Chapter 2
Selection of Chemical Modifications in the siRNA Seed
Region That Repress Off-Target EffectYoshiaki Kobayashi, Kengo Miyamoto, Misako Aida, and Kumiko Ui-Tei
Abstract
RNA interference mediated by small interfering RNA (siRNA) has been widely used as a procedure to
knock down the expression of an intended target gene with perfect sequence complementarity. However,siRNA often exhibits off-target effects on genes with partial sequence complementarities. Such off-target
effect is an undesirable adverse effect for knocking down a target gene specifically. Here we describe thepowerful strategy to avoid off-target effects without affecting the RNAi activity by the introduction of DNA
or 20 -O-methyl modifications in the siRNA seed region. These two types of chemical modifications repress
off-target effects through different molecular mechanisms.
Key words RNAi, siRNA, Seed region, Off-target effect, 20 -OMe

1

Introduction
RNA interference (RNAi) is induced by small interfering RNA(siRNA), a duplex composed of the guide and passenger strandsof 21-nucleotide (nt)-long RNAs with 2-nt 30 overhangs. The
siRNA is loaded onto Argonaute (AGO) protein, which is a corecomponent of the RNA-induced silencing complex (RISC) [1–3]. Subsequently, one of the two strands (passenger strand) isejected and the other strand (guide strand) remains loading on
AGO. Then, the guide strand base pairs with its target mRNA
with perfect sequence complementarity, leading to repression ofgene expression through the cleavage by AGO (Fig. 1). Synthetic
siRNAs have become a powerful tool not only for studying genefunction but also for applying therapeutics. Because the first siRNAdrug was approved by FDA and EMA in 2018 [4], the developmental research for therapeutic applications expected to become
popular.
RNAi efficiencies of siRNAs are widely different depending on
their sequences, and only a limited fraction of siRNAs, which satisfy
the functional siRNA sequence rules, is effective in mammalianHenrik J. Ditzel et al. (eds.), Design and Delivery of SiRNA Therapeutics, Methods in Molecular Biology, vol. 2282,
https://doi.org/10.1007/978-1-0716-1298-9_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

17

18

Yoshiaki Kobayashi et al.

AGOpassenger

siRNA

guide

guide

mRNA
AAA

Translational repression
Cleavage
Seed region
AAA

AAA
8

RNAi

2

Off-target effectFig. 1 Schematic presentation of the mechanisms of siRNA-mediated RNAi and sequence-dependent
off-target effects. SiRNA, a duplex composed of the guide and passenger strands, is loaded onto AGO protein.
Passenger strand is ejected and guide strand remains loading on AGO. The mRNA with exact complementary
sequence with siRNA guide strand is repressed by RNAi through its cleavage by AGO protein. On the other
hand, the sequence-dependent off-target effect is induced for the unintended mRNAs with sequence
complementarities with siRNA seed region (2–8 nucleotides from the 50 end of the guide strand) in their
30 UTRs through translational repression (see Note 4)cells [5]. Meanwhile, the siRNA often exhibits off-target effects
when the siRNA seed region (2–8 nts from the 50 end of the guidestrand) base pairs with the unintended genes [6–10] (Fig. 1). The
off-target effect is considered to be induced by the similar molecular mechanism with miRNA-mediated RNA silencing, which differsfrom the mechanism of RNAi. Furthermore, the degree ofoff-target effect is correlated positively with the thermodynamic
stability in base-pairing between the seed region of the siRNA
guide strand and the unintended mRNA [11]. Therefore, siRNAwith low seed-target stability may be a promising tool fortarget-specific RNAi with little off-target effect. However, theSiRNA Modifications Avoid Off-Target Effect19

number of the functional siRNA sequences with lower seed-target
stability is substantially small.
Thus, we have established the methods to overcome suchsequence-dependent limitations using two types of chemical modifications (Fig. 2): One type of chemical modification is DNA anda

23-mer sequences in all human mRNAs(56,375,087)
5’

3’

3’

5’

b

100%

Functional siRNA sequence(iv) GC stretch < 9nt(ii) G/C(i) A/U

5’

3’

3’

14.7%

5’

(iii) A/U ≧ 4cdReduction of off-target effectby sequence

Reduction of off-target effectby chemical modifications

Off-target effect reduced siRNA sequence
5’
3’

Off-target effect reduced siRNAwith DNA modification

3’
5’

