ISOLATION AND CHARACTERIZATION OF RNA APTAMERS SPECIFIC FOR THE
HUMAN TOLL-LIKE RECEPTOR 3 ECTODOMAIN
Tomoya Watanabe1, Kiyoharu Ito2, Misako Matsumoto2, Tsukasa Seya2, Kotomi Hatakeyama1, Satoshi
Nishikawa3, Tsunemi Hasegawa1, and Kotaro Fukuda1*
1
Department of Material and Biological Chemistry, Faculty of Science, Yamagata University,
Yamagata 990-8560, Japan.
*
Corresponding author: Kotaro Fukuda
FAX: 023-628-4604; E-mail: kotaro.f@sci.kj.yamagata-u.ac.jp
2
Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University,
Sapporo 060-8638, Japan.
3
Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology(AIST), Tsukuba 305-8566, Japan.
(Received March 10, 2009; Accepted June 1, 2009)(Abstract)
Toll-like receptor 3 (TLR3) detects
double-stranded RNA (dsRNA), known to be auniversal viral molecular pattern, and activates theantiviral
immuneresponse.
While
TLR3
preferentially recognizes polyinosinic-polycytidylicacid (poly(I:C)), no sequence-specific dsRNA has
been shown to activate TLR3. To determine whether
TLR3 preferentially recognizes a specific RNA
sequence or structure that acts upon the TLR3
signaling pathway, in vitro selection against thehuman TLR3 ectodomain (TLR3 ECD) was
performed. After the seventh selection cycle, twomajor classes, Family-I and Family-II, emerged from64 clones with binding constants of about 3 nM. To
examine the structure–function relationship of
Family-I and -II aptamers, mutational analyses and
RNase mapping were carried out. Furthermore, toelucidate the effect of selected aptamers on TLR3
signaling in vivo, a reporter gene assay was
conducted in cells. These aptamers did not have any
agonistic or antagonistic effects on TLR3 signaling in
TLR3-transfected HEK293 cells, although theybound to TLR3 ECD with high affinity in vitro.
These results suggest that selection of RNA aptamers
for TLR3 ECD should be performed underphysiological conditions, since TLR3 ECD localizes
in acidic compartments such as endosomes.
(Keywords)
Toll-like receptor 3, TLR3 ectodomain, RNA aptamer,in vitro selection, SELEX
1. Introduction
Toll-like receptors (TLRs) play central roles in
the innate immune response by recognizing
conserved structural patterns in diverse microbial
molecules [1]. All TLRs are type I integral membrane
glycoproteins composed of an ectodomain (ECD)
linked by a transmembrane domain to a cytoplasmic
signaling Toll/IL-1 receptor (TIR) domain [2]. Ligand
recognition by TLRs is mediated through theirN-terminal ECD, which contains varying numbers ofleucine-rich repeats (LRRs) [3]. More than 10 TLRs
have been identified in human and mouse [4]. TLR1,
TLR2, TLR4, TLR5, and TLR6 recognize bacterialcell wall and cell surface components, such as
lipoproteins, lipopolysaccharide, and flagellin. In
contrast, TLR3, TLR7, TLR8, and TLR9 recognize

