Published online February 9, 2004
936±948 Nucleic Acids Research, 2004, Vol. 32, No. 3
DOI: 10.1093/nar/gkh247

Guidelines for the selection of highly effective siRNAsequences for mammalian and chick RNAinterference
Kumiko Ui-Tei1,2,*, Yuki Naito1, Fumitaka Takahashi1,2, Takeshi Haraguchi1,
Hiroko Ohki-Hamazaki3, Aya Juni2,4, Ryu Ueda4,5 and Kaoru Saigo1
1

Received December 1, 2003; Revised and Accepted January 2, 2004

ABSTRACTIn the present study, the relationship between shortinterfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using
62 targets of four exogenous and two endogenous
genes and three mammalian and Drosophila cells.
We present the rules that may govern siRNAsequence preference and in accordance with whichhighly effective siRNAs essential for systematic
mammalian functional genomics can be readily
designed. These rules indicate that siRNAs which
simultaneously satisfy all four of the following
sequence conditions are capable of inducing highly
effective gene silencing in mammalian cells: (i) A/U
at the 5¢ end of the antisense strand; (ii) G/C at the 5¢
end of the sense strand; (iii) at least ®ve A/U residues in the 5¢ terminal one-third of the antisense
strand; and (iv) the absence of any GC stretch ofmore than 9 nt in length. siRNAs opposite in features with respect to the ®rst three conditions giverise to little or no gene silencing in mammaliancells. Essentially the same rules for siRNA sequence
preference were found applicable to DNA-based
RNAi in mammalian cells and in ovo RNAi using
chick embryos. In contrast to mammalian and chickcells, little siRNA sequence preference could be
detected in Drosophila in vivo RNAi.
INTRODUCTION
RNA interference (RNAi) is the process of double-stranded(ds) RNA-dependent, post-transcriptional gene silencing (1±4). dsRNA introduced into cells is digested by Dicer to yieldshort interfering RNA (siRNA) 21±23 nt in length (5,6).
siRNA thus generated within cells or that synthesized in vitro

and introduced into cells reacts directly or indirectly withPIWI protein and/or relevant proteins to give rise to the RNAinduced silencing complex (RISC), which is responsible formRNA degradation (7±12). eIF2C1, a human counterpart ofDrosophila PIWI protein, Argonaute 1 [Ago 1 (13,14)], waspreviously shown to be essential for siRNA-based RNAi in
mammalian cells (15). Martinez et al. (16) observed eIF2C1 to
be involved in active RISC prepared from HeLa cells. Active
RISC includes siRNA antisense strands (AS) but not sense
strands (SS) (16), thus indicating that double-stranded siRNAundergoes denaturation via helicase (8) either prior to or at an
early stage in RISC formation. Dicer, an enzyme which
possesses both RNase III and helicase domains, may beessential for siRNA-based mammalian RNAi (15). In
Drosophila and Caenorhabditis elegans, spindle E (17) andmut-14 (18), both encoding helicase, have also been shown to
be involved in RNAi. Target mRNA is cleaved at a speci®csite corresponding to the center of the siRNA AS in
mammalian and Drosophila cells (9,16,19,20).
The introduction of long dsRNA into mammalian cells
frequently induces a fatal interferon response (21), and thus
siRNA should be a more promising reagent for mammalianRNAi (19) than long dsRNA (22±26). siRNA-based RNAi,
however, may not be readily usable for the large-scale genesilencing essential for mammalian functional genomics, since
only a limited fraction of siRNAs appear capable of producing
highly effective RNAi in mammalian cells [(27,28) see also
Fig. 2A].
The relationship between the siRNA sequence and its
capability to bring about RNAi in human, Chinese hamsterand mouse embryonic stem (ES) cells as well as Drosophila
cells was examined in detail in the present study. Highly
effective RNAi was found to occur in mammalian cells if
siRNA satisfying the four following sequence conditions at
the same time is used: (i) A/U at the 5¢ end of the AS; (ii) G/C
at the 5¢ end of the SS; (iii) AU-richness in the 5¢ terminal, 7 bp
long region of the AS; and (iv) the absence of any long GC
stretch of more than 9 bp in length. All siRNAs opposite in*To whom correspondence should be addressed. Tel: +3 5841 3044; Fax: +3 5841 3044; Email: ktei@biochem.s.u-tokyo.ac.jp
Correspondence may also be addressed to Kaoru Saigo. Tel: +3 5841 4407: Fax: +3 5841 4400; Email: saigo@biochem.s.u-tokyo.ac.jp

Nucleic Acids Research, Vol. 32 No. 3 ã Oxford University Press 2004; all rights reserved

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Department of Biophysics and Biochemistry, Graduate School of Science and 2UPBSB, School of Science,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, 3Department of Molecular Neuroscience,
Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519,
Japan, 4Mitsubishi-Kagaku Institute of Life Sciences, 11 Minami-ooya, Machida-shi, Tokyo 194-8511, Japan and
5
Genetic Strain Research Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

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MATERIALS AND METHODS
Cell culture
Drosophila S2 cells were cultured in Schneider's Drosophila
medium (Gibco-BRL) at 25°C. Chinese hamster CHO-K1(RIKEN Cell Bank) and human HeLa cells were cultured in
Dulbecco's modi®ed Eagle's medium (DMEM; Gibco-BRL)
at 37°C. Both media were supplemented with 10% heatinactivated fetal bovine serum (FBS; Mitsubishi Kagaku) andantibiotics [10 U/ml of penicillin (Meiji) and 50 mg/ml of
streptomycin (Meiji)]. E14TG2a (mouse ES) cells were
cultured in DMEM supplemented with 20% heat-inactivatedFBS (Hyclone), 0.1 mM 2-mercaptoethanol (Wako), 8 mg/mlof adenosine, 8.5 mg/ml of guanosine, 7.3 mg/ml of cytidine,
7.3 mg/ml of uridine, 2.4 mg/ml of thymidine, 0.1 mM each
non-essential amino acid and 1000 U/ml of leukemia
inhibitory factor (CHEMICON International).
Preparation of siRNA
RNA oligonucleotides were synthesized by Proligo. Doublestranded siRNA was prepared as described previously (22).
The concentration of siRNA is shown based on that of the AS.
When necessary, siRNAs were numbered based on the
nucleotide position within the coding region of the target
mRNA, corresponding to the 3¢ siRNA AS end.
luc RNAi assay
A 1 ml aliquot of S2 (1 3 106 cells/ml), CHO-K1 (3 3 105
cells/ml), HeLa (1 3 105 cells/ml) or E14TG2a (2 3 105 cells/ml) cell suspension was inoculated into a 1.5 cm well 24 h prior
to transfection. Cells were transfected with pGL3-Control
DNA (1 mg, Promega) encoding the ®re¯y luciferase gene, andpRL-TK DNA (0.1±1 mg, Promega) or pRL-SV40 DNA (0.1±
1 mg, Promega), both encoding the Renilla luciferase gene,with or without siRNA. The calcium phosphate precipitation
method (22) was used for transfection for S2, HeLa or CHOK1 cells, while DMRIE C reagent (Invitrogen) was used for
E14TG2a transfection. Cells were harvested 24 h after
transfection and luciferase activity was measured using theDual-Luciferase Reporter Assay System (Promega).