3%

5’
3’

Low base-pairing stability㻔㼀㼙㻞㻙㻤 㻨㻌㻞㻝㻚㻡㻌䉝㻕

≈

3’
5’

14.7%

Low base-pairing stability

Off-target effect reduced siRNAwith 2’-OMe modification
5’
3’

3’

≈

5’

14.7%

Steric hindrance

Fig. 2 Flow for selecting functional siRNAs with reduced sequence-dependent off-target effect by two types ofchemical modifications. (a) From all of human mRNA sequences, 56,375,087 of 23-mer sequences are
selectable. This number of siRNAs is set as 100%. (b) Functional siRNA sequences can be selected by using
the four rules described in Note 1. (c) The siRNAs with sequences containing low seed–target Tm values (seeNote 2) for both strands are selected for reduction of off-target effects. (d) Using DNA or 20 -OMe modificationin the siRNA seed region makes it possible to overcome the sequence limitations of siRNA for reducing seeddependent off-target effects through different molecular mechanisms: reduction of base-pairing stability by
DNA modification, and steric hindrance by 20 -OMe modification

20

Yoshiaki Kobayashi et al.
Passenger effect

Guide effectpsiCHECK-gCM_VIM270

CM target
5’ CDS

3’

psiCHECK-gSM_VIM270

5’
seed

RNAi activity(nM)

siContpsiCHECK-pCM_VIM270

psiCHECK-pSM_VIM270

CM target
SM target x 3
SM target x 3
3’ 5’ CDS
3’ 5’ CDS
3’ 5’ CDS
3’

5’

3’
Off-target activity
(nM)

3’

5’

5’
seed
3’
Off-target activity

(nM)

(nM)

RNAi activity

5’-YYYYYYYYYYYYYYYYYYYYY-3’|||||||||||||||||||
3’-XXXXXXXXXXXXXXXXXXXXX-5’
Passenger seed

Unmodified 5’-CCAUCAACACCGAGUUCAAGA-3’|||||||||||||||||||siRNA
3’-GCGGUAGUUGUGGCUCAAGUU-5’
Guide seed5’-CCAUCAACACCGAGUUCAAGA-3’
DNA|||||||||||||||||||-modified 3’-GCGGUAGUUGUGGCTCAAGTU-5’

5’-CCAUCAACACCGAGUUCAAGA-3’
2’-OMe|||||||||||||||||||-modified 3’-GCGGUAGUUGUGGCUCAAGUU-5’
0

25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
Relative luc act (%)
Relative luc act (%)
Relative luc act (%) Relative luc act (%)

Fig. 3 Measurement of RNAi and off-target activities by dual luciferase reporter assays. (Top) Structures of theconstructs for luciferase reporter assays. psiCHECK-gCM_VIM270 contains a CM sequence of the siRNA guidestrand in the downstream of Renilla luciferase CDS (CDS). psiCHECK-gSM_VIM270, psiCHECK-pCM_VIM270,
or psiCHEK-pSM_VIM270 contains three tandem repeats of the SM sequences of the guide strand, the CM andSM sequences of the passenger strand, respectively. The reporter assays were performed as follows: HeLa
cells were co-transfected with each concentration of unmodified, DNA- or 20 -OMe-modified siVIM-270, witheach psiCHECK construct and pGL3 control vector. Cells were harvested 24 h after transfection and relative
luciferase activities were measured. (Bottom) Unmodified, DNA- or 20 -OMe-modified siVIM-270 sequences(left). Results of RNAi activities on the guide CM target using psiCHECK-gCM_VIM270, off-target activities on
the guide SM target using psiCHECK-gSM_VIM270, RNAi activities on the passenger CM target using
psiCHECK-pCM_VIM270 and off-target activities on the passenger SM target using psiCHECK-pSM_VIM270
by the unmodified or modified siVIM-270s. We used siGY441 as siRNA control (siCont). The data wereaveraged from three independent experiments, and the bar indicates the standard deviation

the other is 20 -O-methyl (20 -OMe). The results of reporter assay(Fig. 3), structural simulation (Fig. 4), and microarray (Fig. 5)
reveal that the seed-dependent off-target effect is able to be
reduced by the introduction of DNA and 20 -OMe modificationsin the siRNA seed region without reducing RNAi activity throughdifferent molecular mechanisms: DNA modification in the siRNAseed region decreased the thermodynamic stability in the seedtarget base-pairing stability [12], and 20 -OMe modificationinduced steric hindrance in the duplex formation on the AGO
protein [13]. These chemical modifications may be useful tools
for potential therapeutic applications.

