Article
pubs.acs.org/ac

Ampliﬁcation and Quantiﬁcation of an Antisense Oligonucleotide
from Target microRNA Using Programmable DNA and a Biological
Nanopore
Moe Hiratani, Masayuki Ohara, and Ryuji Kawano*Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology (TUAT), 2-24-16 Naka-cho
Koganei-shi, Tokyo 184-8588, JapanS Supporting Information*

ABSTRACT: This paper describes a strategy for autonomous
diagnoses of cancers using microRNA (miRNA) and therapy fortumor cells by DNA computing techniques and nanopore
measurement. Theranostics, which involves the combination ofdiagnosis and therapy, has emerged as an approach forpersonalized medicine or point-of-care cancer diagnostics. DNA
computing will become a potent tool for theranostics because it
functions completely autonomously without the need for external
regulations. However, conventional theranostics using DNA
computing involves a one-to-one reaction in which a single
input molecule generates a single output molecule; theconcentration of the antisense drug is insuﬃcient for the therapy
in this type of reaction. Herein we developed an ampliﬁcation
system involving an isothermal reaction in which a large amount of the antisense DNA drug was autonomously generated after
detecting miRNA from small cell lung cancer. In addition, we successfully quantiﬁed the amount of the generated drug moleculeby nanopore measurement with high accuracy, which was more accurate than conventional gel electrophoresis. This autonomous
ampliﬁcation strategy is a potent candidate for a broad range of theranostics using DNA computing.

D

methods have been proposed, among which we selected a DNA
strand polymerization reaction using a nicking endonuclease
and DNA polymerase.8 On the basis of this ampliﬁcation
reaction, we constructed a theranostic system for small cell lungcancer (SCLC). In this study, we prepared a model (Figure 1):
miRNA 20a (miR-20a), excreted from SCLC,9,10 was detected,
and then, “an oligo with the same sequence of bases as
oblimersen (oblimersen)”, an antisense DNA drug,11 wasautonomously released with ampliﬁcation. The mean concentration of exosomal miRNA from SCLC patients is 158.6 ng/
mL (ca. 22.5 nM), and the dosage of oblimersen is 3 mg/kg/
day (ca. 500 nmol/L/day).12 Therefore, oblimersen should be
ampliﬁed at an approximately 20-fold concentration from theinitial miRNA concentration in this model.
To develop this autonomous system, nanopore measurement
was employed to detect the output molecule in real time andwithout the need for labeling. The nanopore technique is apromising, recently developed method for single-molecule
detection in an aqueous solution.13−18 We previously
demonstrated DNA logic operation in a four-droplet device
including a lipid bilayer and nanopore.19 The output DNA
molecule was electrically detected using an α-hemolysin (αHL)

NA computing is a form of molecular computing in a wet
solution. Although early DNA computing studies focusedon parallel and complex calculations, for example, an NPcomplete problem (known as a traveling salesman problem), it
has recently been demonstrated to have a broad range ofapplications, such as in vivo,1,2 analytical,3 and autonomous
theranostic4 applications. Shapiro et al. have reported4−6 anautonomous diagnosis and drug release system using DNA
computing as follows. “If” certain diagnostic conditions are true,such as low expression levels of certain mRNAs and highexpression levels of others, “Then” the antisense drug isreleased. However, this type of system involves a one-to-one
reaction, i.e., a single input molecule generates a single output
molecule. This is incompatible with the requirement in mosttherapies that the concentration of the drug molecule (output)
be much higher than that of the diagnostic molecule (input), so
the need for ampliﬁcation of the input remains a challenge.
Therapy for tumor cells is one case that matches the above
scenario: ﬁrst, a small amount of microRNA (miRNA) secreted
from tumor cells is detected,7 after which a large amount ofantisense DNA to treat the tumor caused by the expression of aharmful gene is applied to the target cells. To construct a
procedure to amplify an antisense drug of this kind using DNA
computing, herein we focused on a DNA−enzyme isothermal
reaction, in which an input molecule is obtained and used toamplify output molecules. Several isothermal ampliﬁcation© XXXX American Chemical Society

