REVIEW ARTICLE
published: 11 June 2012
doi: 10.3389/fgene.2012.00102

siRNA design software for a target gene-speciﬁc RNAinterference
Yuki Naito 1 *† and Kumiko Ui-Tei 1,2
12

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

Edited by:
Akio Kanai, Keio University, Japan
Reviewed by:
Rintaro Saito, Keio University, Japan
Kengo Sato, Keio University, Japan
Motoaki Wakiyama, RIKEN, Japan*Correspondence:
Yuki Naito, Genome Information
Biology, Department of Biophysics
and Biochemistry, Graduate School of
Science, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan.
e-mail: y-naito@RNAi.jp†
Present address:
Yuki Naito, Database Center for Life
Science, Research Organization ofInformation and Systems, Tokyo,
Japan.

RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA)
induces sequence-speciﬁc posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended genewhose transcript possesses perfect complementarity to the siRNA guide strand. Hence,
its silencing effect has been assumed to be extremely speciﬁc. However, accumulated
evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon isreferred to as off-target effect. We have revealed that the capability to induce off-target
effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex.
For understanding accurate target gene function and successful therapeutic application,
it may be critical to select a target gene-speciﬁc siRNA with minimized off-target effect.
Here we present our siRNA design software for a target-speciﬁc RNAi. In addition, we also
introduce the software programs open to the public for designing functional siRNAs.
Keywords: siRNA, seed region, off-target effect, thermodynamic stability

INTRODUCTION
RNA interference (RNAi) is a broadly used technique by whichsmall interfering RNA (siRNA) downregulates a speciﬁc target gene with perfect complementary sequence, and promised
to use in therapeutic application for human diseases (Castanotto and Rossi, 2009; Ketting, 2011). Although human has morethan 20,000 genes, it is desirable to use siRNA which is highly
functional and has no effects on any genes other than its speciﬁc target. In this article, we review an optimized method todesign siRNA based on the mechanism of RNAi. In addition,
we introduce the websites open to the public for selecting siRNAsequences.
OPTIMIZED DESIGN OF siRNA
Duplexes of 21-nucleotide (nt) RNA with 2 nt 3 overhangs(siRNA) is usually used for RNAi experiments. Upon delivery into
the cells, siRNAs are incorporated into the RNA-induced silencing
complex (RISC) as a double-stranded RNA. RISC is the effector
complex containing Argonaute protein (Ago) with slicer activity (Hammond et al., 2001; Martinez et al., 2002). The siRNA
guide strand containing the thermodynamically less stable 5 -end
is preferentially retained by RISC (Khvorova et al., 2003; Schwarz
et al., 2003; Ui-Tei et al., 2004). The passenger strands of mostof the double-stranded siRNAs loaded onto RISC are cleaved by
Ago2 protein and degraded (Matranga et al., 2005; Rand et al.,2005; Leuschner et al., 2006). The retained guide strand pairs target mRNA with perfectly complementary sequence, and represses
it by cleavage by Ago2 protein at nucleotide position 10 of siRNA
guide strand (Elbashir et al., 2001; Hammond et al., 2001; Martinez

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et al., 2002). However, an accumulated evidence from genomewide experiments indicate that a great number of mRNAs withpartial complementarities to the guide strand are also reduced(Jackson et al., 2003, 2006; Lim et al., 2005; Birmingham et al.,2006; Ui-Tei et al., 2008). This phenomenon is referred to as seeddependent off-target effect and preferably observed in mRNA 3
UTRs. The target recognition mechanism of this off-target effectis known to be similar to that of miRNA-mediated gene silencing (Lewis et al., 2005; Lim et al., 2005; Grimson et al., 2007).
The transcripts with sequences complementary to the seed region
positioned 2–8 from the 5 terminal are mainly reduced. The seedregion is known to be situated on the surface of Ago in a quasihelical form to serve as the entry or nucleation site for small RNAsin the RISCs (Ma et al., 2005; Yuan et al., 2005). Thus, the seedregion ﬁrst identiﬁes the target mRNAs, and subsequently form
perfect base-pairing with intended target mRNA and induce RNAiby Ago2.
Based on the mechanism of RNAi, a target gene-speciﬁc siRNAis considered to be selectable according to the following three steps.
STEP 1: SELECTION OF FUNCTIONAL siRNA SEQUENCE