SiRNA Modifications Avoid Off-Target Effect

a

K566

b

c

21

d

A2
A3
R792

A4

Original RNA

Optimized RNA

2’-OMe modified
RNA

RNA / 2’-OMe

Fig. 4 Computational prediction of the steric hindrance of 20 -OMe-modified siRNAs loaded on the AGO protein.
(a) Crystal structure of unmodified 50 -AAA-30 RNA with K566 and R792 of AGO protein shown by Schirle et al.
[28], and the optimized structures of unmodified (b) and 20 -OMe-modified (c) RNAs with K566 and R792.
(d) Superposition at C40 -C30 -O30 of A3 of unmodified RNA with 20 -OMe-modified RNA

2

Materials

2.1 Design
and Preparationof siRNAs

1. siDirect2.0 software: http://siDirect2.RNAi.jp/
2. Guide strand sequence of siRNA against human vimentin gene(siVIM-270): 50 -UUGAACUCGGUGUUGAUGGCG-30 .
3. Passenger strand sequence against human vimentin gene(siVIM-270): 50 -CCAUCAACACCGAGUUCAAGA-30 .
SiRNA against green fluorescent protein (siGY441), which
does not have perfectly complementary sequences in completematch (CM) and seed-match (SM) reporter constructs (seeSubheading 2.2), is used as a negative control.
4. Guide strand of siGY441: 50 -AUGAUAUAGACGUUGUGG
CUG-30 .
5. Passenger strand of siGY441: 50 - GCCACAACGUCUAUAUCAUGG-30 .
6. Annealing buffer (2): 40 mM NaCl, 20 mM Tris–HCl,
pH 7.5.
7. 19% Polyacrylamide gel.
8. TBE buffer.

2.2 Luciferase
Reporter Vector
and Oligonucleotides
with CM and SM
Sequences

1. psiCHECK-1 vector (Promega).
2. Oligonucleotides with CM sequences of siVIM-270 guidestrand (gCM) with cohesive XhoI/EcoRI ends: 50 -tcgaGCG
CCATCAACACCGAGTTCAAGAG -30 and 50 -aatt CTCTTG
AACTCGGTGTTGATGGCGC-30 .
3. Oligonucleotides with three tandem repeats of SM sequencesof siVIM-270 guide strand (gSM) with cohesive XhoI/EcoRI

Fig. 5 Microarray analysis of RNAi activities and off-target effects of unmodified, and DNA- and
20 -OMe-modified siRNAs. (a) Expression levels of the target vimentin gene in the cells transfected with theunmodified and modified siRNAs relative to that in the mock-transfected cells, respectively. (b) MA plots of
microarray data of the cells transfected with unmodified, and DNA- and 20 -OMe-modified siRNAs, respectively. The vertical axis indicates the log2 fold change of signal intensity of each gene in each type of siRNAtransfected cells relative to that of mock (M value), and the horizontal axis indicates the averaged log10 signal
intensity of each gene in mock and siRNA transfection (A value). The dark blue dots indicate the transcripts
with seed-matched (SM) sequences in the 30 UTRs of the mRNAs, and the light blue dots indicate the othertranscripts. (c) Cumulative distribution of microarray data of the cells transfected with unmodified, and DNAand 20 -OMe-modified siRNAs, respectively. The horizontal axis indicates the M value of (b), and the verticalaxis indicates the cumulative fraction of transcripts. The red line indicates the cumulative curve of SM
transcripts, and the black line indicates the cumulative curve of the other non-SM transcripts. (d) Mean log2
fold-change of the downregulated SM transcripts compared to those of non-SM transcripts. The horizontal
axis indicates the mean fold-change of off-target transcripts in the cells transfected with unmodified, andDNA- and 20 -OMe-modified siRNAs, respectively