Vimentin RNAi and immunostaining
A 1 ml aliquot of HeLa cell suspension (1 3 105 cells/ml) was
inoculated into a 1.5 cm well 24 h prior to the ®rst transfection.
Cells were treated with three cycles of transfection carriedout in 24 h intervals with vimentin siRNA at 50 nM.
Lipofectamine 2000 (Invitrogen) was used for transfection.
The estimated transfection ef®ciency was >95%. Cells were®xed with 3.7% formaldehyde in phosphate-buffered saline(PBS) and permeabilized 24 h after the last transfection. Afterwashing with PBS, cells were doubly stained with anti-porcine
vimentin antibody (Oncogene Research Products), Cy3conjugated second antibody (Jackson Immuno Research) andanti-human Yes antibody (Upstate Biotechnology), with Cy5conjugated second antibody (Jackson Immuno Research).
Oct 4 RNAi assay
Using Lipofectamine 2000 (Invitrogen), E14TG2a cells (2 3105 cells/ml) were co-transfected with 50 nM Oct 4 siRNAshown in Figure 3B and pCAGIPuro-EGFP (0.5 mg/ml),
encoding enhanced green ¯uorescent protein (EGFP) andpuromycin-resistant genes. Puromycin (2 mg/ml; Clontech)
was added to the medium 24 h after transfection, andmorphological change was observed under a phase contrast
microscope 3 days after transfection. RNA was also extracted
3 days after transfection using RNeasy (QIAGEN) and was
subjected to RT±PCR using the RNA LA-PCR kit (Takara).
Almost all cells were found to express EGFP 3 days after
transfection. The following primers were used for RT±PCR tomeasure the concentration of glyceraldehyde-3-phosphate
dehydrogenase (Gapd) and Oct 4 mRNA. Gapd, 5¢GCCTCATCCGGTAGACAAAA and 5¢-ACCGTGGTCATGAGTCCTTC; Oct-4, 5¢-AGCTGCTGAAGCAGAAGAGG and 5¢-TGTCTACCTCCCTTGCCTTG.
RNAi assay for EGFP, ECFP and DsRed
HeLa cells (1 3 105 cells/ml) were transfected with pCAGGSEGFP (0.25 mg/well), pCAGGS-DsRed [0.25 mg/well (15)]and siRNA (50 nM) for EGFP RNAi. For enhanced cyan
¯uorescent protein (ECFP) RNAi, HeLa cell transfection was
carried out with pECFP-N1 (0.25 mg/well; Clontech),pCAGGS-DsRed (0.25 mg/well) and siRNA (50 nM).
Transfection was carried out using Lipofectamine 2000
(Invitrogen). RNAi activity was estimated by counting
EGFP- or ECFP-positive cells among DsRed-positive cells
under a ¯uorescence microscope (Zeiss). pCAGGS-EGFP wasconstructed by inserting an EGFP fragment of pEGFP-N1(Clontech) into the EcoRI site of pCAGGS (31).
In ovo electroporation
Fertile chick eggs obtained from a local farm were incubatedat 37°C for 2 days. The eggs were windowed, and 0.1±0.5 ml of
PBS containing pCAGGS-EGFP (0.1 mg/ml) and pCAGGSDsRed (0.1 mg/ml) and siRNA (5 mg/ml) along with 0.01% ofluxol fast blue was injected into the central canal of the spinal
cord at the wing level using a glass capillary with a tip
diameter of 50±100 mm. A pair of platinum electrodes 4 mmapart (Nepagene) was used for electroporation. Transfection
occurred exclusively on the right hemilateral side of the neuraltube. Five timed pulses of 50 ms duration at 20 mV were used.
Embryos were incubated at 37°C for 2 days and killed. EGFP

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sequence features except for the fourth condition brought
about the least levels of RNAi in mammalian cells. Essentiallythe same rules for siRNA sequence preference appeared toapply for siRNA-based RNAi in chick embryos and DNAbased RNAi in mammalian cells.
During the course of the preparation of this manuscript,
Schwarz et al. (29) showed sequence conditions resembling
the above to some extent to be necessary for RISC formationand subsequent target RNA cleavage in Drosophila embryonicextracts. Khvorova et al. (30) indicated that both the enhanced¯exibility at the siRNA end including the 5¢ AS end and low
internal energy across the duplex are strongly correlated withsiRNA and microRNA (miRNA) functions. The similarity in
siRNA sequence requirements for in vivo RNAi in mammaliancells [this work, (30)] and in vitro Drosophila RNAi (29)
could re¯ect underlying similarities between the RNAimechanisms in insects and animals. The ®ndings in thepresent study are thus evaluated and discussed from thestandpoints of RISC formation and siRNA unwinding.