The knockdown efﬁciencies of siRNAs are revealed to be highly
dependent on their sequences. We advocated the empirically based
rule which prescribed the characteristics of highly functional
siRNAs (Ui-Tei et al., 2004), such rule is called as Ui-Tei rule
(Figure 1). The siRNA selected by Ui-Tei rule satisfy the following
four conditions simultaneously: (1) A or U at position 1 from 5
terminus of siRNA guide strand, (2) G or C at position 19, (3) AU
richness (AU ≥4) in positions 1–7, and (4) no long GC stretch

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Naito and Ui-Tei

FIGURE 1 | Schematic representation for selecting functional andoff-target effect-reduced siRNAs. Selection of highly functional siRNAby Ui-Tei, Reynolds, Amarzguioui rules, or the combination of them
(STEP 1). Selection of siRNAs with low stability in the seed-target
duplexes (STEP 2). Elimination of siRNAs with near-perfect matchedsequences to non-target genes (STEP 3). In each rule, the nucleotide
position indicates the number of nucleotide counted from 5 terminal of
the guide strand. Ui-Tei rule :  A/U at position 1.  G/C at position 19.

≥10. Except for (4), our rule indicated that the functional siRNAhas asymmetrical stability in 5 and 3 terminals. Our experimental validation using luciferase reporter assay showed that 98% of
the siRNAs satisfying above conditions reduced the expression of
luciferase reporter below 33% (Ui-Tei et al., 2004). Other groups
also demonstrated the rules of highly functional siRNAs referred
to as Reynolds rule (Reynolds et al., 2004) and Amarzguioui rule
(Amarzguioui and Prydz, 2004) as summarized in Figure 1. These
rules also clearly showed that functional siRNAs are asymmetrical: an RNA strand with unstable 5 terminal was effective as

Frontiers in Genetics | Non-Coding RNA

Software for a target-speciﬁc RNAi

 4 to 7 A/Us in positions 1-7.  No GC stretch ≥ 10. Reynolds rule : GC content (30%-52%).  A/U ≥ 3 at positions 1-5.  Absence ofinternal repeats.  A at position 1.  A at position 17.  U at position 10.
 Not G/C at position 1.
Not G at position 7. Amarzguioui rule :  An
asymmetry in the stability of the duplex ends (measured as the A/U
differential of the three terminal basepairs at either end of the duplex). G or C at position 19.  A or U at position 1.  A or U at position 14. 
Not U at position 19.  Not G at position 1.

a guide strand. Furthermore, common in these rules, 5 terminus of functional siRNA guide strand was preferable to be A
or U. It was revealed later that the results reﬂected the structural features of human Ago2 (Frank et al., 2010). The crystal
structure of a MID (middle) domain from human AGO2 andNMR titration experiments showed that nucleotide monophosphates, AMP and UMP, bind with up to 30-fold higher afﬁnity than either CMP or GMP, providing structural evidence fornucleotide-speciﬁc interactions in the MID domain of eukaryotic
AGO proteins.

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Naito and Ui-Tei

Software for a target-speciﬁc RNAi

A

B

C

D

FIGURE 2 | Screen views of siDirect 2.0 siRNA design software.
(A) Top page (http://siDirect2.RNAi.jp/). (B) Optional parameters for
siRNA design. (C) Result page. (D) Detailed list of off-target

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candidates with near-perfect matches. The alignment between each
off-target transcript and the siRNA sequence visualizes the
positions of mismatches.