SiRNA Modifications Avoid Off-Target Effect23

ends: 50 -tcga GAATGATGCACCAGGAGTTCAAGAAATGATGCACCAGGAGTTCAAGAAATGATGCACCAGGAGTTCAAGAG -30 and 50 -aatt CTCTTGAACTCCTGGTGCATCATTTCTTGAACTCCTGGTGCATCATTTCTTGAACTCCTGGTGCATCATTC-30 .
4. Oligonucleotides with three tandem repeats of CM sequencesof siVIM-270 passenger strand (pCM) with cohesive XhoI/
EcoRI ends: 50 -tcga TCTTGAACTCGGTGTTGATGGCGAATCTTGAACTCGGTGTTGATGGCGAATCTTGAACTCGGTGTTGATGGCGAA-30 and 50 -aatt TTCGCCATCAACACCGAGTTCAAGATTCGCCATCAACACCGAGTTCAAGATTCGCCATCAACACCGAGTTCAAGA-30 .
5. Oligonucleotides with three tandem repeats of CM sequencesof siVIM-270 passenger strand (pSM) with cohesive XhoI/
EcoRI ends: 50 -tcgaAATGATGCACCAGGAGTTGATGGAATGATGCACCAGGAGTTGATGGAATGATGCACCAGGAGTTGATGG-30 and 50 -aattCCATCAACTCCTGGTGCA
TCATTCCATCAACTCCTGGTGCATCATTCCATCAACT
CCTGGTGCATCATT-30 .
2.3

1. Human HeLa cells or other culture cells.

Cell Culture

2. 24-Well plate (Sumitomo Bakelite).
2.4 Transfection
and Luciferase
Reporter Assay
Reagents

1. pGL3-Control vector (Promega).

2.5

The melting temperature (Tm) values are calculated using the following formula using ΔH and ΔS thermodynamic parameters
shown in Xia et al. [14] and Sugimoto et al. [15].

2. Lipofectamine 2000 reagent (Thermofisher).
3. Dual-Luciferase Reporter Assay System (Promega).
4. 1 passive lysis buffer (Promega).

Calculation of Tm

T m ¼ fð1000  ΔH Þ=½A þ ΔS þ ln ðCt=4Þg  273:15 þ 16:6log½Naþ :
ΔH (kcal/mol), sum of nearest neighbor enthalpy change.
A, helix initiation constant (10.8).
ΔS, sum of nearest neighbor entropy change.
R, gas constant (1.987 cal/deg/mol).
Ct, total molecular concentration of strand (100μM).
Fix [Na+] at 100 mM.
2.6 Microarray
Analysis

1. RNeasy kit (Qiagen).
2. NanoDrop 2000 spectrophotometer (Thermo Scientific).
3. Bioanalyzer (Agilent).

24

Yoshiaki Kobayashi et al.

4. Agilent one-color spike mix kit (Agilent).
5. Agilent SurePrint G3 human GE microarray (8  60 K, ver.
2.0, 3.0) (Agilent).

3

Methods

3.1 Designing
and Preparing siRNAs
with DNA or 20 -OMe
Modificationsin the Seed Region

Highly functional siRNAs for mammalian cells (see Note 1) can be
designed using the web-based online software, siDirect 2.0. In thissection, the procedure for selecting highly functional siRNA for
human vimentin is shown as an example.
1. Open “http://siDirect2.RNAi.jp/” with any appropriate webbrowser.
2. Input the cDNA sequence of vimentin in a nucleotide sequencebox. Or enter the accession number of human vimentin(NM_003380) into the upper blank box.
3. Click “retrieve sequence” to get the nucleotide sequence from
GenBank (https://www.ncbi.nlm.nih.gov/genbank/).
4. Click the “Options: [click here]” button to set optional parameters if necessary (see Note 2).
5. Click “design siRNA,” and you can get result.
6. Synthesize siRNAs shown in Fig. 3 with or without DNA and
20 -OMe modifications in the siRNA seed region chemically,
respectively (see Note 3).
7. Mix the solutions containing same amounts of guide and passenger strand RNAs, and add the same volume of 2 annealing
buffer, and anneal by incubating the mixture at 95  C for
3 min, at 37  C for 30 min, and at 25  C for 60 min.
8. Annealed products may be checked using 19% polyacrylamide
gel electrophoresis in TBE buffer, which can separate 21-nt
double-stranded siRNA with 2-nt 30 overhangs from singlestranded RNA.