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and DsRed expression was observed under a ¯uorescencemicroscope on embryonic day 4.
Construction of siRNA expression plasmids for DNAbased RNAi
Single-stranded DNA oligonucleotides, ~80 nt in length andencoding, in order: (i) a 21 nt siRNA SS; (ii) a human miRNA
loop; and (iii) the 19 nt AS of the identical siRNA, minus 3¢
overhangs, were annealed with corresponding complementary
single-stranded DNA oligonucleotides. The resultant dsDNA
was inserted into the BamHI±HindIII site of pSilencer 3.0-H1(Ambion) to generate FLx-m23L or FLx-m212L plasmids,
where x indicates the position of the corresponding target
sequence in the ®re¯y luc gene. In FL826-m212L, the order ofSS and AS was reversed. As human miRNA loops, m23L andm212L, derived from miR-23 (32,33) and miR-212 (34),respectively, were used. Escherichia coli XL1-Blue competent cells (Gibco-BRL) were transformed with the resultant
plasmids. Plasmid DNA was puri®ed using a commercial
DNA puri®cation kit (QIAGEN). HeLa cells (1 3 105 cells/ml) were transfected with 150 ng of the plasmid DNA alongwith pGL3-Control (1 mg) and pRL-SV40 (0.1 mg, Promega).
pSilencer with no insert was used as a control. Luciferase
activity was measured using the Dual-Luciferase Reporter
Assay System (Promega) 3 days following transfection.
Free energy calculation
Standard Gibbs free energies, which re¯ect the stability of
pentamer subsequences, were calculated from the siRNA
duplex end containing the 5¢ AS end (position 1) according to
the nearest-neighbor method described by Freier et al. (35).
The values from positions 16 to 19 were not calculated
because of the absence of available pentamer subsequences.
RESULTS
Strong siRNA sequence preference in mammalian RNAi
RNAi in mammalian cells was previously noted to varyconsiderably depending on the siRNA sequence (27,28). To
examine this point in greater detail, 16 siRNAs targeting forthe ®re¯y luciferase gene (luc) were prepared (Fig. 1) andassessed for their ability to produce RNAi in human (HeLa),
Chinese hamster (CHO-K1), mouse ES (E14TG2a) andDrosophila (S2) cells by dual luciferase assay (22). Figure 1

shows the nine luc target sequences, corresponding to siRNA
a±i, to be spaced 6 nt apart, while three of the remaining(corresponding to siRNA n±p) are spaced only 1 nt apart. Cells
were simultaneously transfected with plasmid DNA encoding
the ®re¯y luc gene (target), plasmid DNA with the Renillaluciferase gene (reference) and 50 nM cognate siRNA, andluciferase activity was measured 24 h later (Fig. 2A). In
Figure 2A, siRNA sequences are listed in rank, in order of
average RNAi activity in three mammalian cells, so as toobtain some clari®cation of the relationship between siRNAsequence and the resultant reduction in ®re¯y luc geneactivity.
In mammalian cells, RNAi activity varied signi®cantly
depending on the siRNA employed. Use of ®ve highly
effective siRNAs (a, l, k, f and o) resulted in a 70±95%
reduction in relative ®re¯y luciferase activity, while use offour highly ineffective siRNAs (h, m, b and c) resulted in<20% reduction. Even a 1 nt variation in the target sequence
had a considerable effect on RNAi activity in mammalian cells(compare RNAi effects of siRNA n and o). In contrast, ®re¯yluciferase activity was always abolished at >85% upontransfecting Drosophila cells with any siRNA other thansiRNA c. Thus, most, if not all, siRNAs should be capable ofproducing highly effective RNAi in Drosophila cells, at least
under given conditions. Three of the four siRNAs (a, l and k)giving rise to the highest levels of RNAi in mammalian cells
were also noted to bring about the highest levels of RNAi in
Drosophila cells.
siRNA sequence requirement for highly effective andineffective RNAi in mammalian cells
The values in Figure 2A for reduction in relative ®re¯yluciferase activity in CHO-K1, HeLa and E14TG2a cells canbe seen to be virtually the same, suggesting that siRNA-based
RNAi in mammalian cells is in accordance with the same rules
for siRNA sequence preference.
Three immediately apparent features of the siRNAsequence may possibly serve to discriminate highly effective
siRNAs from those that are ineffective. First, the 5¢ AS end of
highly effective siRNAs may always be A or U, with thecounterpart of ineffective siRNAs being G or C. A/U and G/Cresidues were respectively found to be present at the 5¢ AS
ends of all ®ve highly effective and all four ineffective
siRNAs. Secondly, the 5¢ SS ends of highly effective siRNAs

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Figure 1. Location of 16 cognate siRNAs for silencing of the ®re¯y luc gene.

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Figure 2. Relationship between luc siRNA sequence and induced luc gene silencing (RNAi) activity. AS and SS, respectively, in the upper margin, indicate
siRNA ends with the 5¢ antisense strand and 5¢ sense strand ends. (A) Classi®cation of 16 luc siRNAs. siRNA-dependent reduction in ®re¯y luciferase activity
in three mammalian (CHO-K1, HeLa and E14TG2a) and Drosophila (S2) cells was examined using 50 nM of 16 siRNAs, a±p, shown in Figure 1. Sixteen
siRNAs were aligned according to their RNAi activity in mammalian cells from top to bottom. siRNAs were classi®ed into three groups depending on RNAior reduction in relative luciferase activity. (B) RNAi activity caused by siRNAs designed using our sequence preference rules. Using the rules, 15 class Ia and®ve class III siRNAs were designed and their capability to bring about RNAi in CHO-K1, HeLa, E14TG2a and S2 cells was examined. The siRNA number
indicates the nucleotide position within the luc coding region, corresponding to the 3¢ end of the siRNA AS. The concentration of siRNA was 50 nM andRNAi effects were observed 24 h after transfection. Data obtained from 2±4 experiments were averaged and are shown. Thin vertical lines indicate the average of three mammalian cells. The 7 bp terminal region with the 5¢ AS end is boxed. While A/U and G/C in the boxed region are colored in red and blue,
respectively, highly conserved, 5¢-terminal bases are shown on red (A/U) or blue (G/C) backgrounds. Note that, in class Ia and highly effective siRNAs, the5¢ AS and SS ends, respectively, are almost exclusively A/U and G/C.

are preferably G or C, with the counterpart of ineffective
siRNAs being A or U. Thirdly, in the case of highly effective
siRNAs, at least four out of seven nucleotides in the 5¢terminal AS are A or U while the corresponding region of

ineffective siRNAs are GC rich. Most, if not all, siRNAs
associated with mixed features appear to belong to an siRNA
class with intermediate RNAi activity. A possible molecularbasis for the effectiveness of siRNA a is discussed below.

