OPINION ARTICLE
published: 11 June 2013
doi: 10.3389/fgene.2013.00107

Optimal choice of functional and off-target effect-reduced
siRNAs for RNAi therapeutics
Kumiko Ui-Tei 1,2*
1

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*Correspondence: ktei@bi.s.u-tokyo.ac.jp
2

Edited by:Michael Rossbach, Genome Institute of Singapore, Singapore
Reviewed by:David Langenberger, University Leipzig, Germany
Irene Aksoy, Genome Institute of Singapore, Singapore
Prasanna R. Kolatkar, Genome Institute of Singapore, Singapore

INTRODUCTION
Small interfering RNA (siRNA) represses
a specific target gene that has a perfectly
complementary sequence by a pathway
referred to as RNA interference (RNAi).
Researchers have successfully diverted thisnatural pathway to artificially knockdown
intended genes. RNAi shows promise fortherapeutic applications to human diseases (Castanotto and Rossi, 2009; Ketting,2011). Over the past decade, more thantwenty siRNAs have been applied to clinical trials for dozens of diseases, including various cancers, viral infections, andgenetic disorders (Davidson and McCray,
2011; Snead and Rossi, 2012). Ongoing
patient trials are expected to yield success. However, several major problems,such as the efficacy and off-target effectof siRNA, remain to be overcome before
RNAi-based therapeutics can be efficiently
introduced into clinical practice. In thepresent paper, I propose a procedure forselecting sequences of highly functional
siRNAs with few off-target effects.

FUNCTIONAL siRNAs
In RNAi experiments, siRNAs composedof duplexes of 21-nucleotide (nt) RNA
with 2-nt 3 -overhangs are usually used.
As shown in Figure 1, siRNAs that areincorporated into cells are transferred
to an RNAi effector complex called theRNA-induced silencing complex (RISC)(Hutvagner and Simard, 2008; Jinek
and Doudna, 2009), which contains the
Argonaute 2 (Ago2) protein with slicer
activity (Hammond et al., 2001; Martinez
et al., 2002). In the RISC, the siRNA duplex
is unwound into single-stranded RNAs.
The guide strand selectively retained on

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the RISC base-pairs with the target mRNA.
Then, the target mRNA is silenced bycleavage by Ago2 protein at the region
corresponding to the nucleotide positions 10–11 of the siRNA guide strand(Elbashir et al., 2001; Hammond et al.,
2001; Martinez et al., 2002).
However, the efficacy of RNAi in mammalian cells varies considerably dependingon the siRNA sequence, and a limited proportion of siRNAs is functional (Holenet al., 2002; Harborth et al., 2003). We
and others have reported that preferable
incorporation of the siRNA guide strand
into the RISC is due to the asymmetrical features of both siRNA terminals(Amarzguioui and Prydz, 2004; Reynolds
et al., 2004; Ui-Tei et al., 2004). The siRNA
guide strand, which has a thermodynamically less stable 5 -end, is preferentiallyretained on the RISC (Khvorova et al.,
2003; Schwarz et al., 2003; Ui-Tei et al.,2004). In contrast, the passenger strand
is cleaved by the Ago2 protein on the
RISC and degraded (Matranga et al., 2005;
Miyoshi et al., 2005; Rand et al., 2005;
Leuschner et al., 2006). We extensively
examined the relationship between siRNAsequence and its ability to give rise toRNAi in mammalian cells (Ui-Tei et al.,2004), and empirically defined the practical characteristics of highly functional
siRNAs as follows: (1) A or U residues atnucleotide position 1; (2) four to seven
A/Us at nucleotide positions 1–7; and (3)G/C at position 19, with the nucleotide
position measured from the 5 -end of theguide strand (Figure 1). In addition, (4) a
GC stretch of no more than 9 nucleotides
should occur in the siRNA sequence.
Among these rules, Reynolds et al. (2004)