3.2 Preparing
Luciferase Reporters
with CM and SM
Sequences

1. Both strands of DNA oligonucleotides for gCM and gSM
reporters, or pCM and pSM reporters are mixed, respectively,
and add the same volume of 2 annealing buffer. They areannealed by incubating at 95  C for 3 min, at 37  C for 30 min,and at 25  C for 60 min.
2. Insert the annealed oligonucleotides into the XhoI/EcoRI
restriction enzyme sites within 30 UTR region of Renillaluciferase gene in psiCHECK-1, and name each plasmid vector,
suchaspsiCHECKgCM_VIM270,
psiCHECKgSM_VIM270, psiCHECK-pCM_VIM270, and psiCHECKpSM_VIM270, respectively (Fig. 3). RNA silencing activitiesof the guide strand and passenger strand are measured using

SiRNA Modifications Avoid Off-Target Effect25

psiCHECKgCM_VIM270 and psiCHECK-pCM_VIM270,
respectively. The seed-dependent off-target effects of theguide strand and passenger strand are measured using
psiCHECK-gSM_VIM270 and psiCHECK-pSM_VIM270,
respectively (see Note 4).
3.3 RNA Silencing
Activity Assay Using
the Dual Luciferase
Reporter System

RNA silencing activity is measured using dual luciferase reporter
assay system.
1. Inoculate a human HeLa cell suspension (1.0  105 cells/mL)
into a well of 24-well culture plate 1 day before transfection.
2. Transfect 0.0005, 0.005, 0.05, 0.5, 5 nM of each siRNA (seeNote 5), 0.1μg of pGL3-Control vector encoding the firefly
luciferase gene, and 0.1μg of each psiCHECK-gCM_VIM270,
psiCHECK-gSM_VIM270, psiCHECK-pCM_VIM270, andpsiCHECK-pSM_VIM270 reporter construct simultaneously
into HeLa cells using 2μL of Lipofectamine 2000.
3. Lyse the transfected cells with 1 passive lysis buffer 24 h after
transfection (see Note 6).
4. Measure luciferase activity using the Dual-Luciferase Reporter
Assay System.
5. Calculate the percentage of the Renilla luciferase activity normalized by firefly luciferase activity (Renilla luciferase activity/
firefly luciferase activity  100) for unmodified siRNA, andsiRNA with DNA (see Note 7) or 20 -OMe (see Note 8) modification in the seed region.

3.4 Microarray
Analysis

1. Inoculate HeLa cells in each 2 wells of 24-well plate at 1  105
cells/mL.
2. Transfect 50 nM of each siRNA into HeLa cells using 2μL of
Lipofectamine 2000. Prepare mock-transfected cells treatedwith the transfection reagent in the absence of siRNA as acontrol.
3. At 24 h post-transfection, purify total RNA from the transfected cells using an RNeasy kit.
4. Measure the concentration of purified RNA using a NanoDrop
2000 spectrophotometer and confirm its quality by a Bioanalyzer (see Note 9).
5. Synthesize cDNA from 1μg of each total RNA sample using an
Agilent one-color spike mix kit.
6. Hybridize the cDNA product to an Agilent SurePrint G3
human GE microarray according to the manufacturer’s
protocol.

26

Yoshiaki Kobayashi et al.

7. Calculate the reduction of the expression levels of target vimentin mRNAs in the cells transfected with each siRNA relative tothat in the mock-transfected cells, respectively.
8. Generate M (log ratio) and A (mean average) (MA) plot andcumulative distribution using microarray data of each siRNAby package in R-studio software to evaluate the RNA silencing
activity (see Note 10) and off-target effect (see Note 11).

4

Notes
1. SiRNAs satisfied the following four sequence conditions simultaneously are considered to be functional: (a) A/U at the
50 -terminus of the siRNA guide strand, (b) G/C at the
50 -terminus of the siRNA passenger strand, (c) AU richness at
the 50 one-third of the guide strand, (d) absence of a long GC
stretch (>9 nt). Bioinformatics analysis revealed that 14.7% of
all the 23-mer subsequences in human mRNA satisfy functional siRNA sequence rules [16] (Fig. 2a, b).
2. Using options, other siRNA selection algorithms establishedby Reynolds et al. [17] or Amarzguioui and Prydz [18] can beselected. Furthermore, the highly effective siRNA with
reduced seed-dependent off-target effect is selectable using
Tm value in the seed-target duplex below 21.5  C. Tm value isa good benchmark to express the thermodynamic stability ofthe duplex between siRNA seed region and target mRNA.
Calculated Tm value of siRNA seed-target duplex in proteinfree condition discriminates siRNA with almost weak off-target
effect from that with strong off-target effect [11]. Specificity ofsiRNA sequence in human, mouse, or rat can be also checked.
Other options which impose the number of contiguous GC or
AT nucleotides or GC content are also available.
3. DNA is a modified form of RNA with hydrogen at 20 -positionof the pentose sugar. The 50 one-third of siRNA is capable ofreplacement with DNA without substantial loss of RNAi activity [12]. 20 -OMe is a well-known C20 -modified RNA that is
known to increase stability in the serum and thermodynamic
stability in base-pairing, and also considered to abrogate immunogenicity [19–23]. 20 -OMe modification is tolerated at multiple positions in the siRNA guide strand because of its small
size, comparable to the 20 -OH of natural RNA [24].
4. In RNAi, a target mRNA is reduced by cleavage of target
mRNA by AGO2. However, miRNA-mediated RNA silencing
is induced by translational repression [25–27].