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Figure 3. Highly effective silencing of endogenous genes by class Ia siRNAs (A and B), RNAi caused by an non-cognate siRNA (C) and class Ia siRNAdependent RNAi in chick embryos (D). The sequences of siRNAs examined here are depicted in the right margin (see Fig. 2 for coloration and box). (A)Silencing of vimentin, a human endogenous gene, by class Ia and class III siRNAs. Ten class Ia (VIM-270, -368, -596, -812, -857, -1097, -1128, -1148, -1235
and -1298) and three class III siRNAs (VIM-35, -155 and -491) were designed and their RNAi activity was examined in HeLa cells subjected to three cyclesof 50 nM siRNA transfection. On day 3, cells were stained for vimentin (target) and Yes (control). Vimentin and Yes protein signals are colored in yellow
and green, respectively. Note that all class Ia but not class III siRNAs signi®cantly reduced vimentin signals. (B) Effects of siRNA transfection on the expression of Oct 4, a mouse endogenous gene. E14TG2a (mouse ES) cells were transfected with class Ia (Oct-670, -797 and -821) or class III (Oct-161 and -566).
The four pictures on the left are phase contrast photographs. Note that class Ia siRNAs (Oct-670 and -797) induced a ¯at morphology typical of trophectoderm
cells. Class Ia siRNA-speci®c degradation of Oct 4 mRNA visualized with RT±PCR is also shown. Gapd was used as a control. (C) ECFP RNAi caused by
an non-cognate EGFP siRNA. EGFP-441 is a class Ia EGFP siRNA but not identical in sequence to ECFP-441, a class II ECFP siRNA possessing G at the 5¢
AS end. While ECFP-441 did not abolish ECFP signals 2 days after transfection (on day 2 of transfection), non-cognate EGFP-441 virtually completely eliminated ECFP signals. Note that RNAi effects 1 day after transfection (on day 1) indicate that EGFP is a better target for EGFP-441 than ECFP. Blue and red
arrows indicate that EGFP and ECFP mRNA possess a base substitution at the position corresponding to the 5¢ AS end of EGFP-441 or ECFP-441. (D) In
ovo RNAi in chick embryo. EGFP and DsRed expression plasmids were co-electroporated into chick spinal cord with class Ia siRNAs (EGFP-416, -441 and
DsRed-399) or class III siRNAs (EGCFP-666 and DsRed-383). Class Ia siRNAs signi®cantly abolished the targeted ¯uorescence, while class III siRNAs did
not. Expression of EGFP and DsRed is colored in yellow and red, respectively. Dotted lines show left (L) and right (R) portions of the spinal cords. The 5¢
ends of SS and AS, and the 5¢ region of the AS (7 bp) are boxed and colored as described in Figure 2.

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Silencing of mammalian endogenous genes by siRNAtransfection
Examination was carried out to determine whether the rules
for siRNA sequence preference would be applicable fordesigning highly effective and ineffective siRNAs for RNAiof mammalian endogenous genes. The right margin of
Figure 3A and B shows class Ia and class III siRNAs,
designed for highly effective and ineffective RNAi, respectively, of vimentin and Oct 4 in mammalian cells (HeLa and
E14TG2a). Candidate siRNAs designed by the present rules
were further selected by Blast search so that the activity of any
gene other than the target would not be affected by siRNA
introduced into cells. Class Ia siRNAs unique to vimentin andOct 4, respectively, were found to represent 5% (n = 64) and
3% (n = 37) of all possible siRNAs estimated based on
vimentin and Oct 4 gene sequences.
The vimentin gene codes for an intermediate ®lament
protein. It has been reported that reduction in vimentin geneactivity by cognate siRNA transfection is dif®cult (19). Threecycles of siRNA transfection (one transfection/day) were thus
carried out on HeLa cells prior to staining for vimentin andYes (control). All 10 vimentin class Ia siRNAs were found to
signi®cantly reduce vimentin protein, but not Yes signals(Fig. 3A). Little or no reduction in vimentin or Yes signals
could be detected when using class III vimentin siRNAs for
RNAi. RT±PCR results (K. Ui-Tei and K. Saigo, unpublisheddata) indicated 70±95% of vimentin mRNA to be degraded byclass Ia vimentin siRNA, while virtually no vimentin mRNA
cleavage occurred by class III siRNA.
Oct 4 is a POU transcription factor encoded by the Pou5f1(Oct 4) gene and is considered to be a regulator of ES cell
pluripotency (36). A 50±100% increment in Oct 4 expression
may cause the differentiation of pluripotent ES cells into
primitive endoderm and mesoderm, while reduction in Oct 4
expression induces loss of pluripotency to differentiate ES

