Cancer Gene Therapy (2011) 18, 587–597
r 2011 Nature America, Inc.

All rights reserved 0929-1903/11

www.nature.com/cgt

ORIGINAL ARTICLE

Enhanced specificity of HPV16 E6E7 siRNA by RNA–DNA
chimera modification
K Yamato1, N Egawa2, S Endo3, K Ui-Tei4, T Yamada5, K Saigo4, I Hyodo3, T Kiyono2 andI Nakagawa1
1

Section of Bacterial Pathogenesis, Graduate School of Medical and Dental Science, Tokyo Medical and
Dental University, Tokyo, Japan; 2Division of Virology, National Cancer Center Research Institute Tokyo,
Tokyo, Japan; 3Department of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba
Ibaraki, Ibaraki, Japan; 4Department of Biophysics and Biochemistry, Graduate School of Science, Universityof Tokyo, Tokyo, Japan and 5Division of Pathology, Keio University, School of Medicine, Tokyo, Japan
Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering
RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecificcytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1–8
and its complementary part of the passenger strand plus the 30 overhang, which is designated as a double-strand RNA–DNA
chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 andE7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 50 endof the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in
two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition,
silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced
E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show thatdsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.
Cancer Gene Therapy (2011) 18, 587–597; doi:10.1038/cgt.2011.28; published online 10 June 2011
Keywords: siRNA therapy; HPV16; cervical cancer; off-target effectIntroduction

A decade has passed since the discovery of RNAinterference (RNAi) induction by synthetic 21–23-nucleotide double-stranded small interfering RNA (siRNA)in mammalian cells.1,2 Since then, siRNA has becomea powerful tool for studying gene functions, as well as
development of potential drugs for viral infections andcancer treatments. However, the effects of siRNA are notas specific as initially proposed. siRNA silences theexpression of unintended targets with partial sequence
complementarities (off-target effects),3,4 although minimal matching of mRNA and the seed region of thesiRNA guide strand (GS) (nucleotide positions 2–8 fromthe 50 end) may be sufficient for off-target silencing.3,5,6
Nucleotide modifications at specific positions have been
Correspondence: Dr K Yamato, Section of Bacterial Pathogenesis,
Graduate School of Medical and Dental Science, Tokyo Medical
and Dental University, 1-5-45 Yushima, Bunkyo-kyu, Tokyo
113-8549, Japan.
E-mail: yamato.bac@tmd.ac.jp
Received 28 December 2010; revised 2 March 2011; accepted 30
April 2011; published online 10 June 2011

found to overcome the off-target problem.7–9 In addition,
DNA replacement in the seed region of the GS (positions
1–8 from the 50 end) and its complementary sequence ofthe passenger strand (PS), designated as double-stranded
RNA–DNA chimera (dsRDC), abolishes the off-target
effects with some decrease in silencing activity.8 Most
active siRNAs have been shown to induce a cytotoxic
effect by exerting off-target silencing and interfering withthe endogenous RNAi pathway,7,9–12 whereas cytotoxicity might be reduced by use of chemically modified
siRNA with low off-target activity7 or siRNA with highsilencing activity at a minimal concentration.
Cervical cancer is the second most common female
malignancy throughout the world, with high-risk types ofhuman papillomavirus, such as types 16, 18, 33, 45 and31, known to be causative agents.13 Among them, HPV16
is the most prevalent and accounts for about half ofreported cases. High-risk types of human papillomaviruses are small DNA viruses with a circular doublestranded DNA genome of about 8000 base pairs (bp)containing an upstream regulatory region, along with six
early (E6, E7, E1, E2, E4 and E5) and two late (L1 andL2) genes. High-risk types of human papillomaviruses
exert carcinogenic effects through constitutive expressions

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al588

of the E6 and E7 oncogenes.14–17 Uncontrolled expressions of E6 and E7, which are functionally equivalentto P53 and RB mutations in other cancer types, also
contribute to cellular immortalization, genetic instability,
accumulation of additional genetic changes and resultant
malignant transformation. In addition, their expressions
are essential for maintaining a malignant phenotype,whereas their suppression results in apoptosis and cellularsenescence by restoring and activating the functions of
P53 and RB.18–24 Thus, E6 and E7 are considered to beideal therapeutic targets. Prophylactic vaccines covering
HPV16 and 18 are now clinically available, and have beenshown effective for preventing infections as well as
cervical cancer.25 However, these vaccines do not benefit
individuals who are already infected and their long-term
effects remain to be confirmed. Thus, it is necessary todevelop new therapies for patients who have persistentinfections, precancerous lesions or overt cancer.
A large number of siRNA sequences targeting HPV16
E6E7 oncogenes have been found to exert negative
growth effects on HPV16-positive cancer cells.21–23,26
Among them, we previously reported that three siRNAs(497, 573 and 752) were highly potent.27 In this study, weinvestigated the effects of short dsRDC modification(positions 1–6 from the 50 end of the GS and its
complementary parts of the PS) on cytotoxicity and offtarget effects exerted by some of those potent E6E7
siRNAs. We found that most of the nonspecific cytotoxicity was attenuated by dsRDC modification with aconcomitant marginal or moderate reduction in silencing
activity. Among those modified dsRDCs, one with hightarget specificity and preserved silencing activity suppressed the growth of E6E7-immortalized human keratinocytes, an early cancer model, as well as cervical cancer
cells. These findings indicate the potential of E6E7
dsRDC in therapeutic applications for HPV16-relateddiseases.

Materials and methods

Cell lines and plasmid transfection
SiHa (HPV16-positive cervical cancer), HeLa (HPV16negative cervical cancer) and SNU-1 (gastric cancer) celllines were purchased from American Type Culture
Collection (ATCC, Rockville, MD). SiHa and HeLa cells
were cultured in Dulbecco’s modified Eagle’s medium
containing 10% heat-inactivated fetal calf serum. SNU-1
cell lines were cultured in RPMI1640 medium containing
10% fetal calf serum. Primary human keratinocytes(HDK) were purchased from Cell Applications (San
Diego, CA) and immortalized by the catalytic subunit ofhuman TERT basically, as described previously,28 exceptthat the present HDK were transduced with human TERT
alone, and not a combination of human TERT and Bmi-1(HDK1-T). Generation of HPV16 E6E7-immortalized
HDK (HDK1-E6E7) was performed as reported previously.28 HDK, HDK1-T and HDK1-E6E7 were cultured in serum-free keratinocyte media with recombinant
epidermal growth factor (5 ng ml1) and bovine pituitary

Cancer Gene Therapy

extract (5 mg ml1) (Invitrogen, Carlsbad, CA) at 37 1C
in an atmosphere of 5% CO2. Generation of stableclones expressing firefly luciferase (FLuc) (FL-SiHa-2,
FL-HeLa-1) was as reported previously.27 Plasmidtransfection was performed using Lipofectamine 2000
transfection reagent (Invitrogen) according to the manufacturer’s instructions.