SiRNA Modifications Avoid Off-Target Effect

27

Table 1IC50s of unmodified, DNA-modified, or 20 -OMe-modified siVIM-270 for
RNAi activity or off-target effect measured by reporter assay
siRNA

RNAi (pM)

Off-target effect (pM)

RNA

2

10

DNA

4

295

20 -OMe10

1

1 indicates that the IC50 is higher than 5000 pM

5. It is preferable to perform reporter assays at a series of multiple
concentrations of each siRNA to calculate the IC50 of its RNA
silencing activity quantitatively.
6. If the cell debris is formed from transfected cells, it is preferable
to remove the debris clearly before addition of 1 passive lysis
buffer to measure the luciferase activity correctly.
7. Unmodified siVIM-270 repressed the Renilla luciferase activity derived from psiCHECK-gCM for siVIM-270 (psiCHECK-gCM_VIM270) in a dose-dependent manner(Fig. 3). The calculated IC50 was 2 pM (Table 1).
DNA-modified siVIM-270 repressed the Renilla luciferase
activity at the almost equivalent level to that of unmodified
siVIM-270, particularly at high concentrations (0.5 or 5 nM),
and their IC50 was 4 pM (Table 1). The off-target effects on the
SM target of the guide strand were also measured using
psiCHECK-gSM_VIM270 (Fig. 3). Unmodified siVIM-270
showed strong off-target effects (IC50 ¼ 10 pM), but DNAmodified siVIM-270 reduced the off-target effects(IC50 ¼ 295 pM) (Table 1). This may be because thermodynamic stability in base-pairing of DNA-RNA duplex is weaker
than that of RNA duplex [13]. In fact, the calculated Tm valueof the RNA duplex between the seed region of unmodified
siVIM-270 and target RNA was 31.4  C, and that of
DNA-RNA heteroduplex between the seed region of
DNA-modified siVIM-270 and target RNA was 3.1  C. Furthermore, it is possible that the passenger strand also induces
unintended RNAi and seed sequence-dependent off-target
effects. Then dual luciferase reporter assays were also performed using psiCHECK-pCM_VIM270 and psiCHECKpSM_VIM270, respectively. However, almost no or little
inhibitory effects on both CM and SM targets of the passenger
strand were observed (Fig. 3). Because we used siRNA satisfying the four functional siRNA sequence conditions, such
siRNA is expected to be easily unwound from the 50 terminusof the guide strand, but not that of the passenger strand.

28

Yoshiaki Kobayashi et al.