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Frontiers in Genetics | Non-Coding RNA

http://optirna.unl.edu/

http://rnai.nci.nih.gov/

OptiRNA

OptiRNAi 2.0

form.cgi

server_exe/oligowalk/oligowalk_

HealthRationally designed siRNA

possible secondary structuresfeatures considering all

National Institutes ofCalculating thermodynamic

Nebraska-Lincoln

parameterson thermodynamicmRNA are considered based

the oligomer and target

The secondary structures oflibrary

Design a genome-wide siRNA

University of

Medical Center

http://rna.urmc.rochester.edu/cgi-bin/ University of Rochester

Research Center (DKFZ)

wiki/display/nextrnai/NEXT-RNAi

OligoWalk

German Cancer

datasets were used

(esiRNAs)

small interfering RNAs

endoribonuclease-preparedParis

http://b110-wiki.dkfz.de/signaling/

polymorphism (SNP),

secondary structure

The publicly available siRNA

NEXT-RNAi

and FASTAof single nucleotide

alternative splicing and mRNA

Design and quality control ofsearch by BLAST

consecutive bases, existence

Elbashir rule, Reynolds ruleNot considered

Modiﬁed Reynolds rule

rule

Reynolds rule, Amarzguioui

Reynolds rule, Ui-Tei rule,strandC/G at the 3 end of the guidewith A/U at the 5 end andmotifs in the siRNA sequence

presence of asymmetric short

Regarding importance of thesiRNA

self-alignment energy of

Homology

G/C content, maximum

algorithm considering

search by BLAST

Homology

Mathews

Cui et al. (2004)

Ladunga (2007)(2008)

Not consideredLusearch by BLAST

andHorn et al. (2010)

Vert et al. (2006)

Henschel et al. (2004)

Park et al. (2008)

Reference

Homology

search by Bowtie

Homology

search by BLAST

Homology

search by BLAST

Homology

off-target effectExon-based siRNA design

Elimination of

Rules

Features

Ecole des Mines dehttp://biodev.cea.fr/DSIR/DSIR.html

DSIR

bioinformatics/DEQOR.html

MPI-AGEKRIBB

http://bioinformatics.age.mpg.de/Research Center,

DEQOR

Bioinformatics

http://sysbio.kribb.re.kr:8080/

AsiDesigner/menuDesigner.jsf

AsiDesigner

Developer

URL

WebsiteTable 1 | Small interfering RNA design software programs.

Naito and Ui-Tei
Software for a target-speciﬁc RNAi

June 2012 | Volume 3 | Article 102 | 4

www.frontiersin.org

http://biotools.swmed.edu/siRNA

http://sirna.wi.mit.edu/sIRsiRNA Selector

SpeciﬁcityServer

WHITEHEAD)

au/SpeciﬁcityServer

http://informatics-eskitis.grifﬁth.edu.

http://siRecords.umn.edu/siDRM/

siDRM(siRNA at

http://sidirect2.rnai.jp/

Grifﬁth University

target gene for candidate

A speciﬁcity scoring scheme

user-adjustable rules

siRNA sequences that satisfy

The siRNA design tool scans aBiomedical ResearchPre-designed siRNAs

used

and off-target activities, were

responses, cell toxic effects,

including innate immune

unwanted detrimental effects,

and several ﬁlters to check

siRecords were implemented

sets and fast rule sets, links to

A few high sensitivity ruledesigned siRNA

Empirically and rationallyspecify the parameters

rules but relaxes the need to

Whitehead Institute forCenterSouthwestern Medical

University of Texas

University of Minnesota

University of Tokyo

Bayesian algorithm that is

Department of Healthbased on stacking energy

is a unique feature. Uses

New York State

Target accessibility evaluation

Wadsworth Center,

http://sfold.wadsworth.org/cgi-bin/sirna.pl

siDirect 2.0

Sfold 2.2 (Srna)

search by BLAST

WU-BLAST

search by

Homology

and WU-BLAST

Homology
Reynolds rule, Hsieh rulesearch by BLAST
Tuschl rule, Ui-Tei rule,

Homology
Elbashir rule, Amarzguioui ruleseed duplex

stability in thethermodynamic

considersiDirect, andsearch by

Homology

search by BLAST

Homology

Ui-Tei rule, Reynolds rule,

Ui-Tei rule

Reynolds rulemer (2008)