Viva Origino 37 (2009) 10 - 18
ⓒ 2009 by SSOEL Japan

pathogen nucleic acids, such as viral RNAs andbacterial
DNA[4,5].
TLR3
recognizes
double-stranded RNA (dsRNA) derived from viralgenomes and replication intermediates and recruits
TIR-containing adaptor molecule (TICAM)-I (alsocalled TIR-containing adaptor inducing interferon(IFN)-β, TRIF), leading to the production of IFN-β
and inflammatory cytokines [6,7]. As these immune
responses are essential for combating viral invasions,
TLR3 plays a key role in antiviral immunity. On theother hand, it is known that West Nile virus infection
leads to a TLR3-dependent inflammatory response,
which is involved in brain penetration of the virus
and neuronal injury [8]. The three-dimensional
structure of the human TLR3 ECD, which contains
the dsRNA-binding region, has been reported by twogroups [9,10]. TLR3 ECD resembles ahorseshoe-shaped solenoid of 23 LRRs, with each
LRR forming one turn of the solenoid. Furthermore, arecent report revealed the crystal structure of TLR3
ECD bound to a dsRNA ligand [11]. Each TLR3
ECD binds to a dsRNA at two sites located at
opposite ends of the TLR3 horseshoe, and anintermolecular interaction between the two TLR3
ECD C-terminal domains coordinates and stabilizes
the dimer formation.
Nucleic acid-sensing TLRs, TLR3, TLR7,
TLR8, and TLR9, are localized in the endosome[12,13]. It has been reported that TLR7 and TLR8
(receptors for single-stranded RNA), as well as TLR9
(a receptor for CpG oligodeoxyribonucleotide),
respond to their ligands in a sequence-dependent
manner [14]. Although polyinosinic-polycytidylicacid (poly(I:C)), a synthetic dsRNA analogue, is apotentinducerof
TLR3
signaling,nosequence-specific inducer or inhibitor for TLR3 isknown. Anti-inflammatory drugs (TLR3 antagonists)
and adjuvants for vaccines (TLR3 agonists) that
regulate TLR3 signal transduction could be important
for the development of future therapeutic treatments.
Therefore, to investigate whether TLR3 recognizes
and responds to specific dsRNA, in vitro selection
against TLR3 ECD was carried out. In vitro selection(SELEX) is a powerful tool for obtaining RNA
ligands that bind to a target molecule with highaffinity and specificity [15,16]. So far, high-affinity
DNA or RNA ligands for target molecules including
proteins, organic dyes, and amino acids have been
isolated by in vitro selection [17]. Because binding of- 10 -

Viva Origino 37 (2009) 10 - 18
PAGE. The final yield of the random RNA pool
N40H was approximately 70 µg.
Family-I and -II aptamer mutants wereconstructed by PCR mutagenesis using mutagenic
primers and template DNA. The DNA sequences ofthese mutants were confirmed by an Applied
Biosystems model 3100 automatic sequencer(Applied Biosystems).

an aptamer to its target is a highly specific interactionthat involves discrimination between related proteinsthat share common sets of structural domains,
aptamers represent a potentially attractive class ofmolecules for therapeutic compounds. Furthermore,
RNA aptamers against RNA-binding proteins (RBPs)
are useful for dissecting the interactions between
RNA and protein, and such aptamers could
potentially modulate the function of target RBPs.
This report describes the isolation andcharacterization of RNA aptamers that bind with highspecificity to human TLR3 ECD. Application of
TLR3 ECD aptamers in vivo is also described toestimate their agonistic and antagonistic effects on
TLR3 signaling.

2.2. In vitro selection of RNA aptamers for
TLR3-ECD
In vitro selection was carried out essentially asreported previously [19]. For the first cycle of
selection, the N40H RNA pool (500 pmol) was
incubated with 62.5 pmol of TLR-ECD in 50 µl of
binding buffer [2 mM HEPES-NaOH (pH 7.6), 3 mM
MgCl2, and 100 mM NaCl] at room temperature for 1
h. The binding reaction was separated by filtration
through a nitrocellulose filter (HAWP filter, 0.45 µm)
fitted in a pop-top filter holder (Nucleopore) andwashed with 1 mL of binding buffer. The filter was
eluted with 200 µl of elution buffer [0.4 M sodium
acetate (pH 5.5), 5 mM EDTA (pH 8.0), and 7 Murea] at 90°C for 5 min. The eluted RNA wasrecoveredbyethanol
precipitation
andreverse-transcribed using RevertAidTM M-MuLV
Reverse Transcriptase (Fermentas) at 42°C for 1 h.
The cDNA product was amplified by PCR andtranscribed in vitro using the T7 AmpliScribe Kit(Epicentre Technologies). The product of thisreaction was subjected to another cycle of selection
for a total of 10 cycles. To specifically enrich the
RNA pool for high-affinity ligands for TLR-ECD, theconcentration of TLR-ECD and the RNA ligand weremanipulated for each selection cycle (Table 1). YeasttRNA (Boehringer Mannheim) was used as anon-specific competitor during the selection process.
The PCR product of the seventh selection
cycle was introduced into the TA cloning vector(Invitrogen). After subcloning and transformation
into E. coli, plasmid DNA was isolated from