siRNAs and transfection
The sequences of the siRNAs used in this study27
are summarized in Supplementary Table 1. All siRNAs
were designed using the siDirect software (http://sidirect2.rnai.jp/), as reported previously.29 Control
siRNA was an artificial sequence designed to have theleast homology to human and mouse genes. Control
siRNA and its dsRDC-modified form were included in all
experiments. For preparation of dsRDCs, six nucleotides
from the 50 end of GS and eight from the 30 end of PS
were substituted with corresponding DNAs. Twenty-onebase nucleotides were synthesized, 20 -deprotected, desalted and annealed in buffer (100 mM potassium acetate,
30 mM HEPES-KOH at pH 7.4, 2 mM magnesium
acetate).
At 1 day before transfection, cells were plated at adensity of 2–4  104 cells per well in 24-well plates
containing 0.5 ml of Dulbecco’s modified Eagle’s medium
with 10% fetal calf serum. For analysis of the effects ofsiRNA on cell growth and viability, cells were plated at alow density (1–2  103 cells per well) in 96-well plates
containing 0.1 ml of Dulbecco’s modified Eagle’s medium
containing 10% fetal calf serum. Various amounts ofsiRNA and Lipofectamine RNAiMAX (0.6 ml) (Invitrogen), both of which were added along with Opti-MEM I
reduced serum medium (Opti-MEM, Invitrogen) up to
50 ml, were combined, incubated at room temperature for
20 min and then added to 0.5-ml cultures. For transfection of SiHa cells and their derivatives, 1 ml of
Lipofectamine RNAiMAX was used. For transfectionof immortalized keratinocytes, the culture medium was
replaced at 5 h after transfection. For co-transfection of
HeLa cells with the plasmid and siRNA or dsRDC,
Lipofectamine 2000 was used.
Plasmid construction
Renilla luciferase (RLuc) cDNA fused with HPV16 E6(231–559) (RLuc-DNE6) and RLuc-E7 (562–858)
were isolated from 16DNE6/psiCheck-2 and 16E7/
psiCheck-2,27 respectively, and then cloned into pZeoSV2(Invitrogen) at the NheI and NotI sites. The resultant
plasmids were designated as 16DNE6/pZeoSV2 and 16E7/
pZeoSV2, respectively. Construction of RLuc-DNE6E7/
psiCheck-2 was reported previously.27 Next, 19-nucleotide human gene sequences carrying 3-nucleotide mismatches to GS of each E6E7 siRNA were selected using
the siDirect software29 (Table 1). The sense and antisense
oligoDNAs of these sequences are summarized in
Supplementary Table 2. Sense and antisense oligoDNAs
were phosphorylated, annealed and subcloned at the XhoI
and NotI sites of psiCheck-2 (Promega, Madison, WI).

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al

589

Table 1 Off-target candidate human genes carrying 3-nucleotide mismatches to GS of siRNAs targeting HPV16 E6E7
Sequence nameOT gene accession number

Gene definition and gene symbol

GS sequence (3 0 ’5 0 )OT mRNA (5 0 -3 0 )497OT-1

BX647877

cDNA clone DKFZp313E2325

497OT-2

NM_006237.2

POU4F1573OT-1

NM_000922.2

PDE3B

573OT-2a

AL832149

cDNA clone DKFZp686P15111

573OT-3

NM_001517.4

GTF2H4573OT-4a

BF693154

cDNA clone IMAGE:4244422 50

573OT-5b

AF138859

cDNA clone FLB2932

573OT-6b

BC036590

cDNA clone IMAGE:4801089

573OT-7

NM_004582.2

RABGGTB

573OT-8

AA861522

cDNA clone IMAGE:1407653 30

573OT-9

F03276

cDNA clone c-1sa08 30

752OT-1

BQ054887

cDNA clone IMAGE:5806941 50

GAUCUGUCGAUGUAUGUUU||X|X||||||||||||X|
CUGGCCAGCUACAUACAGAUACCUGUCCAUGUAUGUCU
X|||X|||X||||||||||CUGGCCAGCUACAUACAGACACAUACAUUGAAUAAAUA|||X|||||||X||X||||GUGGAUGUAACGUACUUAUCACCUACACAGCAUGAAUU||||||||XX||||||||X
GUGGAUGUAACGUACUUAUCACCUACAAUGCAGGAAUC||||||||X||||X||||X
GUGGAUGUAACGUACUUAUCACCUACACAGCAUGAAUU||||||||XX||||||||X
GUGGAUGUAACGUACUUAUCAACUACAUUACAUUAAUA||X|||||||X|||X||||GUGGAUGUAACGUACUUAUCAACUACAUUACAUUAAUA||X|||||||X|||X||||GUGGAUGUAACGUACUUAUCAACUUCAUCGCAUGAAUA||X||X|||X|||||||||GUGGAUGUAACGUACUUAUCUCCUACAUUGUAGGAAUA|X|||||||||X|X|||||GUGGAUGUAACGUACUUAUCACCUCCAUUUAAUGAAUA|||||X||||XX|||||||GUGGAUGUAACGUACUUAUCUUCGGCUGCGCGUAAAAA||||||X||X|||||X|||
GAAGCCAACACGCAUGUUUAbbreviations: GS, guide strand; HPV, human papillomavirus; OT, off-target candidate; PS, passenger strand; siRNA, small interferingRNA.
All human genes with 3-nucleotide mismatches to the GS of each E6E7 siRNA were selected using the siDirect software.
Matched and mismatched base pairs between off-target candidate sequences and GSs are visualized by vertical lines and crossmarks, respectively.
a
The sequence of 573OT-4 is identical to that of 573OT-2.
b
The sequence of 573OT-6 is identical to that of 573OT-5.

Immortalized human keratinocytes stably expressing
FLuc
FLuc cDNA was amplified using a polymerase chain
reaction (PCR) method with Platinum PCR Supermix
High Fidelity (Invitrogen) and sense and antisense
primers, with psiCheck2 utilized as a template. The
PCR product was cloned into pCR8/GW/TOPO(Invitrogen) using Topoisomerase I (Invitrogen) andsequenced (FLuc/pCR8/GW). FLuc cDNA was thensubcloned in a lentivirus expression plasmid (pLenti6.3/
V5-DEST; Invitrogen) with LR Clonase II (Invitrogen)and the resultant lentivirus plasmids were designated as
FLuc/pLenti6.3. An infectious recombinant virus wasproduced by co-transfection of 293FT cells (Invitrogen)with FLuc/pLenti6.3 and packaging plasmids (pLP1,

pLP2 and pLP/VSVG) (Invitrogen) using Lipofectamine
2000, according to the manufacturer’s instructions.
The culture medium was changed at 24 h and collected
at 96 h after transfection. HDK1-T and HDK1-E6E7
were infected with a recombinant lentivirus for 6 h in thepresence of polybren (6 mg ml1), and then selected using
2.5 mg ml1 of blasticidin (Invitrogen), and FLuc-expressing cells were designated as HDK1-T-luc and HDK1E6E7-luc, respectively.