also showed that A is preferable at position 1 and not G/C, and Amarzguioui
and Prydz (2004) also recommended A/U
but not G at this position. Furthermore,Amarzguioui and Prydz also recommend
G/C but not U at the position 19.
All three features from (1) to (3) may be
involved in the thermodynamic asymmetry of both terminals in the selection of thefunctional guide strand RNA. However,feature (1) should also be involved in theother property that strongly contributes to
the RNAi activity. The 5 -end of the siRNA
guide strand is reported to be anchoredin the binding pocket of the Mid domainof the Archaeoglobus fulgidus Ago-like protein (Ma et al., 2005; Parker et al., 2005).
The crystal structure of a MID domain ofhuman Ago2 and NMR titration experiments showed that nucleotide monophosphates, AMP and UMP, bind with up to
30-fold higher affinities than either CMP
or GMP, providing structural evidence
for preferential interactions of the A/Uresidues in the MID domain of eukaryotic Ago proteins (Frank et al., 2010). In
the RNAi pathway, thermodynamic asymmetry due to features (2) and (3) may beessential for dissociating double-stranded
siRNA into single-stranded guide RNA
from its 5 -terminal. Then, the 5 -endof the siRNA guide strand should beanchored in the Ago2 pocket due to feature (1), immediately after unwinding,since the passenger strand in the siRNA
duplex is cleaved by the catalytic activityof Ago2 protein and degraded (Figure 1)(Kawamata et al., 2009; Yoda et al., 2010).
However, in the case of siRNA with long
GC stretches, the 10-nt and 11-nt fragments of the passenger strand cleaved byJune 2013 | Volume 4 | Article 107 | 1

Ui-Tei

FIGURE 1 | Functional siRNA sequence and the mechanism of RNAi
without off-target effects in mammalian cells. An RNA strand with A or
U at position 1 measured from the guide strand 5 end (1), four to seven
A/Us in positions 1–7 (2) and G/C at position 19 (3) are easily unwoundfrom the 5 end and retained in the RISC. The passenger strand is cleaved
in the Ago2-containing RISC, but dissociated from the Ago1, 3, or
4-containing RISC following unwinding. In the RNAi pathway (left column),
the guide strand recognizes target mRNA with completely complementary

Frontiers in Genetics | Non-Coding RNA

Functional and off-target effect-reduced siRNAsequences, and the target mRNA is cleaved by the Ago2 protein.
However, off-target transcripts with partial complementary sequences tothe seed region positions 2–8 are downregulated or not downregulated
according to the thermodynamic stability of the duplex formed between
the siRNA seed region and target mRNA. The siRNAs with weakseed-target base-pairing are not reduced by the off-target effect (middle
column), but those with strong base-pairing are downregulated by theoff-target effect (right column).

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Ago2 cannot be dissociated from the guidestrand due to the strong base-pairing by
the long GC stretch. This may explain why
feature (4) is a preferable condition for afunctional siRNA.

MECHANISM TO AVOID
SEED-DEPENDENT OFF-TARGET
EFFECTSRNAi has been assumed to be an extremely
specific procedure to knockdown a particular target gene. However, accumulated
evidence from genome-wide experiments
has revealed that numerous non-targeted
mRNAs with partially complementary
sequences in their 3 -UTRs to the guidestrand seed region (positions 2–8) are also
reduced (Jackson et al., 2003, 2006; Linet al., 2005; Birmingham et al., 2006; UiTei et al., 2008) through a mechanism
similar to that of miRNA silencing (Lewis
et al., 2005; Lim et al., 2005; Grimson
et al., 2007). This phenomenon is referred
to as the off-target effect, and it is likely tobe due to the fact that the seed region issituated on the surface of Ago in a quasihelical form, serving as the entry or nucleation site for small RNAs in the RISCs(Ma et al., 2005; Yuan et al., 2005). In thepathway of the off-target effect, most of
the siRNA duplexes may be loaded onto
Ago proteins other than Ago2, such as
Ago1, 3, and 4, which lack slicer activities (Figure 1). Thus, the thermodynamic
asymmetry of both terminals due to features (1)–(3) should be essential for siRNA
unwinding, as the passenger strand in thesiRNA duplex is not cleaved by Ago2 protein, in contrast to the case of RNAi.
The off-target effect is induced by only
7-nt base-pairing between the siRNA seedregion and the mRNA 3 -UTRs. If such
7-nt base-pairing is a unique factor toinduce the off-target effect, a target genespecific RNAi is not possible, since random 7-mer sequences are stochastically
distributed at about ≥1000 sites in 3 UTR regions. Comparing the efficiencies of off-target effects using various
reporter plasmids with different 7-nt seedsequences, we found that the ability to
induce the off-target effect strongly correlated with the thermodynamic stability ofthe duplex formed between the siRNA seedregion and target mRNAs (Ui-Tei et al.,2008, 2012). This result was confirmed bygenome-wide expression profiling usingwww.frontiersin.org