2. Materials and Methods
2.1. Protein and nucleic acids
TLR3 ECD with an N-terminal FLAG tag andC-terminal His tag (TLR3-ECD) was expressed using
a baculovirus system and purified as described
previously [18]. The starting RNA pool of N40H forin
vitro
selectionhasthesequence5'-GGUAGAUACGAUGGA–(N40)–CAUGACGCG
CAGCCA-3'. The antisense DNA template,
5'-TGGCTGCGCGTCATG–(N40)–TCCATCGTATC
TACC-3' (10 pmol), was converted to dsDNA by
PCR
with
a
5'-endprimer,5'-TGTAATACGACTCACTATAGGTAGATACGAT
GGA-3' (40 pmol). Underlined letters in the 5'-endprimer indicate the T7 RNA promoter sequence. PCR
was carried out for six cycles, and dsDNA wasrecovered by ethanol precipitation. The dsDNA (50pmol) was reamplified by PCR with the 5'-end primer
and a 3'-end primer, 5'-TGGCTGCGCGTCATG -3'(100 pmol each). After 20 cycles of PCR, theamplified DNA pool was recovered by ethanol
precipitation. T7 RNA transcription was performed
with at least 100 pmol of the DNA pool using the T7
AmpliScribe Kit (Epicentre Technologies). The
transcribed RNA was then purified on 8% denaturing

Table 1. TLR3-ECD aptamer selection and binding analysis. TLR3-ECD,
RNA pool, and competitor tRNA concentrations used during the selection
step (total volume, 50 µL) of each generation are shown. The binding
activity of the RNA pool at each cycle was analyzed by a filter binding
assay with competitor tRNA (TLR3-ECD : RNA pool: tRNA = 0.05 :0.05 : 2.5 µM) as described in the Materials and Methods. Binding
activities were calculated as the percent of input RNA.

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Viva Origino 37 (2009) 10 - 18
2.6. Reporter gene assay
HEK293 cells were seeded in 24-well plates (5x 105 cells/well). Twenty-four hours later, the cells
were transiently transfected with pEFBOS/TLR3 (0.1µg) together with a p125-luc reporter (0.1 µg) and
Renilla luciferase reporter (2.5 ng) in the presence of
Lipofectamine 2000 (Invitrogen). The total amount oftransfected plasmid (0.8 µg) was kept constant byadding empty vector. Twenty-four hours after
transfection, the medium was replaced by fresh
medium. RNA aptamer, poly(I:C) or both, was mixed
with DOTAP Liposomal Transfection Reagent (3µg/well; Roche) in OPTI-MEM (7 µl/well;
Invitrogen) and pre-incubated for 15 min at R.T. The
DOTAP/RNA complex was then transfected into cells
and incubated. Cells were collected and washed twicewith 1 ml of PBS buffer. The collected cells werethen lysed using Passive Lysis Buffer (Promega), andthe cell lysates were assayed for the dual luciferase
activities (Promega). Data are expressed as mean
relative stimulation SD for a representative
experimentfromindependent
experiments,
performed in duplicate.

individual clones and the DNA sequences of 64
clones were analyzed with an Applied Biosystems
model 3100 automatic sequencer (Applied
Biosystems). The secondary structure models of
selected aptamers, Family-I and -II, were drawn by
the Mulfold program based on the Zuker algorithm[20].
2.3. Filter binding assay
The binding activity of the RNA pool was
analyzed after each selection cycle. PCR products
were internally transcribed in vitro with [α-32P]CTP(Amersham), and the transcription product was tested
by a filter binding assay using equimolar TLR-ECD
and RNA and a 50-fold molar excess of non-specificcompetitor tRNA (TLR-ECD and RNA pool, 50 nM;
tRNA, 2.5 µM) in binding buffer [2 mM
HEPES-NaOH (pH 7.6), 3 mM MgCl2, and 100 mM
NaCl]. Radioactivity retained on the filter wascounted with a BAS2000 image analyzer (Fuji Film).
The binding activity was evaluated by calculating thepercent of the input RNA retained on the filter incomplexes with TLR-ECD (Table 1).
The equilibrium dissociation constants (KD)for the selected aptamers, Family-I and -II, were
determined by using a constant amount of internallylabeled RNA aptamer (1 nM) in binding reactions
with increasing concentrations of TLR-ECD (0.25–64nM). Mixtures containing RNA and TLR-ECD werepassed through a nitrocellulose filter and the filter
was washed with 1 mL of binding buffer. The data
points were fitted to a Scatchard plot to determine theequilibrium dissociation constant by Prism 4 ver 4.0b
(GraphPad Software Inc.).
To analyze the binding ability of Family-I and-II mutants to TLR3-ECD, each 32P-labeled mutant
RNA (50 nM) was incubated with TLR3-ECD (50nM) in 50 µl of binding buffer at 37°C for 1 h. The
protein/RNA complex was separated by filtration ona nitrocellulose filter as described above.
Radioactivity remaining on the filter was measured
using a BAS2000 image analyzer (FujiFilm), and theamount of Family-I and -II mutants bound to
TLR3-ECD was calculated as a percentage of the
RNA input prior to filtration.