SiHa cells stably expressing FLuc and RLuc-E6, or
RLuc-E7, and luciferase assay
SiHa cells stably expressing both FLuc and RLuc-E6(RL-E6-FL-SiHa-10), and those expressing both FLucand RLuc-E7 (RL-E7-FL-SiHa-2) were obtained by

Cancer Gene Therapy

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al590

transfection of FL-SiHa-2 cells27 with 16DNE6/pZeoSV2
and 16E7/pZeoSV2, respectively, and antibiotic selection using both zeocin (1 mg ml1; Invitrogen) andG418 (1 mg ml1; Invitrogen). For luciferase assays,
cells were harvested at 48 h after transfection with siRNAor dsRDC, and then luciferase activity was determined using a Dual Luciferase Reporter Assay System(Promega).

Real-time quantitative reverse transcription-PCR
Total RNA was extracted from cells using a High PureRNA Isolation Kit (Roche Diagnostics, Mannheim,
Germany), according to the manufacturer’s instructions.
cDNA was synthesized using a Transcriptor First Strand
cDNA Synthesis Kit (Roche Diagnostics). Real-timequantitative reverse transcription-PCR assays were performed using a LightCycler 480 System (Roche Diagnostics) in 96-well plates. Each reaction was carried out in
20 ml containing 10 ml of LightCycler 480 Probes Master(Roche Diagnostics), 5 ml of cDNA from a 10  dilutionof the reverse transcriptase reaction (equivalent to 25 ngof reverse-transcribed RNA), 900 nM of a forward andreverse prime pair and 250 nM of 6-carboxyfluoresceinlabeled probe. Serial dilutions of plasmid DNA(10–0.01 ng) were used to generate a standard curve.
The LightCycler protocol was used as follows: initial cycleof 95 1C for 10 min, followed by 40 cycles of 95 1C for 15 s
and 60 1C for 1 min. Water was used as the template fornegative control amplifications included with each PCR
run. All reactions were performed in triplicate in each
96-well plate. Data were analyzed using the Roche
LightCycler 480 software and the crossing point (Cp)value was calculated with the Second Derivative Maximum Method. The amount of target mRNA was
examined and normalized to that of 18S ribosomal RNA.
The primers and probes for E6-E7 and E6*I-E7 havebeen described by others.30 Those for 18S ribosomal
RNA and AL832149 were designed by Sigma Aldrich
Japan (Tokyo, Japan) and Applied Biosystems (Foster
City, CA), respectively. Primers and probes for POU4F1(NM_006237.3) and GTF2H4 (NM_001517.4) wereobtained from Applied Biosystems (POU4F1, Assay ID
Hs00366711_m1; GTF2H4, Assay ID Hs00231008_m1). Allprobes were labeled at the 50 end with the reporterdye 6-carboxyfluorescein and at the 30 end with a nonfluorescent quencher. The PCR primer and probe sequences
are listed in Supplementary Table 3.
Analysis of cell growth and senescence-associatedb-galactosidase staining
Cell viability was examined with a colorimetric assay
using WST-8 (Cell Counting Kit 8; Dojin Laboratories,
Kumamoto, Japan), according to the manufacturer’s instructions. Senescence-associated b-galactosidase (SAb-gal)
staining was performed using a Senescent Cell Staining Kit(Sigma-Aldrich). The viability of HDK1-T-luc and HDK1E6E7-luc cells was determined using a Luciferase Assay
System (Promega).

Cancer Gene Therapy

Statistical analysis
The statistical significance of differences was determinedusing an unpaired two-tailed Student’s t-test. Differenceswere considered statistically significant at Po0.05.

Results

Silencing activities of E6E7 siRNAs and their dsRDC
counterparts
In this study, we examined whether dsRDC modificationof HPV16 E6E7 siRNAs with high silencing activity(497, 573 and 752; Figure 1a) would increase their target
specificity and reduce nonspecific cytotoxicity. dsRDCs
with an 8-nucleotide DNA substitution often show lower
silencing activity than those with a 6-nucleotide DNA
substitution (data not shown). We used the shorter DNA
substitution in this study (Figure 1b).
HPV16 E6E7 siRNAs (497, 573 and 752) and theirdsRDC forms were compared with regard to theirsilencing activity using HeLa cells transiently expressing
RLuc-E6E7. HeLa cells were co-transfected with RLucE6E7-expressing plasmids along with siRNAs (control,
497, 573 and 752) and their dsRDC counterparts. At 48 h
after transfection, a dual luciferase assay was performed.
As shown in Figure 1c, siRNAs 497, 573 and 752
exhibited potent silencing activity (8.4, 11.6 and 5.6%,
respectively), whereas control siRNA showed very mild
suppression (72.1%). dsRDC modification of thesesiRNAs slightly reduced the silencing activities of siRNAs
497 and 573 (16.2 and 13.6%), and moderately that of 752(22.0%). SiHa cells stably expressing both RLuc-E6 andFLuc (RL-E6-FL-SiHa-10) were used for determining
the half-maximal inhibitory concentration (IC50) valuesof siRNA-497 and its dsRDC. Various concentrationsof siRNA and dsRDC were transfected into RL-E6-FLSiHa-10 cells, and then a dual luciferase assay was
performed 48 h after transfection. siRNA-497 showedmaximal suppression of RLuc activity to 4.6% at 5 nM
with an IC50 of 0.015 nM, whereas dsRDC modificationslightly decreased the silencing activity by 6.8-fold(IC50, 0.105 nM) (Figure 1d). Similarly, siRNAs targeting
E7 (573 and 752) were analyzed with RL-E7-FL-SiHa-2
cells expressing RLuc-E7 and FLuc. siRNA-573 had ahigh silencing activity with an IC50 of 0.019 nM anddsRDC modification increased the IC50 by 5.2-fold(0.098 nM; Figure 1e). In contrast to the other two
dsRDCs, dsRDC-752 had a low level of silencing activity
and was 32-fold less active than its siRNA form (IC50
1.774 vs 0.055 nM) (Figure 1f). Both control siRNA anddsRDC had negligible effects at 1 nM on RLuc expression
in RL-E6-FL-SiHa-10 and RL-E7-FL-SiHa-2 cells (datanot shown).
Effects of E6E7 dsRDC on endogenous E6E7 mRNAs
The effects of dsRDCs targeting E6E7 on endogenous
E6-E7 and E6*I-E7 mRNA expressions were comparedwith their siRNA counterparts using HPV16-positve
cervical cancer cells. FL-SiHa-2 cells were transfectedwith siRNAs (control, 497, 573 and 752) or their dsRDC