Received: September 29, 2016
Accepted: January 27, 2017
Published: January 27, 2017
A

DOI: 10.1021/acs.analchem.6b03830
Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Biolabs Japan Inc. (Japan). Nt.AlwI, which is a nicking
endonuclease that cleaves only one strand of DNA on adouble-stranded DNA substrate, was also purchased from thesame company. dNTPs were from Takara Bio Inc. (Shiga,
Japan). 10× TBE buﬀer was obtained from Takara Bio Inc.
(Japan) and was 10-fold diluted for gel electrophoresis. The
power supply and an LED transilluminator were obtained from
Bio Craft Co., Ltd. (Tokyo, Japan) and Optocode Corporation(Tokyo, Japan), respectively.
DNA design and free energy calculation. To conﬁrm
that DNA and miRNA hybridized as intended, thermodynamic
analysis was performed using NUPACK (California Institute ofTechnology, http://www.nupack.org/).27 Because NUPACK
cannot simulate hybridization between DNA and RNA, two
hybridization patterns were analyzed: between DNAs only andbetween RNAs only. This analysis was performed at 37 °C
using a DNA or miRNA concentration of 20 nM in 1.0 M KClbuﬀered electrolyte solution. The melting temperature (Tm)
was calculated with Oligo Calculator version 3.27 (Northwestern University, http://biotools.nubic.northwestern.edu/
OligoCalc.html).28
miRNA detection and oblimersen generation basedon the three-way junction structure. Before the enzyme
reactions, the mixture of each miRNA and DNA was heated to
95 °C for 5 min and then rapidly cooled to 0 °C. miR-20a (100nM for gel and 20 nM for nanopore experiments) was added to
10 μL of 1× NE buﬀer 2 containing 20 nM primer DNA, 20
nM template DNA, 1.6 units of Klenow fragment, 4.0 units ofNt.AlwI, and 0.2 mM dNTPs. The solutions were incubated at
37 °C for 30 min for enzyme reactions. The enzyme reaction
was stopped by heating at 80 °C for 20 min prior to nanopore
measurements.
Gel electrophoresis and ﬂuorescence intensity analysis. Products of miRNA-triggered oblimersen ampliﬁcation
were analyzed by 15% denaturing polyacrylamide gel electrophoresis [containing 19/1 acrylamide/bis (w/w)] in 1× TBE
buﬀer (89 mM Tris-borate, 2 mM EDTA, pH 8.3) at a constant
power of 7.5 W for 45 min at room temperature. The gel was
prepared in our laboratory. After electrophoresis, the gel was
stained with diluted SYBR Green II (Takara Bio Inc., Japan)
solution for 30 min, imaged under blue LED irradiation, andﬁnally imaged with a digital camera. Fluorescence intensity was
analyzed by ImageJ 1.50 (National Institutes of Health,Bethesda, Maryland, USA). The ﬂuorescence intensity of eachband was calculated within square regions with the same area.
The measurement of ﬂuorescence intensity was done threetimes for each band.
Bilayer lipid membrane preparation and reconstitution of α-hemolysin. Bilayer lipid membranes (BLMs) wereprepared using a device produced by microfabrication. BLMs
can be simultaneously formed in this device by the droplet
contact method (Figure 2).29−34 In this method, the two lipid
monolayers contact each other and form BLMs on a parylene C
ﬁlm that separates two chambers. BLMs were formed asfollows: the wells of the device were ﬁlled with n-decane (2.7μL) containing DPhPC (10 mg/mL). The recording solutions(4.7 μL) on each side of the BLMs contained 1 M KCl and 1×
NE buﬀer 2 (pH 7.9). αHL was reconstituted in BLMs to form
a nanopore from the ground side, and 60 nM αHL was used fornanopore formation. The reaction solution was also added to
the ground side. Within a few minutes of adding the solutions,
BLMs were formed, and αHL created nanopores within them.
When the BLMs ruptured during this process, they were

Figure 1. (a) Schematic illustration of the theranostic system for small
cell lung cancer. (b) Schematic diagram of oblimersen translocation
through the αHL nanopore.