3. Results
3.1. Sequence and secondary structure of RNA
aptamers specific for TLR3 ECD
To isolate high-affinity RNA ligands for TLR3
ECD, in vitro selection was carried out using an RNA
library containing N40 random core sequences andrecombinant TLR3-ECD protein. Initially, therandomized RNA pool (N40H), which consists ofapproximately 3 x 1014 molecules (500 pmol), wasincubated with TLR3-ECD (62.5 pmol), and the
RNA/TLR3-ECD complexes were separated from theunbound RNA molecules by passage through anitrocellulose filter. The RNA retained on the filter
was recovered, subjected to RT-PCR, transcribed by
T7 RNA polymerase, and used for the next selection
cycle. To specifically enrich the RNA pool forhigh-affinity ligands specific for TLR3-ECD, theconcentrations of TLR3-ECD, RNA ligand, andcompetitor tRNA were manipulated for each selection
cycle (Table 1). To confirm whether the selected
RNA pools bind to TLR3-ECD, the binding activityof the RNA pools was evaluated by a filter binding
assay after each selection cycle. The RNA pool fromthe seventh selection cycle (G7) had the highest
affinity; about 17% of the total RNA pool bound to
TLR3-ECD (Table 1).
To analyze the sequence and structural motifs
in the G7 pool, we cloned the seventh-cycle PCR
product into the TA vector and sequenced 64 clones.
Although there were no conserved sequences among
any of the clones, we were able to categorize theminto 11 classes, from Family-I to -XI as shown in
Table 2. Of these, two major classes emerged withentirely identical sequences: Family-I with 12 clones,
followed by Family-II with 11 clones. We therefore
focused on these Family-I and -II aptamers andproceeded to characterize them. When the secondary
structures of the two aptamer classes were analyzed
by the Mulfold program [20], we found that both2.4. Structural analysis of RNA aptamers
For enzymatic probing, 5'-end-labeled
Family-I and -II were partially digested with RNases
T1 or A as previously reported with slight
modifications [19], and the cleaved products wereanalyzed by 12% PAGE (7 M urea) with alkaline
ladder of Family-I and -II.
2.5. Cell culture
For the expression of plasmids encoding
human TLR3, HEK293 (human embryonic kidney)cells were used and cultured in DMEM cell culture
medium (GIBCO) supplemented with 10%
heat-inactivated fetal bovine serum (BIOSOURCE)
and antibiotics (penicillin/streptomycin).

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Table 2. Nucleotides sequences of RNA aptamers categorized into Family-I – Family-XI.
Selected sequences from the G7 RNA pool are shown in uppercase. The fixed sequences
for PCR primers are indicated in lowercase. Numbers in parentheses indicate thenumber of identical clones.