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al

591
A

573

497

97

E6-E7 mRNA
(E6 coding)

AA
AA

752

AA

AA

E6*I-E7 mRNA 97(E7 coding)97
E6*II-E7 mRNA

226

A
AA

AA

409

A

A
AA

A
AA

526

226

556

104

562

855
E7

E6

E6 E7 ORFs

siRNA
5'
3'

5'
dsRDC

3' PS
GS

8 nt DNA

5'

3' PS
5'
GS

3'
6 nt DNA

relative RLuc activity (%)

125
dsRDC

siRNA
100
75
50

†
*

*

25
***

* **

497

573

**

0
control
497

IC50 0.105nM

75
50
siRNA
IC50 0.015nM
00.0001 0.001 0.01

0.1

1

10

siRNA/dsRDC concentration (nM)

dsRDC
IC50 0.098nM

100
75
50
25

752

125
relative RLuc activity (%)

dsRDC
100

25

573

125
relative RLuc activity (%)

relative RLuc activity (%)

125

siRNA
IC50 0.019nM

00.0001 0.001 0.01

0.1

1siRNA/dsRDC concentration (nM)

10

752

dsRDC
100

IC50 1.774nM

75
50
siRNA

25

IC50 0.055nM

0
0.001

0.01

0.1

1

10

siRNA/dsRDC concentration (nM)

Figure 1 RNA–DNA chimera modification of small interfering RNA (siRNA) targeting human papillomavirus 16 (HPV16) E6E7. (a) E6E7 mRNAand splicing variants, and E6E7 siRNAs. siRNAs targeting E6 and E7 are depicted as short lines (top). Open bars represent mRNAs coding E6
and E7 with introns within E6 (middle). Gray area indicates cellular sequence derived from the chromosomal integration site. Closed boxes show
locations of the E6 and E7 coding regions of the HPV16 prototype (accession number K02718) (bottom). Numbers refer to the locations of thetranscription initiating site and splicing junctions. ORF, open reading frame. (b) Structure of double-strand RNA–DNA chimera (dsRDC).
Conventional siRNA is a 19-bp long double-stranded RNA with 2-nucleotide 30 overhangs. The dsRDCs used in this study contained 6-nucleotide
DNA in the 50 region of the guide strand and 8-nucleotide DNA in the 30 region of the PS. Open and closed bars represent ribonucleic acid anddeoxyribonucleic acid, respectively. PS, passenger strand; GS, guide strand. Silencing activities of control and HPV16 E6E7 targeting siRNAs(497, 573 and 752), and their dsRDC forms were determined by transient transfection of HeLa cells with a Renilla luciferase (RLuc)-E6E7/
psiCheck-2 plasmid, as well as siRNAs and dsRDCs. At 48 h after transfection, a dual luciferase assay was performed. RLuc activities are shown
as relative to mock-transfected cells (c). The half-maximal inhibitory concentration (IC50) values of siRNA and dsRDC targeting E6 (497) were
determined using SiHa cells stably expressing firefly luciferase (FLuc) and artificial fusion mRNA of RLuc-E6 (RL-E6-FL-SiHa-10) (d), whereas
those targeting E7 (573 and 752) were analyzed using SiHa cells stably expressing FLuc and RLuc-E7 (RL-E7-FL-SiHa-2) (e, f). Cells weretransfected with various concentrations of siRNAs or dsRDCs, and then analyzed using a dual luciferase assay 48 h after transfection. RLucactivities were normalized to those of FLuc. RLuc activities are shown as relative to mock-transfected cells. Experiments were performed intriplicate and error bars represent the standard deviation from the mean. Data points marked with asterisks are statistically significant ascompared with control transfection (*Po0.001; **Po0.01; ***Po0.05). Data points marked with crosses are statistically significant (wPo0.01).

counterparts at 5 nM for 72 h, and then analyzed for levels
of mRNAs coding E6 and E7 by quantitative reverse
transcription-PCR. As shown in Table 2, transfection
with siRNAs 497, 573 and 752 decreased the levels of both
E6-E7 and E6*I-E7 mRNA to 13.5–27.6 and 3.2–6.4%,
respectively. Their dsRDC counterparts 573 and 752 wereless active, although they still suppressed those levels to
30.9–32.7 and 17.2–31.2%, respectively. dsRDC-497 also

suppressed E6*I-E7 mRNA expression to 24.9%, however, its effect on E6-E7 mRNA expression was indistinguishable from that of control dsRDC (64.7 vs 67.2%).
Thus, dsRDC modification of most E6E7 siRNAs slightly
decreased the silencing activity towards E6-E7 and E6*IE7 mRNA expressions, except for dsRDC-497, which
exerted a silencing effect only on E6*I-E7 mRNA and not
on E6-E7 mRNA.

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DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al

Effects of HPV16 E6E7 siRNAs and dsRDCs on growthof HPV16-positive and -negative cancer cells
To examine if dsRDCs targeting E6E7 are capable ofinducing specific growth inhibition of HPV16-related
cancer cells, the effects of dsRDC on cell growth were
examined using HPV16-positive (FL-SiHa-2) and -negative cells (FL-HeLa-1). Control and E6E7 dsRDCs (497,573 and 752) as well as their siRNA counterparts weretransfected into FL-SiHa-2 cells at 2 nM. All E6E7
dsRDCs tested in this study suppressed the growth of
FL-SiHa-2 cells, although the effects of two of thedsRDCs (497 and 752) were less potent as comparedwith those of their siRNA counterparts (Figure 2a).
HeLa and FL-HeLa-1 cells are highly vulnerable tosiRNA-mediated nonspecific cytotoxicity.9,27 By taking
advantage of this characteristic, E6E7 dsRDC and siRNA(497, 573 and 752) were evaluated for their nonspecificcytotoxicity by determining cell viability 7 days after
transfection. As shown in Figure 2b, all siRNAs including
the control showed moderate to strong growth suppression toward FL-HeLa-1 cells. dsRDC modification ofthese siRNAs (control, 497 and 752), except for 573,
also decreased cytotoxicity as compared with theirsiRNA counterparts. These results suggest that mostsiRNAs induce nonspecific cytotoxicity9 and that dsRDC
modification reduces the level of cytotoxicity.
The negative growth effect of E6E7 siRNA on E6E7positive cancer cells is mediated in part by inducing
cellular senescence.24,26,27 We examined if E6E7 dsRDCs
capable of specific growth suppression (497 and 752) also
induced senescence in FL-SiHa-2 cells. siRNA-573 and its
dsRDC were omitted, because of their strong nonspecifictoxicity. Cells were transfected with either control siRNA,
E6E7 siRNA or their dsRDC counterparts for 7 days, andthen tested for the expression of SAb-gal, a hallmark of
cellular senescence. The proportions of SAb-gal-positive
cells transfected with control siRNA and dsRDC was 8.6
and 3.5%, respectively, whereas those of siRNA-497- anddsRDC-497-induced cells were 64.1 and 70.3%, respectively. dsRDC-752 induced a slightly lower percentage ofSAb-gal-positive cells (46.6%) than its siRNA counterpart (78.8%) (Figure 2c). Thus, it was shown that E6E7
dsRDC exerts growth suppression in HPV16-positivecancer cells through a mechanism similar to that ofsiRNA.

