programming language: Perl19.

Any restrictions to use by non-academics: Contact
license@RNAi.jp
20.

Authors' contributions
YN developed the webserver and performed the computational analyses. JY and SM developed the core frameworkof the software. KU-T supervised the entire study. YN and
KU-T drafted the manuscript. All authors read andapproved the final manuscript.

21.

22.

Acknowledgements
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to YN, SM and KUT. KU-T is a member of the Genome Network Project (MEXT).

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