cells into trophectoderm, which is characterized by ¯atmorphology and induced expression of Hand 1 and Psx(36,37). Three class Ia siRNAs (Oct-670, -797 and -821) andtwo class III siRNAs (Oct-161 and -566) for Oct 4 RNAi wereprepared and examined for change in cell morphology andgene expression 3 days following transfection of 50 nM
cognate siRNA. As partly shown in Figure 3B, the pluripotent
ES cells treated with cognate class Ia siRNAs, Oct-670, -797
and -821, had ¯attened out over the culture surface, withenlarged nuclei acquired in many cases. Oct 4 expression was
virtually eliminated (Fig. 3B) while the expression oftrophectoderm markers, Hand 1 and Psx, was induced(K.Ui-Tei, unpublished data). In contrast, no apparent change
in morphology or gene expression could be found to result
from class III Oct 4 siRNAs, Oct-566 and -161 (Fig. 3B). Ourrules for siRNA sequence preference are thus shown to servequite well for identifying highly effective and ineffective
siRNAs for RNAi of endogenous genes in mammals.
Thirty-two class Ia siRNAs for ®re¯y luc, vimentin andOct 4 were examined, 31 (97%) of which were found to becapable of giving rise to highly ef®cient RNAi in human,
Chinese hamster and mouse cells. Virtually all of theinvestigated class Ia siRNAs were thus shown to be highlyef®cient RNAi reagents for mammalian cells. Thus, it isconcluded that our rules for siRNA sequence preference
may be highly useful for the design of effective siRNAs for
RNAi of both exogenous and endogenous genes in mammalian cells.
siRNAs with long stretches of G/C residues areincapable of bringing about high levels of RNAi in bothmammalian and Drosophila cells
siRNA n may be an exceptional member of class Ia siRNAs in
that, unlike any others which we evaluated, it was incapable ofgiving rise to high levels of RNAi in mammalian cells whentransfected at 50 nM (see Fig. 2A). An investigation was thus
undertaken to clarify in greater detail relationships among thesiRNA sequence, siRNA concentration and RNAi activity in
CHO-K1 or S2 cells using the 16 siRNAs shown in Figure 2A(Fig. 4A). With siRNA at 0.005±5 nM, most graph points
for siRNAs which gave rise to effective RNAi in CHO-K1
or S2 cells after transfection at 50 nM overlapped or weresituated near the shaded area bounded by two lines, intersecting, respectively, with the horizontal axis at 0.5 and 5
and the 50% line of relative luciferase activity at 0.05 and 0.5.
The vertical bars in Figure 4A show the relative luciferase orRNAi activity range for siRNAs which give rise to effective
RNAi in CHO-K1 or S2 cells subsequent to transfection at
50 nM. siRNAs that bring about highly effective RNAi on
transfection at 50 nM would thus appear comprised ofheterogenous members with over 10 times the capacity tobring about RNAi.
A comparison of RNAi effects due to individual siRNA in
CHO-K1 and S2 cells is presented in each of the 11 pictures in
Figure 4B. The pictures are arranged according to siRNA
classi®cation and order of RNAi activity. Maximum levels ofRNAi resulted from the transfection of siRNA l, a class Ia
siRNA, in both CHO-K1 and S2 cells. Note that suppression
due to siRNA l in S2 cells was virtually the same as in CHOK1 cells. We interpret this ®nding as suggesting that virtually
all siRNA l molecules incorporated into cells become fully

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siRNAs may be grouped into three classes, I±III, based on
combinations of terminal base sequences. Class I consists ofsiRNAs possessing A/U at the 5¢ AS end, G/C at the 5¢ SS end
and at least four A/U nucleotides in a 7 nt 5¢-terminal end of
the AS, whereas those with opposite features are class III
siRNAs. All other siRNAs are considered to belong to class II.
Class I siRNAs may be subdivided into two classes, Ia and Ib.
Class I siRNAs with 5±7 A/U residues in a 7 nt 5¢-terminal endof the AS are presumed to belong to class Ia siRNAs; theremainder belong to class Ib.
It is possible to generate 1631 different siRNAs based onthe ®re¯y luc coding sequence. The number of class I siRNAs
was calculated as 275 (17% of the total) and that of class Ia
siRNAs as 154 (9%). To test the validity of the above rules for
siRNA sequence preference, assessment was made of theability of 15 different class Ia and ®ve class III siRNAs to giverise to RNAi using three mammalian and Drosophila S2 cells(Fig. 2B). All class Ia siRNAs brought about highly effective
RNAi in all three mammalian cells as well as Drosophila cells,
while little or no effective RNAi resulted via transfection ofclass III siRNAs in the mammalian cells. We thus conclude
that the rules stipulated here for siRNA sequence preference
predict sequences for highly effective and ineffective siRNAs
for mammalian RNAi at least in the case of the exogenous®re¯y luc gene.

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Nucleic Acids Research, 2004, Vol. 32, No. 3functional in both Drosophila and mammals. Hardly any
RNAi occurred with transfection in siRNA c, a class III
siRNA, to S2 and CHO-K1 cells. Mammalian and Drosophila
cells would thus appear to possess virtually the same capacityof siRNA-mediated RNAi induction, the maximum and theminimum limits of which are determined by the transfection ofsiRNA l and c, respectively. Although within each class,siRNA-dependent RNAi activity in S2 cells increases withincreasing RNAi activity in CHO-K1 cells, our rules for
siRNA sequence preference may not be applicable forpredicting highly effective and ineffective siRNAs for RNAiin S2 cells. RNAi-inducing capability in S2 cells was much thesame for two class Ia siRNAs (o and n) and two class III
siRNAs (b and h). Three class II siRNAs (a, i and g) were
found to be much more effective in S2 cells compared withtwo class Ia siRNAs (o and n).

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Figure 4. Dose dependency of RNAi effects in CHO-K1 and S2 cells. The shaded area is the region bounded by two lines, intersecting, respectively, with thehorizontal axis at 0.5 and 5 and the 50% line of luc activity at 0.05 and 0.5. The thick vertical bar at the right of each panel indicates the region with >77%
reduction in luc activity. (A) Change in luc gene silencing activity with siRNAs ranging from 0.005 to 50 nM in CHO-K1 (left) and S2 (right) cells. siRNAsa±p in Figure 1 are grouped into three classes, I (open circles), II (open triangles) and III (closed circles). (B) Direct comparisons of RNAi activity curves in
S2 (open circles) and CHO-K1 cells (®lled circles). The sequences of corresponding or similar siRNAs are schematically shown in the lower margin. siRNAs
i, e, j, m and h behave the same way as their corresponding examples above. Filled circles, G/C; open circles, A/U. The 7 bp duplex region containing the5¢ end is boxed. Eleven pictures are positioned based on siRNA classi®cation and induced RNAi activity levels.

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Figure 5. GC content distribution of highly effective class Ia siRNAs. The
distribution of the GC content of 31 highly effective class Ia siRNAs shownin Figures 2 and 3 is presented. Position 1 corresponds to the siRNA duplex
end including the 5¢ AS end. The 5¢ ends of AS and SS of all class Ia
siRNAs causing highly effective RNAi in mammalian cells were A/U and
G/C, respectively. The average GC content in the region 2±7 was 19%,
while that of the region 8±18 was 52%.