Chalk and Sonnham-

Yuan et al. (2004)

Shah et al. (2007)

Gong et al. (2008)

Naito et al. (2009)

Ding et al. (2004)

Naito and Ui-Tei
Software for a target-speciﬁc RNAi

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Naito and Ui-Tei

Software for a target-speciﬁc RNAi

STEP 2: SELECTION OF siRNA SEQUENCE WITH REDUCED OFF-TARGET
EFFECTSTo avoid seed-dependent off-target effects, one approach may be to
select the siRNA guide strand whose seed sequence is not complementary to any sequences in the 3 UTR of all non-targeted genes.
However, this approach is proved to be impossible, because humansiRNAs with the most infrequent seven-nt seed sequence still had
seed-complementarities with several non-targeted mRNAs. So, we
have looked for the rules that govern the capability of siRNAs
to induce seed-dependent off-target effect, and revealed that the
efﬁciency of off-target effect is highly correlated to the thermodynamic stability of the duplex formed between the seed region ofsiRNA guide strand and its target mRNA (Ui-Tei et al., 2008). The
melting temperature (T m ), one of the thermodynamic parameters
for the formation of RNA duplex, showed strong positive correlation with the induction of seed-dependent off-target effects. Thus,
selecting the siRNAs with low T m of the seed-target duplex should
minimize seed-dependent off-target silencing (Figure 1). The T mof 21.5˚C may serve as the benchmark, which discriminates thealmost off-target-free seed sequences from the off-target-positive
ones. Furthermore, since the off-target effect may be caused by
not only by the guide strand but also by the passenger strand, siRNAs whose seed-target T m is sufﬁciently low for both strands arefavorable.
STEP 3: ELIMINATION OF NEAR-PERFECT MATCHED OFF-TARGET
GENES

Even when the T m value of the seed-target duplex is sufﬁciently
low, the target gene silencing can still take place if the non-seedregion is completely complementary. Therefore, in the third step,siRNAs that have near-perfect matches to any other non-targeted
transcripts were eliminated (Figure 1).

siRNA DESIGN SOFTWARE
We presented siRNA design software, siDirect 2.0 (http://siDirect2.
RNAi.jp/; Figure 2), which provides functional, target-speciﬁc
siRNA design software according to the procedures mentioned
above (Naito et al., 2009). In default parameter, siRNAs satisfying Ui-Tei rule can be selected. When the candidate functional
siRNAs could form seed-target duplexes with T m values below
21.5˚C, and their 19-nt regions spanning positions 2–20 of bothstrands have at least two mismatches to any other non-targeted

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This work was partially supported by grants from the Ministryof Education, Culture, Sports, Science and Technology of Japan(MEXT), and the Cell Innovation Project (MEXT), and Core
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Conﬂict of Interest Statement: The
authors declare that the research was
conducted in the absence of any commercial or ﬁnancial relationships that
could be construed as a potential conﬂict of interest.
Received: 26 April 2012; accepted: 20 May
2012; published online: 11 June 2012.
Citation: Naito Y and Ui-Tei K (2012)
siRNA design software for a target genespeciﬁc RNA interference. Front. Gene.
3:102. doi: 10.3389/fgene.2012.00102
This article was submitted to Frontiers in
Non-Coding RNA, a specialty of Frontiersin Genetics.
Copyright © 2012 Naito and Ui-Tei. Thisis an open-access article distributed under
the terms of the Creative Commons Attribution Non Commercial License, which
permits non-commercial use, distribution, and reproduction in other forums,
provided the original authors and source
are credited.

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