8. The 20 -OMe-modified siVIM-270 repressed the Renilla luciferase activity derived from psiCHECK-gCM for siVIM-270(psiCHECK-gCM_VIM270) at the almost equivalent level tothat of unmodified siVIM-270, and its IC50 was 10 pM(Table 1). The off-target effects on the SM target of theguide strand of 20 -OMe-modified siVIM-270 measured using
psiCHECK-gSM_VIM270 was significantly reduced, eventhough this modification is well-known modification toenhance base-pairing stability [19]. It is known that the phosphates of RNA oligonucleotides interact with the amino acidside chains of human AGO protein [28, 29]. Therefore, 20-OMe-modified single-stranded RNA structure on the AGO
protein was examined computationally (Fig. 4). The 20 -OMemodification in the siRNA seed region at position 3 was essentially stable on the AGO protein. However, RNA structure of
the nucleotide adjacent to the 20 -OMe-modified nucleotide
was changed from that of unmodified RNA (Fig. 4a–d), suggesting that 20 -OMe modification in the siRNA seed region atleast at position 3 from 50 end may disturb base-pairing
between siRNA guide strand and its target or off-target
mRNAs. But, as shown in Fig. 3, the expression of the CMtarget was significantly repressed, suggesting that the non-seedregion may compensate the incomplete seed-target base pairing for inducing RNAi on the CM target.
9. Particularly, it is recommended that the value of RNA Integrity
Number (RIN), a quantitative measurement of RNA quality, of
total RNA sample for microarray application is high. Highquality samples with RIN values higher than 9 are best for
microarray application, but the reliable results are also obtained
from the sample with the value as low as 7.0.
10. Unmodified siVIM-270 repressed the expression level ofvimentin mRNA to 11%. DNA- and 20 -OMe-modified
siVIM-270 repressed vimentin mRNA expression at almost
equivalent level to 13% and 19%, respectively (Fig. 5a).
11. The seed-dependent off-target effect of each siRNA using
microarray data can be evaluated by analyzing the expression
profiles of genes with seed matched sequences in their 30 UTRs,
since siRNA off-target effect is mainly induced by the mechanism similar to miRNA-mediated RNA silencing [25–27]. Off-target effects on mRNAs having complementary
sequences with siRNA seed region in their 30 UTRs were examined. The obvious off-target effects were observed by the
introduction of unmodified siVIM-270. However, DNA-modified siVIM-270 showed reduced off-target effects. On theother hand, 20 -OMe-modified siRNA showed little off-target
effects (Fig. 5b–d).

SiRNA Modifications Avoid Off-Target Effect29

Acknowledgment
This work was financially supported by the Grants-in-Aid for Scientific Research (B) (No. 15H04319) and on Innovative Areas(No. 26102713) from the Ministry of Education, Culture, Sports,
Science and Technology and Japan Society for the Promotion of
Science, and by the grant from the Suzuken Memorial Foundation
to Kumiko Ui-Tei. We used figure data rearranged with original
previous report [13].
References
1. Wilson RC, Doudna JA (2013) Molecularmechanisms of RNA interference. Annu Rev
Biophys 42:217–239
2. Nakanishi K (2016) Anatomy of RISC: how dosmall RNAs and chaperones activate Argonaute
proteins? Wiley Interdiscip Rev RNA
7:637–660
3. Sheu-Gruttadauria J, MacRae IJ (2017) Structural foundations of RNA silencing by Argonaute. J Mol Biol 429:2619–2639
4. Adams D, Gonzalez-Duarte A, O’Riordan
WD, Yang C-C, Ueda M, Kristen AV,
Tournev I, Schmidt HH, Coelho T, Berk JL
et al (2018) Patisiran, an RNAi therapeutic, forhereditary transthyretin amyloidosis. N Engl J
Med 379:11–21
5. Ui-Tei K, Naito Y, Takahashi F, Haraguchi T,
Ohki-Hamazaki H, Juni A, Ueda R, Saigo K(2004) Guidelines for the selection of highly
effective siRNA sequences for mammalian andchick RNA interference. Nucleic Acids Res
32:936–948
6. Jackson AL, Bartz SR, Schelter J, Kobayashi
SV, Burchard J, Mao M, Li B, Cavet G, Linsley
PS (2003) Expression profiling reveals
off-target gene regulation by RNAi. Nat Biotechnol 21:635–637
7. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ,
Wolfsberg TG, Umayam L, Lee JC, Hughes
CM, Shanmugam KS, Bhattacharjee A,
Meyerson M, Collins FS (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted
proteins in mammalian cells. Proc Natl Acad
Sci USA 101:1892–1897
8. Lin X, Ruan X, Anderson MG, McDowell JA,
Kroeger PE, Fesik SW, Shen Y (2005) siRNA
mediated off-target gene silencing triggered by
a 7 nt complementation. Nucleic Acids Res
33:4527–4535
9. Birmingham A, Anderson EM, Reynolds A,
Ilsley-Tyree D, Leake D, Fedorov Y,
Baskerville S, Maksimova E, Robinson K,