Family-I and -II aptamers were composed of threestem-loop (SL) structures, but we did not observe any
sequence conservation or structural similarity
between the two Families (Fig. 1).
3.2. Binding activity of Family-I and -II aptamers to
TLR3 ECDThe equilibrium dissociation constant (KD) of
Family-I and -II aptamers was determined by a filter
binding assay. Each aptamer (1 nM) was internallylabeled and incubated with increasing concentrationsof TLR3-ECD (0.25–64 nM) in the binding buffer.
When the molar ratio of the RNA aptamers and
TLR3-ECD was 1:64 (1 nM : 64 nM), approximately
27% maximal binding activity was observed (Fig. 2).
The data points were fitted to a Scatchard plot todetermine the equilibrium dissociation constant using
Prism 4 ver 4.0b (GraphPad Software Inc.). The
apparent KD values for the aptamers wereapproximately 3 nM (Family-I, 2.1 nM; Family-II,
3.6 nM). Based on the KD values for TLR3-ECD,
selected Family-I and -II aptamers harbor highaffinity for TLR3-ECD.
3.3. Enzymatic probing of Family-I and -II aptamers
The secondary structure models for Family-I
and -II aptamers shown in Fig. 1 are based on
Mulfold analysis of the RNA sequences. To confirm
whether the solution structures of the two aptamergroups are consistent with the proposed secondary
structure models, Family-I and -II aptamers were
subjected to enzymatic probing using RNases T1 andA, which cleave adjacent to the 3'-phosphate of G

Figure 1. The secondary structure models of Family-I and-II aptamers against TLR3-ECD predicted by the Mulfold
program [20]. Selected sequences are shown in normal
letters. Lowercase letters indicate the constant sequence
regions flanking the randomized N40 core. SL, stem–loop
structure.

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Viva Origino 37 (2009) 10 - 18
andpyrimidine
nucleotides,
respectively.
5'-end-labeled Family-I and -II RNAs were partiallydigested, and the cleavage products were analyzed bydenaturing 12% PAGE (Fig. 3A and 3C). The
secondary structures of the Family-I and -II aptamers,
cleavage sites were observed mainly at unpaired
residues, corresponding to loop regions and spacer
sequences flanked by stem–loop structures (Fig. 3B
and 3D). In general, the cleavage patterns of the two
enzymes appear to be consistent with the proposed

Figure 2. Binding curves of Family-I and -II aptamers for
TLR3-ECD. Data points were obtained by a filter binding
assay (50 µ l reaction) as described in the Materials andMethods (Family-I, closed circles with solid line;
Family-II, open circles with dotted line). The
concentration of labeled aptamers was 1 nM. The values
represent means of three independent experiments. RNAbinding shows the percentage of RNA retained on thefilter in protein/RNA complex against input RNA.

Figure 3. Enzymatic probing of Family-I and -II aptamers.
5'-end-labeled Family-I (A) and Family-II (C) aptamers
were partially digested with RNase T1 (T1) or RNase A(A) and analyzed by 12% PAGE containing 7 M urea
together with an alkaline ladder of them (OH).
Nuclease-accessible sites in Family-I (B) and in Family-II(D) aptamers are shown by arrows (RNase T1) and open
triangles (RNase A), respectively.