We noted that siRNA n, the most ineffective class Ia
siRNA, possesses a long GC stretch extending from the 5¢ endof the SS and that class Ia siRNA-dependent RNAi activity inS2 and CHO-K1 cells is negatively correlated with the lengthof the GC stretch extending from the 5¢ end of the SS. Similar
negative effects of a long GC stretch on RNAi were also
evident in class II- or class III-dependent RNAi in CHO-K1
and S2 cells. In contrast, the average GC content in the 11 bp
region adjacent to the 5¢ SS end was ~50% in the case of the 31
highly effective class Ia siRNAs (Fig. 5). It may thus followthat a long GC stretch in the siRNA sequence serves as a
suppressor of RNAi, the extent depending on the length of thestretch.
Possible dual functions of the 5¢ end of the siRNA
antisense strand
During RNAi of EGFP and ECFP (a derivative of EGFP),EGFP-441, an siRNA homologous in sequence to the EGFP
but not completely to the ECFP gene, was noted to be capableof effectively inactivating ECFP. HeLa cells were transfected
simultaneously with DsRed plasmid DNA (control), EGFP or
ECFP plasmid DNA (target) and siRNA, and the relative
number of target gene-expressing cells was counted at various
times. As shown in Figure 3C, nearly all EGFP signals from
EGFP-expressing cells were abolished 24 h after transfection,
when EGFP-441, a cognate class Ia siRNA, was transfected,
while EGCFP-666, a class III siRNA completely homologous
in sequence to EGFP and ECFP genes, could reduce only a
few EGFP signals 2 days following transfection. EGFP-441 ishomologous in sequence to ECFP mRNA except for the
position corresponding to the 5¢ AS end (see the right marginof Fig. 3C). Figure 3C shows that EGFP-441 is capable ofmore effectively bringing about ECFP RNAi than ECFP-441,
a class II siRNA completely identical in sequence to the target(ECFP mRNA). EGFP-441 abolished nearly 70% of ECFP
signals at 24 h following transfection and the rest was almost
entirely eliminated at 2 days after transfection. On challenging
ECFP with the cognate siRNA, ECFP-441 (class II), most of
the ECFP signals could still be detected 2 days following
transfection. The presence of A/U at the 5¢ end of the siRNA

siRNA sequence requirement for DNA vector-basedRNAi
To determine whether target sequence preference in mammalian siRNA-based RNAi is intrinsic to the RNAi mechanism, astudy was carried out to clarify whether similar rules for target
sequence preference would hold for DNA-based mammalianRNAi, in which siRNA is produced via cleavage of hairpintype RNA ®rst transcribed and then transported from nuclei(38±40). pSilencer and ®re¯y luc were used as vector andtarget genes, respectively. The pro®les of RNAi activity
change in DNA-induced RNAi can be seen from Figure 6 to bebasically the same as siRNA-based RNAi. That is, all the
pSilencer with the DNA insert encoding hairpin-type class Ia
siRNA (shRNA) induced highly ef®cient RNAi in mammaliancells 3 days following transfection. In contrast, little or no
RNAi was induced by transfection of pSilencer with the DNA
insert encoding the hairpin of class III siRNA (FL14-m23L).
siRNA sequence preference in mammalian siRNA-based
RNAi may thus be concluded to hold for DNA-based RNAiin mammalian cells and, accordingly, should be a re¯ection of
the intrinsic features of RNAi.
siRNA sequence requirement for RNAi in chick embryos
The siRNA sequence preference rules presented here may be
applicable to RNAi in vertebrates other than mammals andmay prove useful in the design of siRNAs for gene silencing inindividuals. To con®rm these possibilities, siRNAs designedby the present rules were introduced into the right half of the
spinal cord of day 2 chick embryos by in ovo electroporation,and the change in target gene activity on embryonic day 4 was
examined (Fig. 3D). EGFP and DsRed expression served as
criteria for assessing RNAi effects brought on by transfected
siRNAs. EGFP-441, EGFP-416, DsRed-399 (Fig. 3D) and
DsRed-231 (data not shown), all being class Ia siRNAs, wereclearly shown to be capable of bringing about highly effective
RNAi in the spinal cord of chick embryos. EGCFP-666,
DsRed-140 (Fig. 3D) and DsRed-383 (data not shown), allbelonging to class III, were found to be ineffective in thisregard. Thus, our rules for siRNA sequence preference would
certainly appear quite useful for the design of effective
siRNAs in chick embryos.
Free energy calculation of siRNAs
The enhanced ¯exibility at the siRNA end containing the 5¢
AS end and low internal energy across the duplex (especiallyat the region 9±14) have recently been shown to be strongly

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AS would thus appear essential for some RNAi process otherthan mRNA recognition. That EGFP mRNA is a better target
for EGFP-441 than ECFP would indicate that the 5¢ end of thesiRNA AS is also involved in hydrogen bonding between thetarget mRNA and the siRNA AS. Accordingly, the 5¢ end of
the AS would probably be involved in two separate RNAiprocesses, RISC formation, which includes siRNA unwinding,and mRNA recognition.
The time course of RNAi, as followed using several highly
effective EGFP or ECFP siRNAs, showed target gene activity
abolishment to remain at >70% for 7 days, at least startingfrom day 2. In contrast, little or no RNAi effects were evident
on using ineffective class III siRNAs (data not shown).