nanopore, which is utilized for the detection of single DNA/RNA molecules because the sizes of the pore and nucleotides
are comparable.20−26 In this droplet system, we conﬁgured
outputs “1” and “0” as single-stranded DNA (ssDNA) that is or
is not translocated through a nanopore, respectively; as a result,a NAND logic gate could be operated with electrical nanopore
measurements. Building on this previous work, in this study, weconstructed an autonomous ampliﬁcation system based on
DNA computing with nanopore measurements. The antisense
drug oblimersen could be ampliﬁed after detecting miR-20a by
isothermal ampliﬁcation using a three-way junction (3WJ)
structure, and then, the output molecule oblimersen wasquantiﬁed by nanopore measurement, instead of by conventional gel electrophoresis.

■

EXPERIMENTAL SECTION
Reagents and chemicals. All aqueous solutions wereprepared with ultrapure water from a Milli-Q system (Millipore,
Billerica, MA, USA). The reagents were as follows: 1,2diphytanoyl-sn-glycero-3-phosphocholine (DPhPC; Avanti
Polar Lipids, Alabaster, AL, USA), n-decane (Wako Pure
Chemical Industries, Ltd., Osaka, Japan), and potassium
chloride (KCl; Nacalai Tesque). Buﬀered electrolyte solutions[1.0 M KCl, 1× NE buﬀer 2 (New England Biolabs Japan Inc.,Tokyo, Japan), pH 7.9] were prepared from ultrapure water.
Wild-type alpha-hemolysin (Sigma-Aldrich, St. Louis, MO,
USA, and List Biological Laboratories, Campbell, CA, USA)was obtained as the monomer polypeptide, isolated from
Staphylococcus aureus in the form of a powder and dissolved at aconcentration of 1 mg/mL in ultrapure water. For use, samples
were diluted to the designated concentration using a buﬀered
electrolyte solution and stored at 4 °C. High-performanceliquid chromatography-grade DNA oligonucleotides andmiRNA were synthesized by FASMAC Co., Ltd. (Kanagawa,Japan) and stored at −20 °C and −80 °C, respectively. The
Klenow fragment (3′ → 5′ exo-) is an N-terminal truncation of
DNA polymerase I, and it was obtained from New England
B

DOI: 10.1021/acs.analchem.6b03830
Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 2. Photograph of the device for measuring channel currents.
Lipid bilayers are prepared by the droplet contact method, and αHL isreconstituted in the lipid bilayer.

recreated by tracing with a hydrophobic stick at the interface ofthe droplets.
Channel current measurements and data analysis.
The channel current was recorded with an Axopatch 200B
ampliﬁer (Molecular Devices, USA), ﬁltered with a low-passBessel ﬁlter at 10 kHz at a sampling rate of 50 kHz, or with
Pico2 (Tecella, CA, USA), ﬁltered with a low-pass Bessel ﬁlter
at 20 kHz at a sampling rate of 40 kHz. A constant voltage of+120 mV was applied from the recording side, and the groundside was grounded. The recorded data from Axopatch 200B
were acquired with Clampex 9.0 software (Molecular Devices,
USA) through a Digidata 1440A analog-to-digital converter(Molecular Devices, USA). The recorded data from Pico2 weremonitored through TecellaLab v0.98 (Tecella, USA). Data
from both ampliﬁers were analyzed using Clampﬁt 10.6
(Molecular Devices, USA), Excel (Microsoft, Washington,
USA), and R software (the R Foundation). DNA or miRNAtranslocation and blocking were detected when >60% of open
αHL channel currents were inhibited. Between 200 and 500
translocating events were recorded. All data are presented asmean ± S.E., and the diﬀerences were considered statisticallysigniﬁcant at P < 0.05 using Welch’s t test. The event frequency
was counted in each 10-s interval when one pore was open.
Subsequently, event frequency was analyzed using the central
limit theorem. Nanopore measurements were conducted at 22± 2 °C.

Figure 3. Schematic illustration of miRNA detection and oblimersen
generation.