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Viva Origino 37 (2009) 10 - 18
in binding to TLR3-ECD.
On the other hand, four mutants wereconstructed in the Family-II aptamer sequence asfollows: 1) exStem I (three duplex sequences at Stem
I
interchanged),
2)
mLoop
II
(CGGGUCC→CaaaUCC), 3) inStem II (several
duplex sequences at Stem II inserted), and 4) mSp II(ACGUUAAUUGAG→ACGUaAAUaGAG;
thespacer sequence between SL II and SL III was
mutated) (Fig. 4C).
In a filter binding assay, exStem I retained
binding ability as well as wild type (Fig. 4D).
However, a drastic decrease of binding was observedin mLoop II, inStem II, and mSp II mutants (Fig. 4D).
These results suggest that both SL II structure and thespacer sequence between SL II and SL III in
Family-II aptamers are indispensable for binding to
TLR3-ECD.
secondary structures of the Family-I and -II aptamers,
which are composed of three stem–loop structures.
Furthermore, sites that were not susceptible tocleavage were noted in the loop regions in both
aptamers, suggesting that these loop structures mightinteract with other regions to form a tertiary structure,such as a pseudoknot. Taken together, these results
provide experimental evidence to support the
proposed secondary structures of Family-I and -II
aptamers determined by the Mulfold program.
3.4. Structural and binding analyses of Family-I and-II aptamers
As mentioned above, no conserved sequenceor structural similarity was observed between
Family-I and -II aptamers (Fig. 1). To investigate therole of stem–loop structures in binding to TLR3-ECD,
mutagenesis on Family-I and -II aptamers was carriedout. Seven mutants were constructed in the Family-I
aptamer sequence as follows (small letters indicate
changednucleotides):
1)
dLoop
I
(ACGAUGG→ACGAU; GG at 3’ end of Loop I
deleted), 2) exStem I (two duplex sequences at Stem I
interchanged),
3)
mLoop
II
(GCCCGUCAA→GaaaGUCAA), 4) exStem II(several duplex sequences at Stem II interchanged),5) dLoop III (AUGACGCGCA→aug; deletion ofmost of Loop III), 6) exStem III (two duplex
sequence at Stem III interchanged), and 7) mSp I(AGGGUAC→AuuuUAC; the spacer sequence
between SL I and SL II were mutated) (Fig. 4A).
The binding of 32P-labeled mutant forms of
Family-I aptamers to TLR3-ECD was examined by afilter binding assay as described in the Materials andMethods (Fig. 4B). We observed that the binding
levels of dLoop I, exStem I, dLoop III, and exStem
III, in which mutations were introduced into SL I andSL III, respectively, were almost comparable to thatof wild type. However, mLoop II, exStem II, andmSpI dramatically lost the ability to bind to
TLR3-ECD. These results suggest that in addition to
the spacer sequence between SL I and SL II, the SL II
structure in Family-I aptamers plays a significant role-15-

3.5. Selected aptamers have no effect on TLR3
signaling
To clarify the effect of Family-I and -II
aptamers on TLR3 signaling, a reporter gene assay
was performed as previously reported [21].
TLR3-negative HEK293 cells were transiently
transfected with a human TLR3 expression plasmid,
together with a reporter plasmid containing aluciferase gene under the control of the human IFN-β
promoter; selected aptamers were then introduced
into the cells. TLR3 preferentially recognizes
poly(I:C) as an RNA ligand, therefore it was used asa positive control. Although TLR3-mediated IFN-β
promoter activation by poly(I:C) was clearlyobserved, both Family-I and -II aptamers failed toinduce luciferase gene expression (Fig. 5A). Next, we
introduced both the selected aptamer and poly(I:C)
into HEK293 cells in which TLR3 was transiently
expressed to examine the competitive effect ofFamily-I and -II aptamers against poly(I:C) on TLR3
signaling. Although the competitive effects ofFamily-I and -II aptamers on the poly(I:C) induction
were noticeable, the G0 RNA pool (N40H RNA pool)for the negative control also produced the same effect(Fig. 5B). Taken together, these selected aptamers
showed neither agonistic, nor antagonistic effects inhuman TLR3-transfected HEK293 cells in spite of
the high affinity binding to TLR3 ECD in vitro.
4. Discussion
TLR3 has an essential role in the innate
immuneresponseandrecognizes
polyinosinic-polycytidylic acid (poly(I:C)), a
synthetic double-stranded (ds) RNA analog, as well
as viral dsRNA, presumably formed during viral
infection. The TLR3-mediated immune response
induces the activation of NF-κB and the productionof type I interferons, and thus TLR3 plays a key
function in antiviral immune responses. On the other
hand, several reports have shown that TLR3 does not
display a protective function against some viral
infections in spite of its anti-viral immune responses[8, 22]. Although poly(I:C), or a synthetic dsRNA
analog, are potent inducers of TLR3 signaling, aViva Origino 37 (2009) 10 - 18

Figure 4. Binding analyses of Family-I and -II aptamer mutants to TLR3-ECD. Secondary structures ofmutants in Family-I (A) and -II aptamers (C) are shown. Mutated regions are circled and boxed; small
letters indicate the exchanged or introduced bases. The binding activity of Family-I (B) and -II (D) aptamer
mutants were examined by a filter binding assay as described in the Materials and Methods. The values
represent the means of three independent experiments and the relative RNA binding activity (wildtype=100%) shows the percentage of RNA retained on the filter in protein/RNA complex against inputRNA.