Table 2 qRT-PCR analysis of E6-E7 and E6*I-E7 mRNA levels in
FL-SiHa-2 cells transfected with siRNAs and dsRDCs targeting
HPV16 E6E7
E6-E7 mRNA (%)

E6*I-E7 mRNA (%)

Mock

100.0±5.0

100.0±10.8

Control
siRNA
dsRDC

53.8±4.1
67.2±1.355.3±8.1
72.2±3.6

497
siRNA
dsRDC

27.6±0.764.9±4.2

5.9±0.624.9±1.9

573
siRNA
dsRDC

21.4±1.130.9±0.9

6.4±0.717.2±0.9

752
siRNA
dsRDC

13.5±1.1
32.7±0.9

3.2±0.431.2±2.9

Abbreviations: dsRDC, double-strand RNA–DNA chimera;FL, firefly luciferase; HPV, human papillomavirus; qRT-PCR,
quantitative reverse transcription-polymerase chain reaction;s.d., standard deviations; siRNA, small interfering RNA.
E6-E7 and E6*I-E7 mRNA levels were analyzed by qRT-PCR
and were normalized to that of 18S rRNA. Mean values and s.d.
of relative mRNA levels (%) to mock-transfected cells areshown. Experiments were carried out in triplicate.

100

125

125

siRNA

dsRDC

siRNA

100
%Cell viability

100
75
50
†

†
25

**
*

0

*
**

**

*

573

752

†

**

75
50
25
***
0

control

497†

control

497

** *
573

dsRDC
*

siRNA

dsRDC
%SA-b gal potive cell

siRNA/dsRDC%Cell viability

592

*

80

*
**

60
40
20
0

752

cont

497

752

Figure 2 Effects of E6E7 double-strand RNA–DNA chimera (dsRDCs) on growth of human papillomavirus 16 (HPV16)-positive and -negative
cervical cancer cells. HPV16-positive FL-SiHa-2 (a) and HPV16-negative (FL-HeLa-1) cells (b) were transfected with either mock, smallinterfering RNA (siRNA) (control, 497, 573 and 752) or their dsRDC counterparts at 2 nM. Cell viability was determined using a WST-8 assay 5–7
days after transfection. Cell viability relative to the mock-transfected cells is shown. (c) Induction of senescence-associated b-galactosidase(SAb-gal) activity in FL-SiHa-2 cells by dsRDCs targeting E6E7. FL-SiHa-2 cells were transfected with mock, siRNA (control, 497 and 752) (1 nM)
or dsRDC (control, 497 and 752) (2 nM) for 7 days, and then stained for SAb-gal activity. Values for SAb-gal-positive cells are shown aspercentages. All assays were performed in triplicate and error bars represent the standard deviation from the mean. Data points marked withasterisks are statistically significant as compared with control transfection (*Po0.001; **Po0.01; ***Po0.05). Data points marked with crosses
are statistically significant (wPo0.01).

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DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al593
relative RLuc activity (%)

140

siRNA target
Cont

(-)

RLuc

GS

RLuc

sense
orientation

PS

RLuc

antisenseorientation

siRNA

120

dsRDC

100
80
60

**

40
20

***

***

0

Cont

497GS

497PS

Target sequence

120

140

siRNA

dsRDC

100
80
60
**

40
20
*

*

0
Cont

573GS

573PS

Target sequence

relative RLuc activity (%)

relative RLuc activity (%)

140

siRNA
120

dsRDC

100
80
60
40

*
20

*

*

752GS

752PS

0

Cont

Target sequence

Figure 3 Lack of passenger-strand mediated RNA interference (RNAi) in E6E7 double-strand RNA–DNA chimera (dsRDC). Plasmids ligatedwith a small interfering RNA (siRNA) target sequence in a sense orientation (497GS, 573GS and 752GS) expressed targets of the guide strand(GS)-loaded RNA-induced silencing complex (RISC), whereas those in an antisense orientation (497PS, 573PS and 752PS) expressed targetsof the passenger strand (PS)-loaded RISC (a). HeLa cells were co-transfected with Renilla luciferase (RLuc)-GS (497GS, 573GS and 752GS) or
RLuc-PS expression plasmids (497PS, 573PS and 752PS), along with respective siRNA and dsRDC (497 (b), 573 (c) and 752 (d)). Data points
marked with asterisks are statistically significant as compared with control transfection (*Po0.001; **Po0.01; ***Po0.05). All siRNAs examined
here exhibited PS- and GS-mediated RNAi, whereas all examined dsRDCs showed only GS-mediated RNAi.

Lack of PS-mediated RNAi in E6E7 dsRDCs
To examine the differences between E6E7 siRNAs andtheir dsRDC forms with regard to PS-mediated silencing
activity, we used an expression plasmid comprised of
RLuc fused with 23-bp siRNA target sequences in sense
and antisense directions as GS and PS targets, respectively(Figure 3a). As shown in Figure 3b–d, all siRNAs tested(497, 573 and 752) suppressed their respective RLuc-GS
as well as RLuc-PS expression, whereas their dsRDC
counterparts suppressed only RLuc-GS.
Effects of HPV16 E6E7 siRNAs and dsRDCs on off-target
candidate genes
We also examined whether 6-nucleotide DNA substitution in the seed region (nucleotides 1–6 from the 50 end ofGS) could alter the off-target property of siRNA towards
human gene sequences with 3-nucleotide mismatches toeach E6E7 siRNA GS (Table 1), using plasmids of RLuc
fused with 19-bp off-target candidate gene sequences(OT). HeLa cells were transfected with siRNA or dsRCD(2 nM) along with a corresponding RLuc-OT plasmid,and then analyzed using a dual luciferase assay. E6E7
siRNAs and dsRDCS showed no or very mild inhibitory effects on 7 of 10 sequences with less than 35%

suppression (data not shown). Furthermore, 497OT-2 wasinhibited by siRNA-497 to 23.6%, whereas its suppression by dsRDC-497 was significantly less (65.6%,
Po0.001; Figure 4a), indicating that dsRDC modification of 497 reduced off-target activity. In addition,
573OT-2 and 573OT-3 expressions were suppressed bysiRNA-573 to 20.3 and 49.5%, respectively. Surprisingly,
dsRDC modification of siRNA-573 did not reduce theinhibitory effect on 573OT-2 or OT-3 (Figure 4b and c).
Next, we examined whether E6E7 siRNA (497 and 573)
could suppress mRNA expression of the off-target candidate genes (497OT-2, POU4F1; 573OT-2, AL832149;
573OT-3, GTF2H4) using quantitative reverse transcription-PCR. As neither SiHa nor HeLa cells expressed
POU4F1, we used SNU-1 cells, which expressed all three
OT genes. SNU-1 cells were transfected with siRNA-497
or dsRDC-497 for 2 days, and then analyzed for mRNA
expression of POU4F1 (Figure 4d). siRNA-497 suppressed the POU4F1 mRNA level to 57.7%, whereas
dsRDC-497 had no effect. The mRNA levels were parallel
to RLuc activities of 497OT-2 (Figure 4a). Similarly, theeffects of siRNA-573 and its dsRDC form on AL8321449
mRNA level were examined using SNU-1 cells(Figure 4e). siRNA-573 decreased its level to 65.4% andCancer Gene Therapy