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Nucleic Acids Research, 2004, Vol. 32, No. 3

correlated with siRNA function (30). Thus, internal stability
re¯ecting the stability of pentamer subsequences wasestimated in each of the 16 luc siRNAs shown in Figure 2A,
using the nearest-neighbor method (35). DG° at position 1 of®ve highly effective siRNAs varied from ±3.6 to ±7.2 kcal/mol(Fig. 7B), whereas for seven siRNAs causing intermediatelevels of RNAi, from ±4.5 to ±10.3 kcal/mol (Fig. 7C) and forhighly ineffective siRNAs, the values exceeded ±9.8 kcal/mol(Fig. 7D). These values would support the notion that the
duplex end containing the 5¢ AS end of highly effective
siRNAs is considerably less thermostable. However, our data
disclosed no clear reduction in the absolute values of DG° in
the region 9±14. To further examine this point, value
distribution across the duplex was studied using 32 highly
effective siRNAs shown in Figures 2 and 3, but again there
was no apparent low internal energy across the duplex(Fig. 7A). Thus, the notion proposed by Khvorova et al. (30)
was partly supported by our study.
The experimental results in Figure 7B and C indicate DG° at
position 1 of three siRNAs that give rise to intermediate levelsof RNAi in mammalian cells (p, n and d) to be within the rangeof those of ®ve highly effective siRNAs (a, f, k, l and o). Thus,
based on thermodynamic stability calculation, the selection ofhighly effective siRNAs from a random siRNA set may bequite possible, but only at a probability of 60%.
DISCUSSION
The relationship between siRNA sequence and its ability togive rise to RNAi in mammalian cells was extensively
examined here and, on the basis of the results, rules were
established for siRNA sequence preference and are schematically presented in Figure 8A. The rules predict that siRNAs
satisfying all the four following sequence conditions at thesame time give rise to highly effective RNAi in mammaliancells and possibly also in chick embryos: A/U at the 5¢ AS end;
G/C at the 5¢ SS end; at least ®ve A/U residues in the 5¢
terminal one-third of the AS, and the absence of any GC
stretch of >9 nt in length. siRNAs opposite in features withrespect to the ®rst three conditions bring about little or no geneDownloaded from nar.oxfordjournals.org at Ekigaku-Kyoshitsu (UNIV OF TOKYO) on October 17, 2010

Figure 6. Comparison of siRNA-based RNAi and DNA-based RNAi in HeLa cells. The predicted sequences of hairpin-type transcripts are shown on the left,
while induced RNAi activity (reduction in relative luciferase activity) is shown by open boxes on the right. Stippled boxes indicate relative luciferase activity
reduction due to cognate siRNA in HeLa cells. On the left, predicted AS are shaded. Note that the sequence preference of DNA-based RNAi is essentially
identical to that of siRNA-based RNAi. Data obtained from 2±4 experiments were averaged and are shown.

Nucleic Acids Research, 2004, Vol. 32, No. 3945

silencing. A total of 57 highly effective and 16 ineffective
siRNA candidates have been designed for four exogenous and
23 endogenous genes to date based on these rules (this workand our unpublished data), and all have been found to produce
the anticipated RNAi activity in mammalian cells and chickembryos.
Recently, Holen et al. pointed out that siRNA-based RNAiin mammalian cells varied considerably depending on target
sequences (27). Their experimental results are clearly
explained based on our rules. They showed that only four of
11 siRNAs examined could give rise to effective RNAi in
HeLa, 293, Cos-1 and HaCaT cells. Our rules show that only
these four effective siRNAs belong to class Ia or Ib, highly
effective siRNA classes. Thus, the rules here may beconcluded to be very useful for designing highly effective
and ineffective siRNAs for silencing of mammalian and chickgenes. However, it should be pointed out that, while the four
conditions above are almost entirely suf®cient for highly
effective gene silencing, some may possibly be replaced byother functionally redundant conditions (see below for
example).
The secondary structure of target RNA has been shown to
be important for target mRNA recognition by siRNAs (41,42).
However, at variance with these considerations, our results
would indicate that target sequences are much more essential
for target recognition by siRNAs than the secondary structure.

No special secondary structure of the target can be deduced
from our rules. Possibly, the frequency of serious secondary
structure occurrence may be quite low in protein-coding
regions of mRNA used here as targets.
EGFP/ECFP RNAi experiments (see Fig. 3C) indicated thepresence of A/U at the 5¢ AS end to possibly be required not
only for target recognition but RISC formation as well, which
includes siRNA unwinding. The step size of unwinding for
UvrD DNA helicase is 5 bp (43) and thus a one-step motor
function of putative siRNA helicase may unwind several basepairs from one of the two siRNA ends at the earliest stage in
RISC formation. The 7 bp AS terminal duplex regions ofhighly effective and ineffective siRNAs are AU rich and GC
rich, respectively, and 5¢ AS ends of highly effective andineffective siRNAs are A/U and G/C, respectively. It would
thus follow that the putative siRNA helicase preferably
initiates unwinding of the RNA duplex in an AU-rich terminal
region with A/U at its 5¢ free end, while RNA duplex
unwinding from the GC-rich terminal region with G/C at its 5¢
free end is blocked. Our unpublished experiments (Y.Naito,
K.Ui-Tei and K.Saigo) have indicated that while virtually no
degradation of the sense target RNA (vimentin mRNA) isbrought about by VIM-35, a class III vimentin siRNA, ~80%of antisense target RNA is cleaved by the same siRNA, which
serves as class Ib siRNA for antisense target silencing. These
considerations would appear consistent with the asymmetric

Downloaded from nar.oxfordjournals.org at Ekigaku-Kyoshitsu (UNIV OF TOKYO) on October 17, 2010

Figure 7. Internal stability pro®les of siRNAs. Thermodynamic pro®les of highly effective 32 siRNAs (A), and 16 luc siRNAs shown in Figure 1 are composed of three groups giving rise to highly effective, intermediate and ineffective RNAi in mammalian cells (B±D). The vertical bars indicated in (A) show
the standard deviation of 32 highly effective siRNAs. Thick and open vertical bars, respectively, indicate free energy change ranges at position 1 of highly
effective and ineffective siRNAs.