Functional and off-target effect-reduced siRNA

siRNA with high or low seed-target stability (Ui-Tei et al., 2008). It was revealed
that siRNA with high stability (benchmark
melting temperature [Tm ] ≥ 21.5◦ C) in
the 7-bp duplex is a good inducer ofoff-target activity, whereas siRNA withlow stability in the 7-bp duplex (Tm ≤21.5◦ C) is a poor inducer. Approximately
22% of the 16,384 sequences of 7-nt had
Tm -values ≤ 21◦ C, indicating that limited
seed sequences are available for selecting
siRNAs with reduced off-target effects.
It was shown that siRNA off-target
effect is induced by a similar mechanism
to miRNA-mediated gene silencing (Lewis
et al., 2005; Lim et al., 2005; Grimson et al.,2007). Thus, it was expected that miRNAsilencing efficacy is also determined by
the thermodynamic stability in the duplex
formed between the miRNA seed region
and the target mRNA. We carried out
reporter experiments using various miRNAs to evaluate their silencing efficacies. However, unlike the siRNA off-target
effect, the silencing efficacy of miRNA wasnot simply determined by seed-target stability, but also affected by the instabilityof the 5 region of the miRNA duplex(Hibio et al., 2012). MiRNAs with an
unstable 5 terminal duplex and stableseed-target duplex exhibit strong silencing activity. The RNA strand in a small
RNA duplex containing the thermodynamically less stable 5 -end is preferentially loaded onto the RISC (Khvorova
et al., 2003; Schwarz et al., 2003; Ui-Tei
et al., 2004). Although siRNA is simply composed simply of perfectly complementary double-stranded RNA, miRNA
has specific structural features, such as
an internal bulge/mismatch. Because the5 region and seed region overlap eachother, the stabilities of these regions
in siRNA are similar. These regions in
siRNA may function in a similar manner, and the stability of seed region alone
is enough to define the efficacy of siRNA
off-target effect. However, the efficacy ofmiRNA-mediated silencing activity may
be defined by the combination of thenucleotide sequence in the seed regionand structure of 5 region of miRNA
duplex. Although the structure depends
directly on the nucleotide sequence, thesequence and the structure may contributeto silencing efficacy as distinguishable
parameters.

CONCLUSION AND OPTIMAL CHOICE
OF FUNCTIONAL siRNA WITH
REDUCED OFF-TARGET EFFECT
For safe RNAi therapeutics, it is important to select highly functional siRNAs thathave reduced off-target effects. I proposethat those siRNAs that simultaneously satisfy the following five conditions should begood candidates:(1) A or U residues at nucleotide position 1 measured from the 5 -end of theguide strand(2) Four to seven A/Us in nucleotide positions 1–7 (A/U ≥ 57%)
(3) G/C at position 19(4) No long GC stretch (≤9 GC)(5) Low thermodynamic stability in the
duplex formed between the siRNA
7-nt seed region and the target mRNA(Tm ≤ 21.5◦ C)
To facilitate the selection of such siRNAs,
we constructed the siRNA design webserver siDirect 2.0 (http://siDirect2.RNAi.
jp/) (Naito et al., 2009; Naito and Ui-Tei,2012). siRNAs for >94% of human mRNA
sequences in RefSeq can be successfully
designed using our procedure.

ACKNOWLEDGMENTS
This work was supported by the Ministryof Education, Culture, Sports, Scienceand Technology of Japan (MEXT) [grant
numbers 21115004, 21310123, 23651200],
the Cell Innovation Project (MEXT), andthe Core Research Project for Private
University: matching fund subsidy to
Kumiko Ui-Tei.

REFERENCES
Amarzguioui, M., and Prydz, H. (2004). An algorithm for selection of functional siRNA sequences.
Biochem. Biophys. Res. Commun. 316, 1050–1058.
doi: 10.1016/j.bbrc.2004.02.157
Birmingham, A., Anderson, E. M., Reynolds, A.,
Ilsley-Tyree, D., Leake, D., Fedorov, Y., et al. (2006).
3 UTR seed matches, but not overall identity, areassociated with RNAi off-targets. Nat. Methods 3,
199–204. doi: 10.1038/nmeth854
Castanotto, D., and Rossi, J. J. (2009). The
promises and pitfalls of RNA-interferencebased therapeutics. Nature 457, 426–433. doi:
10.1038/nature07758
Davidson, B. L., and McCray, P. B. Jr. (2011).
Current prospects for RNA interference-based
therapies. Nat. Rev. Genetics 12, 329–340. doi:
10.1038/nrg2968
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin,
A., Weber, K., and Tuschl, T. (2001). Duplexes ofJune 2013 | Volume 4 | Article 107 | 3