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al

594
dsRDC

siRNA

***

50
*

25

75
50
*

*

25

dsRDC

POUF1

†

75
**
50
25

control

497

75
*

50

*

25

control

dsRDC

573

siRNA

dsRDC

125
GTF2H4

AL832149

100

†
**

75

*

50
25

100
75
50
25
0

0

0

100

573

siRNA
125

100

dsRDC
573OT-30
control497

relative mRNA levle (%)

relative mRNA levle (%)

573OT-2
1000control

siRNA
125

siRNA
125
relative RLuc activity (%)

75

relative RLuc activity (%)†

relative mRNA levle (%)

relative RLuc activity (%)

497OT-2
100

0

dsRDC

siRNA
125

125

control

573

control

573

Figure 4 Effects of human papillomavirus 16 (HPV16) E6E7 small interfering RNA (siRNA) and double-strand RNA–DNA chimera (dsRDC) on
off-target candidate sequences. All human gene sequences carrying 3-nucleotide mismatches to the guide strand (nucleotides 1–19 from the 50end) of E6E7 siRNAs are listed in Table 1. The 23-nucleotides oligoDNAs encompassing these 19-nucleotide sequences were synthesized andligated downstream to the stop codon of Renilla luciferase (RLuc) in a psiCheck2 plasmid. HeLa cells were co-transfected with RLuc-497OT-2(a) or RLuc-573OT-2 (b), or an RLuc-573OT-3 expression plasmid (c), along with respective siRNAs and their dsRDC counterparts (2 nM). After48 h of incubation, cells were analyzed using a dual luciferase assay. RLuc activities were normalized to those of firefly luciferase (FLuc). RLucactivities relative to mock-transfected cells are shown. SNU-1 cells transfected with siRNAs (473 and 573) and their dsRDC counterparts at
100 nM were incubated for 48 h, and then analyzed for 497OT-2 (POU4F1, d), 573OT-2 (AL832149, e) and 573OT-3 (GTF2H4, f) expressions
using quantitative reverse transcription-PCR (qRT-PCR). mRNA levels were normalized to that of 18S rRNA and those relative to mocktransfected cells are shown. All experiments were performed in triplicate and error bars represent the standard deviation from the mean. Data
points marked with asterisks are statistically significant as compared with control transfection (*Po0.001; **Po0.01; ***Po0.05). Data points
marked with crosses are statistically significant (wPo0.01).

dsRDC modification partially relieved the suppression(80.8%). GTF2H4 mRNA expression was unaffected bysiRNA-573 and its dsRDC form (91.9 and 93.4%, respectively; Figure 4f ), whereas expression of 573OT-3 carrying the same target sequence was inhibited by more than
twofold (Figure 4c), suggesting that GTF2H4 mRNAmight have lower accessibility to RNA-induced silencing
complex than artificial 573OT-3 mRNA. These results
showed that E6E7 siRNA exerts off-target effects toward
some human genes carrying 3-nucleotide mismatches tosiRNA and that dsRDC modification can reduce theoff-target effects of some, if not all, mRNAs.

Effects of HPV16 E6E7 siRNAs and dsRDC on growthof E6E7-positive and -negative keratinocytes
Finally, we examined whether E6E7 dsRDC (497)suppressed the growth of E6E7-immortalized keratinocytes (HDK1-E6E7) with features of early cervical cancer
cells.31 TERT-immortalized keratinocytes (HDK1-T)
were used as an E6E7-negative control. The vulnerabilityof these cells to FLuc siRNA transfection was tested using
HDK1-E6E7-luc and HDK1-T-luc. The dose effect ofFLuc siRNA on FLuc activity was similar between thecells at concentrations of 0.004–1.0 nM (data not shown),

Cancer Gene Therapy

indicating that these cells could be transfected withsiRNA to induce target gene silencing with comparable
efficacy.
HDK1-E6E7-luc and HDK1-T-luc cells were transfected with control and E6E7 siRNAs, as well as theirdsRDC-modified forms (497 and 752) at 0.5 nM for
6 days. A WST8 assay did not reflect a viable cell numberof E6E7 siRNA-treated HDK1-E6E7 cells (data notshown), thus we took advantage of the constitutive
FLuc expression of these cells and relative cell viability
was determined based on FLuc activity, which paralleledcell viability. As shown in Figure 5a and b, siRNA-497
suppressed the growth of TERT-immortalized keratinocytes as well as E6E7-immoratalized keratinocytes,
indicating that siRNA-497 had nonspecific growth suppression, as also seen in FL-HeLa-1 cells (Figure 2b).
When siRNA-497 was modified to a dsRDC form, nonspecific growth inhibition of HDK1-T-luc was reduced
and cell viability recovered to 62.5%, with a marginalloss of growth suppression of E6E7-immortalized cells(19.1%). siRNA-752 also showed specific growth suppression as compared with siRNA control and siRNA-497.
However, dsRDC modification compromised the E6E7specific growth suppression of siRNA-752. Taken together,

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al
siRNA

siRNA

dsRDC

125

125
HDK1-T-luc

100

relative cell viablity (%)

relative cell viability (%)

HDK1-E67E7-luc

75
50
**

25
*
0

dsRDC

*

100
†
75
50
*

25
0

control

497

752

control

497

752

Figure 5 Specific growth suppression of E6E7-immortalized human keratinocytes by E6E7 double-strand RNA–DNA chimeras(dsRDCs). Human papillomavirus 16 (HPV16) E6E7-immortalized
keratinocytes (HDK1-E6E7) and TERT-immortalized (HDK1-T)
stably expressing luciferase were obtained, and designated as
HDK1-E6E7-luc and HDK1-T-luc, respectively. HDK1-E6E7-luc (a)and HDK1-T-luc (b) cells were transfected with mock, smallinterfering RNA (siRNA) (control, 497 and 752) or their dsRDC
counterparts at 0.5 nM. At 5 days after transfection, relative cell
viability was determined using a luciferase assay. Data points
marked with asterisks are statistically significant as compared with
control transfection (*Po0.001; **Po0.01). Data points marked withcrosses are statistically significant (wPo0.01).