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Nucleic Acids Research, 2004, Vol. 32, No. 3

RISC formation model recently proposed by Schwarz et al.
(29) for in vitro RNAi in Drosophila embryonic extracts. Thismodel predicts that siRNA unwinding preferably occurs at an`easier' duplex end, possessing A:U, G:U or unpaired bases at
its 5¢ end position and being thermodynamically less stable,
and that the strand with the 5¢ end serves as a single-stranded
guide RNA assembled into RISC. The importance ofthermodynamically unstable or ¯exible base pairs at or near
the AS end for siRNA unwinding in HEK 293 cells has also
been pointed out by Khvorova et al. (30). A RISC formation
mechanism similar to that proposed for the Drosophila in vitro
system should thus also be applicable to mammalian and chickin vivo RNAi (see Fig. 8A).
According to the rules established here, 5¢ AS and SS endsof highly effective siRNAs should be A/U and G/C, respectively, with the counterparts of ineffective siRNAs being G/C
and A/U (see Fig. 8B). This terminal base compositional
asymmetry may be important for determining the direction ofsiRNA unwinding. Recently, two Drosophila PIWI proteins
have been shown to be capable of binding to a 5 bp singlestranded RNA or siRNA duplex (44±46). We found that thePAZ domain of eIF2C1, a human PIWI protein, binds todsRNA with a 2 nt 3¢ overhang but not to those with blunt or 5¢
overhang ends (N. Doi, K. Ui-Tei and K. Saigo, unpublisheddata). In plant cells infected with tombusvirus, p19 may bindto siRNA ends and inhibit post-transcriptional gene silencing

Downloaded from nar.oxfordjournals.org at Ekigaku-Kyoshitsu (UNIV OF TOKYO) on October 17, 2010

Figure 8. Possible models of siRNA-based RNAi in mammalian cells. The
rules for siRNA sequence preference are schematically shown in (A).
siRNA unwinding might be effectively initiated from the AU-rich AS end
in the case of class Ia siRNA, lacking a long GC stretch. On the other hand,siRNA duplex unwinding might be suppressed from the GC-rich class III
AS end. G/C at the 5¢ SS end of class Ia and the 5¢ AS end of class III
siRNAs might provide a site for binding of an unidenti®ed protein possibly
suppressing siRNA unwinding (B). Alternatively, A/U at the 5¢ SS end ofclass III and the 5¢ AS end of class Ia siRNAs might serve as a binding site
for putative unwinding stimulation factors other than helicase. A long GC
stretch such as that found in siRNA n (see Fig. 4B) might prevent theelongation of siRNA duplex denaturation from the AS end (C). A/U at the5¢ AS and SS ends and their counterparts in the SS and AS, respectively,
are shown as hatched circles; G or C, closed circles. Terminal AU-rich or
GC-rich regions are boxed. Open arrows indicate the direction of siRNA
unwinding due to a hypothetical siRNA helicase.

(47). Thus, a protein or protein complex, possibly not relevant
to helicase but capable of binding preferentially to G/C or A/Uat siRNA ends, might be involved in early strand separation ofsiRNA so as to either suppress or stimulate siRNA duplex
unwinding.
Helicase functions might be doubly suppressed by G/C at
the 5¢ AS end position and an adjacent GC-rich sequence inhighly ineffective siRNAs, while helicase functions appear
blocked only by a single G/C pair at the 5¢ SS end position(Fig. 8), suggesting that a single G/C pair at the 5¢ SS end
position and a GC-rich sequence near the 5¢ SS end might befunctionally redundant with each other and, accordingly, thelatter might serve as a substitute for the former. We consider
that this might be the reason why siRNA a (a class II siRNA) is
capable of acting as a highly effective siRNA (see Figs 2A and
4B).
The results in Figure 2A indicate that siRNA n, possessing a
10 bp G/C stretch extending from the SS end, is incapable ofgiving rise to highly effective RNAi in mammalian cells,
although it belongs to class Ia. Complete strand separation ofsiRNA appears to be required for active RISC formation (16)and, consequently, a long G/C stretch extending from the SS
end may prevent helicase from unwinding not only from the
SS end but from the AS end as well in a G/C stretch lengthdependent manner (Fig. 8C).
In contrast to in vitro RNAi in Drosophila (29), in vivo
Drosophila RNAi was far less sensitive to the siRNAsequence (see Fig. 2); virtually all siRNAs gave rise toeffective RNAi in S2 cells when used at 50 nM. Our siRNAsequence preference rules established based on mammalianRNAi data were found to be not directly applicable to in vivo
Drosophila (Fig. 2). Unlike mammalian cells, Drosophila
cells might produce more protein components required for
RISC formation and, hence, be capable of accumulating aconsiderable amount of RISC with a less ef®cient siRNA
strand, i.e. asymmetric RISC formation may possibly not be arate-limiting step in RNAi in Drosophila cells.
Figure 4 also indicates highly effective class Ia siRNAs to
be comprised of heterogeneous members with over 10 times
the capacity to bring about RNAi and maximum genesilencing activity to be induced by siRNA l transfection to
CHO-K1 and S2 cells. Schwarz et al. (29) indicated genesilencing activity of siRNAs in the Drosophila in vitro system
to be improved by the introduction of a U:G pair or unpaired
bases at the 5¢ AS end position. There may thus be thepossibility of converting almost all class Ia siRNAs to siRNAs
capable of inducing maximum levels of RNAi or RNAi levels
brought about by siRNA l in mammalian cells via a change interminal base pairing.
In a separate study, 19 986 human and 16 256 murine
sequences registered in the NCBI Reference Sequence(RefSeq) database were examined using the siRNA sequence
preference rules established here, and 92 and 99% of humanand mouse sequences, respectively, were noted to possess atleast one unique potential target for class Ia siRNA without along G/C stretch (Y. Naito, K. Ui-Tei and K. Saigo,
unpublished data). Our rules should thus ®nd a wide scopeof application to the design of siRNAs that are highly effective
for mammalian RNAi including systematic mammalianfunctional genomics.

Nucleic Acids Research, 2004, Vol. 32, No. 3

ACKNOWLEDGEMENTS
We thank H. Sasaki and T. Hamada for supplying HeLa cells,
K. Nakamura for ES cells, J. Miyazaki for pCAGGS vector,
S. Zenno, T. Noce, H. Niwa and N. Doi for helpful discussions
and comments, H.Kaji for experiments at the initial stages, and
R. Eda and A. Tanaka for technical assistance. We also thankJohn Rose for critical reading of the manuscript and for
discussion. This work was partially supported by a Special
Coordination Fund for promoting Science and Technology toK.S., and grants from the Ministry of Education, Culture,
Sports, Science and Technology of Japan to K.S. and K.U.-T.

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