Ui-Tei

21-nucleotide RNAs mediate RNA interference in
cultured mammalian cells. Nature 411, 494–498.
doi: 10.1038/35078107
Frank, F., Sonenberg, N., and Nagar, B. (2010).
Structural basis for 5 -nucleotide base-specificrecognition of guide RNA by human AGO2.
Nature 465, 818–822. doi: 10.1038/nature09039
Grimson, A., Farh, K. K., Johnston, W. K., GarrettEngele, P., Lim, L. P., and Bartel, D. P. (2007).
MicroRNA targeting specificity in mammals:
determinants beyond seed pairing. Mol. Cell 27,
91–105. doi: 10.1016/j.molcel.2007.06.017
Hammond, S. M., Boettcher, S., Caudy, A. A.,
Kobayashi, R., and Hannon, G. J. (2001).
Argonaute 2 link between genetic and biochemical
analyses of RNAi. Science 293, 1146–1150. doi:
10.1126/science.1064023
Harborth, J., Elbashir, S. M., Vandenburgh, K.,
Manninga, H., Scaringe, S. A., Weber, K., et al.
(2003). Sequence, chemical and structuralvariation of small interfering RNAs and short
hairpin RNAs and the effect on mammalian genesilencing. Antisense Nucleic Acid Drug Dev. 13,
83–105. doi: 10.1089/108729003321629638
Hibio, N., Hino, K., Shimizu, E., Nagata, Y., and UiTei, K. (2012). Stability of miRNA 5 terminaland seed regions is correlated with experimentallyobserved miRNA-mediated silencing efficacy. Sci.
Rep. 2:996. doi: 10.1038/srep00996
Holen, T., Amrzguioui, M., Wiiger, M. T., Babaie, E.,
and Prydz, H. (2002). Positional effects of shortinterfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res. 30,
1757–1766. doi: 10.1093/nar/30.8.1757
Hutvagner, G., and Simard, M. J. (2008). Argonaute
proteins: key players in RNA silencing. Nat. Rev.
Mol. Cell Biol. 9, 22–32. doi: 10.1038/nrm2321
Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi,
S. V., Burchard, J., Mao, M., et al. (2003).
Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotech. 21, 635–637. doi:
10.1038/nbt831
Jackson, A. L., Burchard, J., Schelter, J., Chau, B.
N., Cleary, M., Lim, L., et al. (2006). WidespreadsiRNA “off-target” transcript silencing mediatedby seed region sequence complementarity. RNA
12, 1179–1187. doi: 10.1261/rna.25706
Jinek, M., and Doudna, J. A. (2009). A threedimensional view of the molecular machinery
of RNA interference. Nature 457, 405–412. doi:
10.1038/nature07755
Kawamata, T., Seitz, H., and Tomari, Y. (2009).
Structural determinants of miRNAs for RISC
loading and slicer-independent unwinding.
Nature Struct. Mol. Biol. 16, 953–960. doi:
10.1038/nsmb.1630
Ketting, R. F. (2011). The many faces of RNAi. Dev.
Cell 15, 148–161. doi: 10.1016/j.devcel.2011.01.012