RNA aptamers specific for TLR3-ECD, designated as
Family-I and -II. Although both show high affinity
for the target with KD values of about 2 to 4 nM, they
do not harbor any conserved sequences between them.
Mutational analyses on Family-I and -II aptamers
revealed that particular regions, such as SL II
structures and spacer sequences, are indispensable forbinding to TLR3-ECD. The binding site of theselected RNA aptamers on TLR3 ECD is not known,sequence-specific inducer, or inhibitor, has not been
identified.
It is important to develop novel RNAs thatfunction as anti-inflammatory drugs (TLR3
antagonists) and adjuvants for vaccines (TLR3
agonists). For this purpose, in vitro selection against
TLR3 ECD was performed using a recombinant
TLR3-ECD protein and a synthetic N40 random
RNA pool. We succeeded in obtaining two classes of-16-Viva Origino 37 (2009) 10 - 18

although one possibility is that the two aptamers
might interact with an electropositive region of theprotein through ion pairs. Indeed, two patches,
including several positively charged residues, arefound on the glycosylation-free surface of TLR3
ECD [9, 10].
Two selected aptamers, which bind to
TLR3-ECD with high affinity in vitro, had noinfluence on the TLR3 response. It has been reported
that TLR3 localizes to acidic compartments and thatacidic conditions are necessary for TLR3 signaling[12, 23]. Recent reports show that four highly
conserved histidine residues, H39, H60, H108, andH539, that are protonated under acidic conditions,play a key role in recognizing dsRNA in vitro and invivo [18, 24]. The RNA aptamers were isolated under
neutral conditions; therefore we checked the binding
ability of Family-I and –II under acidic conditions(pH 4.2) using a filter binding assay. From the results,
both aptamers bound to TLR3-ECD as shown in thesame experiment under neutral conditions (pH7.6)(data not shown). Furthermore, the G0 RNA pool,which was the non-selected initial RNA pool, also

Figure 5. A reporter gene assay was carried out asdescribed in the Materials and Methods. (A) HEK293 cells
transfected with empty vector (pEF-BOS) or TLR3
expression vector (pEF-BOS/TLR3) were stimulated with
Family-I and -II aptamers (0.2 and 1.0 µ g/ml), the G0
RNA pool (N40H RNA pool; 0.2 and 1.0 µ g/ml), or 1.0µg/ml of poly(I:C) complexed with DOTAP. (B) Family-I
and -II aptamers and the G0 RNA pool (2, 6, and 10µg/ml) were introduced into HEK293 cells transfectedwith pEF-BOS or pEF-BOS/TLR3, in the presence, orabsence, of 2 µg/ml of poly(I:C) complexed with DOTAP.
The firefly luciferase activity was normalized to Renillaluciferase activity. Relative luciferase units (RLU) were
calculated by dividing the normalized luciferase activity
by the result obtained in the absence of RNA ligands (–).

revealed the same binding behavior (data not shown).
Taking into account all of these data, the specificinteractions of the two aptamers may be diminishedunder acidic conditions due to the protonation offunctional histidine residues in TLR3 ECD. This-17-

Viva Origino 37 (2009) 10 - 18
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speculation is supported by the results, which showedthat Family-I and –II aptamers mimicked theantagonistic effect as observed in the G0 RNA pool(Fig. 5B). In addition, the length of the
double-stranded region of the RNA is also importantfor binding to TLR3 [21, 25]. Therefore, the binding
region of the RNA aptamers may be too short, or
their binding may not be specific to the functional
regions of TLR3 ECD for proper signaling.
Furthermore, steric hindrance by higher structure
glycosylation, or the presence of co-receptors, oraccessory molecules, may affect the precise
interaction of ligands.
In this study, we obtained RNA aptamers(Family-I and -II) that bind to TLR3 ECD with highaffinity. These aptamers showed neither agonistic,
nor antagonistic effects on TLR3 signaling in acell-based assay. However, the function of TLR3
might be manipulated by designing novel RNA
constructs based on these two aptamers, or a new invitro selection procedure under acidic conditions
could create functional aptamers that regulate TLR3
signaling in vivo.
Acknowledgement
We thank T. Tokisue for helpful discussions.
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