these results showed that dsRDC-497 and siRNA-752
are able to induce specific growth suppression of E6E7expressing keratinocytes.
Discussion

In this study, we assessed the off-target activities of E6E7
siRNAs (497, 573 and 752) and their dsRDC modified
forms using 10 potential human off-target sequencescarrying 3-nucleotide mismatches to the GS of E6E7
siRNA (nucleotides 1–19 from the 50 end). In most cases,
3-nucleotide mismatches were adequate to avoid suppression of artificial mRNA of RLuc fused with a single copyof the off-target candidate sequence. However, thesilencing effect was significant when targeted mRNA
contained a single copy of the 19-nucleotide sequence witha completely matched seed region as well as one of threemismatches located at either nucleotide 1 or 19 from the
50 end of the GS. These sequences (497OT-2, 573OT-2
and 573OT-3) were found to have mismatches to theirrespective siRNAs at position 10, or at positions 10 and
11 from the 50 end of the GS. Pairing of these GS
nucleotides to target mRNA is essential for mRNA
cleavage and mismatch of these positions is a hallmark of
miRNA-mediated translational inhibition.32–34 Thus, theoff-target effects observed in our study might have been
mediated through miRNA-like activity without mRNA
cleavage.
Perfect RNA–RNA duplex formation between thesiRNA seed region (GS positions 2–8 from the 50 end)
and unintended target mRNA is essential for off-target
silencing.3,5,6 Using unmodified authentic siRNA, we
previously found that the melting temperatures of seed–mRNA duplexes (Tm 2–8) are divergent and positively
correlated to the extent of off-target suppression.35 We
also reported that this rule is applicable to the off-target
effects mediated by dsRDCs.36 Substitution of seed
RNA by DNA lowers Tm 2–8, which explains why mostdsRDCs, if not all, might have reduced off-target effects
as compared with siRNA.8 In this study, dsRDC
modification reduced the inhibitory effects of siRNA497 on RLuc-497OT-2 and POU4F1 mRNA expressions,which was consistent with our previous observation.8,36 In
contrast, similar DNA substitution of siRNA-573 did notcompromise the suppression of RLuc-573OT-2. According to results of our calculation formula,35 the Tm 2–8
change induced by dsDNA modification was highest insiRNA-497 (11.5 1C), followed by siRNA-752 (7.7 1C)
and lowest in siRNA-573 (6.9 1C), which was wellcorrelated with their reduction of off-target effects.
All HPV16 E6E7 siRNAs (497, 573 and 752) showed
potent nonspecific cytotoxicity in HeLa-derived cells
cultured at a low density. As HeLa cells express HPV18
E6E7 mRNAs with a relatively low homology to HPV16
E6E7 mRNAs, we analyzed the HPV18 E6E7 region andfound no sequences perfectly matching the seed regions ofthese siRNAs, which is the minimal requirement forknockdown of HPV18 E6E7 (data not shown).3,5,6
Control siRNA also exhibited cytotoxicity to FL-HeLa1 cells under the same conditions. Furthermore, siRNA(497) was highly toxic to TERT-immortalized keratinocytes lacking E6E7 expression. Thus, these results
suggested that the nonspecific cytotoxicity of thesesiRNAs was not mediated through suppression of E6E7
in FL-HeLa-1 cells. Reduction of off-target effects by
dsDNA modification was correlated with their reductionof cytotoxicity. These results favor the hypothesis that
nonspecific cytotoxicity is induced by off-target suppression of genes that regulate cell growth and viability.7
Alternatively, such cytotoxicity might be mediated by
disturbing the endogenous miRNA pathways throughcompetition of molecules involved in RNAi by exogenous
siRNA.10–12 It remains to be determined how siRNA
exerts toxic effects in transduced cells.
We previously reported that most E6E7 siRNAs, with afew exceptions including 573,8 exerted PS-mediatedsilencing toward mRNA containing inverted E6E7(628 bp fragment). However, we detected PS-mediatedsilencing in all siRNAs (497, 753 and 573) tested in thisstudy when mRNAs containing inverted short target
sequences were used as targets. Detection of PS RNAimight be influenced by the secondary structure of thetarget mRNA. Our results suggest that PS RNAi is
unavoidable in conventional siRNAs. Thus, modifications such as dsRDC would be applicable for avoiding
unwanted PS-induced silencing.
Deregulation of E6 and E7 expression is an initial event
during multistep carcinogenesis in cervical cancer. In
addition to expressions of these oncogenes, progression
into overt cancer requires additional genetic changes.
Human keratinocytes immortalized with HPV16 E6E7
were shown to be less invasive and have a less tumorigenic
phenotype.31 Using E6E7-immortalized keratinocytes as

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DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al596

an early disease model and TERT-immortalized keratinocyte s as normal keratinocytes, we examined the effectsof E6E7 siRNA and dsRDC on their growth andspecificity, and found that dsRDC-497 and siRNA-752
suppressed the growth of E6E7-positive keratinocytes, but
not that of E6E7-negative keratinocytes. Thus, thosemight be useful for the treatment of pre-cancer and earlycancer lesions.
In summary, we found that dsRDC modification
enhanced the specificity of HPV16 E6E7 siRNA by reducing its nonspecific cytotoxicity and off-target property.
E6E7 dsRDC inhibited the growth of E6E7-expressing
human keratinocytes as well as HPV16-positive cervicalcancer cells; however, not that of non-expressing keratinocytes. Taken together, our results indicate a large
potential for HPV16 E6E7 dsRDC in treatments for
HPV16-related cancer and early disease stages.
Conflicts of interest

The authors declare no conflicts of interest.
Acknowledgements

We thank Yukikazu Natori (Tokyo Institute of Technology) for the helpful discussion and suggestions. This work
was supported in part by Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of
Science (KY) and a Grant-in-Aid for Cancer Researchfrom the Ministry of Health Labor and Welfare ofJapan (KY).
References
1 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K,
Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells. Nature 2001; 411:
494–498.
2 Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specificinhibition of gene expression by small double-stranded
RNAs in invertebrate and vertebrate systems. Proc Natl
Acad Sci USA 2001; 98: 9742–9747.
3 Jackson AL, Bartz SR, Schelter J, Kobayashi SV, BurchardJ, Mao M et al. Expression profiling reveals off-target gene
regulation by RNAi. Nat Biotechnol 2003; 21: 635–637.
4 Saxena S, Jonsson ZO, Dutta A. Small RNAs with imperfectmatch to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in
mammalian cells. J Biol Chem 2003; 278: 44312–44319.
5 Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE,
Fesik SW et al. siRNA-mediated off-target gene silencing
triggered by a 7 nt complementation. Nucleic Acids Res 2005;
33: 4527–4535.
6 Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D,
Leake D, Fedorov Y et al. 30 UTR seed matches, but notoverall identity, are associated with RNAi off-targets. Nat
Methods 2006; 3: 199–204.
7 Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J,
Guo J et al. Position-specific chemical modification ofsiRNAs reduces ‘off-target’ transcript silencing. RNA 2006;
12: 1197–1205.