Frontiers in Genetics | Non-Coding RNA

Functional and off-target effect-reduced siRNA

Khvorova, A., Reynolds, A., and Jayasena, S. D.
(2003). Functional siRNAs and miRNAs exhibit
strand bias. Cell 115, 209–216. doi: 10.1016/S00928674(03)00801-8
Leuschner, P. J., Ameres, S. L., Kueng, S., andMartinez, J. (2006). Cleavage of the siRNA
passenger strand during RISC assembly inhuman cells. EMBO Rep. 7, 314–320. doi:
10.1038/sj.embor.7400637
Lewis, B. P., Burge, C. B., and Bartel, D. P.
(2005). Conserved seed pairing, often flanked
by adenosines, indicates that thousands ofhuman genes are microRNA targets. Cell 120,
15–20. doi: 10.1016/j.cell.2004.12.035
Lim, L. P., Lau, N. C., Garrett-Engele, P.,
Grimson, A., Schelter, J. M., Castle, J.,et al. (2005). Microarray analysis showsthat some microRNAs downregulate large
numbers of target mRNAs. Nature 433,
769–773. doi: 10.1038/nature03315
Lin, X., Ruan, X., Anderson, M. G., McDowell, J. A.,
Kroeger, P. E., Resik, S. W., et al. (2005). siRNAmediated off-target gene silencing triggered by
a 7-nt complementation. Nucleic Acids Res. 33,
4527–4535. doi: 10.1093/nar/gki762
Ma, J.-B., Yuan, Y. R., Meister, G., Pei, Y., Tuschl,
T., and Patel, D. J. (2005). Structural basis for 5 end-specific recognition of guide RNA by the A.
fulgidus piwi protein. Nature 434, 666–670. doi:
10.1038/nature03514
Martinez, J., Patkaniowska, A., Urlaub, H., Lührmann,R., and Tuschl, T. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in
RNAi. Cell 110, 563–574. doi: 10.1016/S00928674(02)00908-X
Matranga, C., Tomoari, Y., Shin, C., Bartel, D. P.,
and Zamore, P. S. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2containing RNAi enzyme complexes. Cell 123,
607–620. doi: 10.1016/j.cell.2005.08.044
Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H.,
and Siomi, M. C. (2005). Slicer function of
Drosophila Argonautes and its involvement in
RISC formation. Genes Dev. 19, 2837–2848. doi:
10.1101/gad.1370605
Naito, Y., and Ui-Tei, K. (2012) siRNA design software
for a target gene-specific RNA interference. Front.
Genet. 3:102. doi: 10.3389/fgene.2012.00102
Naito, Y., Yoshimura, J., Morishita, S., andUi-Tei, K. (2009) siDirect 2.0: updated software for designing functional siRNA with
reduced seed-dependent off-target effect. BMC
Bioinformatics 10:392. doi: 10.1186/1471-210510-392
Parker, J. S., Roe, S. M., and Barford, D. (2005).
Structural insights into mRNA recognition from a
PIWI domain-siRNA guide complex. Nature 434,
663–666. doi: 10.1038/nature03462

Rand, T. A., Petersen, S., Du, F., and Wang, X. (2005).
Argonaute2 cleaves the anti-guide strand of siRNAduring RISC activation. Cell 123, 621–629. doi:
10.1016/j.cell.2005.10.020
Reynolds, A., Leake, D., Boese, Q., Scaringe, S.,
Marshall, W. S., and Khvorova, A. (2004).
Rational siRNA design for RNA interference. Nat.
Biotechnol. 22, 326–330. doi: 10.1038/nbt936
Schwarz, D. S., Hutvágner, G., Du, T., Xu, Z.,
Aronin, N., and Zamore, P. D. (2003). Asymmetry
in the assembly of the RNAi enzyme complex.
Cell 115, 199–208. doi: 10.1016/S0092-8674(03)00759-1
Snead, N. M., and Rossi, J. J. (2012). RNA interference trigger variants: getting the most out of RNAinterference-based therapeutics. Nucleic Acid Ther.
22, 139–146. doi: 10.1089/nat.2012.0361
Ui-Tei, K., Naito, Y., Nishi, K., Juni, A., andSaigo, K. (2008). Thermodynamic stability andWatson-Crick base pairing in the seed duplex
are major determinants of the efficiency of thesiRNA-based off-target effect. Nucleic Acids Res. 36,
7100–7109. doi: 10.1093/nar/gkn902
Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi,T., Ohki-Hamazaki, H., Juni, A., et al. (2004).
Guidelines for the selection of highly effective
siRNA sequences for mammalian and chick RNAinterference. Nucleic Acids Res. 32, 936–948. doi:
10.1093/nar/gkh247
Ui-Tei, K., Nishi, K., Takahashi, T., and Nagasawa,T. (2012). Thermodynamic control of small RNAmediated gene silencing. Front. Genet. 3:101. doi:
10.3389/fgene.2012.00101
Yoda, M., Kawamata, T., Paroo, Z., Ye, X., Iwasaki, S.,
Liu, Q., et al. (2010). ATP-dependent human RISC
assembly pathways. Nature Struct. Mol. Biol. 17,
117–123. doi: 10.1038/nsmb.1733
Yuan, Y.-R., Pei, Y., Ma, J. B., Kuryavyi, V., Zhadina,M., Meister, G., et al. (2005). Crystal structureof A. aeolicus argonaute, a site-specific DNAguided endoribonuclease, provides insights
into RISC-mediated mRNA cleavage. Mol.
Cell 19, 405–419. doi: 10.1016/j.molcel.2005.
07.011
Received: 29 April 2013; accepted: 24 May 2013;
published online: 11 June 2013.
Citation: Ui-Tei K (2013) Optimal choice of functionaland off-target effect-reduced siRNAs for RNAi therapeutics. Front. Genet. 4:107. doi: 10.3389/fgene.2013.00107
This article was submitted to Frontiers in Non-Coding
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Copyright © 2013 Ui-Tei. This is an open-access article distributed under the terms of the Creative Commons
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