Karpilow J, Marshall WS, Khvorova A (2006)
30 UTR seed matches, but not overall identity,
are associated with RNAi off-targets. Nat
Methods 3:199–204
10. Jackson AL, Burchard J, Schelter J, Chau BN,
Cleary M, Lim L, Linsley PS (2006) Widespread siRNA “off-target” transcript silencing
mediated by seed region sequence complementarity. RNA 12:1179–1187
11. Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K(2008) Thermodynamic stability and WatsonCrick base pairing in the seed duplex are major
determinants of the efficiency of the siRNAbased off-target effect. Nucleic Acids Res
36:7100–7109
12. Ui-Tei K, Naito Y, Zenno S, Nishi K,
Yamato K, Takahash F, Juni A, Saigo K(2008) Functional dissection of siRNAsequence by systematic DNA substitution:
modified siRNA with a DNA seed arm is a
powerful tool for mammalian gene silencing
with significantly reduced off-target effect.
Nucleic Acids Res 36:2136–2151
13. Iribe H, Miyamoto K, Takahashi T,
Kobayashi Y, Leo J, Aida M, Ui-Tei K (2017)
Chemical modification of the siRNA seedregion suppresses off-target effects by sterichindrance to base-pairing with targets. ACS
Omega 2:2055–2064
14. Xia T, SantaLucia J Jr, Burkard ME, Kierzek R,
Schroeder SJ, Jiao X, Cox C, Turner DH(1998) Thermodynamic parameters for an
expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick basepairs. Biochemistry 37:14719–14735
15. Sugimoto N, Nakano S, Katoh M,
Matsumura A, Nakamuta H, Ohmichi T,
Yoneyama M, Sasaki M (1995) Thermodynamic parameters to predict stability of
RNA/DNA hybrid duplexes. Biochemistry34:11211–11216
16. Naito Y, Yoshimura J, Morishita S, Ui-Tei K(2009) siDirect 2.0: updated software for30

Yoshiaki Kobayashi et al.

designing functional siRNA with reduced seeddependent off-target effect. BMC Bioinformatics 10:392
17. Reynolds A, Leake D, Boese Q, Scaringe S,
Marshall WS, Khvorova A (2004) Rational
siRNA design for RNA interference. Nat Biotechnol 22:326–330
18. Amarzguioui M, Prydz H (2004) An algorithm
for selection of functional siRNA sequences.
Biochem
BiophysRes
Commun316:1050–1058
19. Grünweller A, Wyszko E, Bieber B, Jahnel R,
Erdmann VA, Kurreck J (2003) Comparison ofdifferent antisense strategies in mammaliancells using locked nucleic acids, 20 -O-methyl
RNA, phosphorothioates and small interfering
RNA. Nucleic Acids Res 31:3185–3193
20. Chiu YL, Rana TM (2003) siRNA function in
RNAi: a chemical modification analysis. RNA
9:1034–1048
21. Czauderna F, Fechtner M, Dames S, Aygun H,
Klippel A, Pronk GJ, Giese K, Kaufmann J(2003) Structural variations and stabilising
modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res 31:2705–2716
22. Choung S, Kim YJ, Kim S, Park HO, Choi YC(2006) Chemical modification of siRNAs to
improve serum stability without loss of efficacy.
Biochem Biophys Res Commun 342:919–927

23. Ge Q, Dalla A, Ilves H, Shorenstein J, Behlke
MA, Johnston BH (2010) Effects of chemical
modification on the potency, serum stability,
and immunostimulatory properties of short
shRNAs. RNA 16:118–130
24. Lee JH, Pardi A (2007) Thermodynamics andkinetics for base-pair opening in the P1 duplexof the Tetrahymena group I ribozyme. Nucleic
Acids Res 35:2965–2974
25. Iwakawa HO, Tomari Y (2015) The functionsof microRNAs: mRNA decay and translational
repression. Trends Cell Biol 11:651–665
26. Treiber T, Treiber N, Meister G (2019) Regulation of microRNA biogenesis and its crosstalkwith other cellular pathways. Nat Rev Mol Cell
Biol 1:5–20
27. Gebert LFR, MacRae IJ (2019) Regulation of
microRNA function in animals. Nat Rev Mol
Cell Biol 1:21–37
28. Schirle NT, MacRae IJ (2012) The crystal
structure of human Argonaute2. Science
336:1037–1040
29. Elkayam E, Kuhn CD, Tocilj A, Haase AD,
Greene EM, Hannon GJ, Joshua-Tor L(2012) The structure of human argonaute2 in complex with miR-20a. Cell 150:100–110