Cancer Gene Therapy

8 Ui-Tei K, Naito Y, Zenno S, Nishi K, Yamato K, Takahashi F
et al. Functional dissection of siRNA sequence by systematic
DNA substitution: modified siRNA with a DNA seed arm isa powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Res 2008; 36:
2136–2151.
9 Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C,
Langkjaer N et al. A large-scale chemical modification screen
identifies design rules to generate siRNAs with high activity,
high stability and low toxicity. Nucleic Acids Res 2009; 37:
2867–2881.
10 Castanotto D, Sakurai K, Lingeman R, Li H, Shively L,
Aagaard L et al. Combinatorial delivery of small interfering
RNAs reduces RNAi efficacy by selective incorporation into
RISC. Nucleic Acids Res 2007; 35: 5154–5164.
11 Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K,
Davis CR et al. Fatality in mice due to oversaturation ofcellular microRNA/short hairpin RNA pathways. Nature
2006; 441: 537–541.
12 Khan AA, Betel D, Miller ML, Sander C, Leslie CS, Marks
DS. Transfection of small RNAs globally perturbs gene
regulation by endogenous microRNAs. Nat Biotechnol 2009;
27: 549–555.
13 Bosch FX, de Sanjose S. Chapter 1: Human papillomavirus
and cervical cancer—burden and assessment of causality.
J Natl Cancer Inst Monogr 2003; 31: 3–13.
14 zur Hausen H. Papillomaviruses causing cancer: evasion
from host-cell control in early events in carcinogenesis.
J Natl Cancer Inst 2000; 92: 690–698.
15 Longworth MS, Laimins LA. Pathogenesis of humanpapillomaviruses in differentiating epithelia. Microbiol Mol
Biol Rev 2004; 68: 362–372.
16 Munger K, Baldwin A, Edwards KM, Hayakawa H,
Nguyen CL, Owens M et al. Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004; 78: 11451–11460.
17 Yugawa T, Kiyono T. Molecular mechanisms of cervicalcarcinogenesis by high-risk human papillomaviruses: novel
functions of E6 and E7 oncoproteins. Rev Med Virol 2009;
19: 97–113.
18 Chen Z, Kamath P, Zhang S, St John L, Adler-Storthz K,
Shillitoe EJ. Effects on tumor cells of ribozymes that cleave
the RNA transcripts of human papillomavirus type 18.
Cancer Gene Ther 1996; 3: 18–23.
19 Venturini F, Braspenning J, Homann M, Gissmann L,
Sczakiel G. Kinetic selection of HPV 16 E6/E7-directed
antisense nucleic acids: anti-proliferative effects on HPV
16-transformed cells. Nucleic Acids Res 1999; 27:
1585–1592.
20 DeFilippis RA, Goodwin EC, Wu L, DiMaio D. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis
in HeLa cervical carcinoma cells. J Virol 2003; 77:
1551–1563.
21 Jiang M, Milner J. Selective silencing of viral gene expressionin HPV-positive human cervical carcinoma cells treated withsiRNA, a primer of RNA interference. Oncogene 2002; 21:6041–6048.
22 Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T,
Ikeda Y et al. In vitro and in vivo growth suppression ofhuman papillomavirus 16-positive cervical cancer cells by E6
siRNA. Mol Ther 2003; 8: 762–768.
23 Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M,
Hoppe-Seyler F. siRNA targeting of the viral E6 oncogene
efficiently kills human papillomavirus-positive cancer cells.
Oncogene 2003; 22: 5938–5945.

DNA–RNA chimera siRNA targeting HPV16 oncogenes
K Yamato et al

24 Hall AH, Alexander KA. RNA interference of humanpapillomavirus type 18 E6 and E7 induces senescence in
HeLa cells. J Virol 2003; 77: 6066–6069.
25 Bosch FX, Castellsague X, de Sanjose S. HPV and cervicalcancer: screening or vaccination? Br J Cancer 2008; 98:
15–21.
26 Putral LN, Bywater MJ, Gu W, Saunders NA, Gabrielli BG,
Leggatt GR et al. RNA interference against humanpapillomavirus oncogenes in cervical cancer cells results inincreased sensitivity to cisplatin. Mol Pharmacol 2005; 68:
1311–1319.
27 Yamato K, Yamada T, Kizaki M, Ui-Tei K, Natori Y,
Fujino M et al. New highly potent and specific E6 and E7
siRNAs for treatment of HPV16 positive cervical cancer.
Cancer Gene Ther 2008; 15: 140–153.
28 Haga K, Ohno S, Yugawa T, Narisawa-Saito M, Fujita M,
Sakamoto M et al. Efficient immortalization of primary
human cells by p16INK4a-specific short hairpin RNA or
Bmi-1, combined with introduction of hTERT. Cancer Sci
2007; 98: 147–154.
29 Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K.
siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res
2004; 32: W124–W129.

30 Lanham S, Herbert A, Watt P. HPV detection and measurement of HPV-16, telomerase, and survivin transcripts incolposcopy clinic patients. J Clin Pathol 2001; 54: 304–308.
31 Narisawa-Saito M, Yoshimatsu Y, Ohno S, Yugawa T,
Egawa N, Fujita M et al. An in vitro multistep carcinogenesis
model for human cervical cancer. Cancer Res 2008; 68:
5699–5705.
32 Hutvagner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002; 297: 2056–2060.
33 Doench JG, Petersen CP, Sharp PA. siRNAs can function as
miRNAs. Genes Dev 2003; 17: 438–442.
34 Chiu YL, Rana TM. RNAi in human cells: basic structuraland functional features of small interfering RNA. Mol Cell
2002; 10: 549–561.
35 Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K. Thermodynamic
stability and Watson–Crick base pairing in the seed duplex
are major determinants of the efficiency of the siRNA-based
off-target effect. Nucleic Acids Res 2008; 36: 7100–7109.
36 Ui-Tei K, Nishi K, Naito Y, Zenno S, Juni A, Saigo K.
Reduced base–base interactions between the DNA seed andRNA target are the major determinants of a significant
reduction in the off-target effect due to DNA-seed-containing siRNA. Proceedings of the 2009 Micro-Nano Mechatronics and Human Science; 2009, pp 298–304.

Supplementary Information accompanies the paper on Cancer Gene Therapy website (http://www.nature.com/cgt)

Cancer Gene Therapy597