DNA to produce new 3′-OH, which induced the next
polymerization by a Klenow fragment. (iii) The next polymerization started with strand displacement of oblimersen. By
repeating these cycles of nicking and strand displacement, alarge amount of oblimersen was autonomously generated.
Both template DNA and primer DNA contain a partial
sequence complementary to miR-20a and can also join together
to form double-stranded DNA. When miR-20a was not present
in the solution, they did not form double-stranded structureswith each other because polymerase reaction (i) undesirably
occurred despite the absence of miR-20a. To design theappropriate sequence of template DNA and primer DNA, we
ﬁrst designed at the stem region of 3WJ because other parts
were ﬁxed as the complementary sequence of oblimersen andmiR-20 (Figures 4a and 4b). Thermodynamic simulation (using
NUPACK) allowed us to design a sequence associated with a
Tm appropriate for the above conditions (see also the detailed
protocol in the SI). The length of the stem region of the 3WJ
was changed as its Tm became much lower than the reaction
temperature (37 °C).35 After designing the stem region, theappropriate stem sequence for the hybridization of the 3WJstructure was determined as the Tm of the stem region being 16°C (Figure 4c). In NUPACK simulation, two diﬀerent patterns
needed to be stimulated, one was the 3WJ formed by DNA
itself and the other was the 3WJ formed by RNA itself, because■

RESULTS AND DISCUSSION
Working principle of autonomous ampliﬁcation anddesign of DNA. To achieve the autonomous ampliﬁcation of
oblimersen after discriminating miR-20a, an isothermal DNA
reaction was employed using enzymes and 3WJ structure,35 thestructure and reaction scheme of which are shown in Figure 3.
The 3WJ was formed after recognizing miR-20a and consistedof template DNA that had a sequence complementary to that ofoblimersen, primer DNA, and a sequence for detecting miR20a. Ampliﬁcation was performed after the formation of the
3WJ structure as follows (see Figure 3): (i) The 3WJ structure
had a site (3′-OH) for recognizing a Klenow fragment at theend of the junction, and the polymerase synthesized oblimersen
along with the complementary sequence in the template DNA.
(ii) Nt.Alwl that cleaves the speciﬁc site nicked the synthesizedC

DOI: 10.1021/acs.analchem.6b03830
Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry(17 nM each). Next, the reaction conditions were optimized for
rapid ampliﬁcation. The reaction eﬃciency depending on the
Nt.AlwI concentration (3.0 U, 3.5 U, 4.0 U, 4.5 U, and 5.0 U)
was tested because this would be the rate-limiting factor of thisenzyme reaction under these conditions. The isothermal
reaction showed the highest activity in the case of 4.0 U
Nt.AlwI (Figure S1); similar enzyme optimization has beenshown for a duplex-speciﬁc nuclease.37 Finally, the quantity ofoblimersen generated was examined depending on the reaction
time using the ﬂuorescence intensity of the bands. The
apparent concentration of the output oblimersen was 34-foldin 30 min and 72-fold in 60 min compared with the initialconcentration of miR-20a (0.1 μM) (Figure S2). This was
suﬃcient for the purpose of theranostics because more than 20fold ampliﬁcation from the input miRNA has been reported tobe required for antisense SCLC therapy.11,12 Therefore, areaction time of 30 min was used in the nanopore experiments.
Nanopore measurement for quantifying autonomous
oblimersen generation. An αHL nanopore enables thedetection of DNA or RNA electrically in real time and without
the need for labeling. The nanopore was embedded in the lipid
bilayer at the interface of two droplets, and it could bemonitored using channel current, reﬂecting the translocation of
the generated oblimersen from one droplet to another. Figures
5a and 5b show the typical current−time traces for the

Figure 4. (a, b) Thermodynamic simulation of the 3-WJ predictedusing NUPACK when DNA was treated as RNA (a) and whenmiRNA was treated as DNA (b). (c) Sequence of template DNA,
primer DNA, and miR-20a. (d) PAGE image for conﬁrmation of
oblimersen generation. Products appeared in the lane of the reaction
solution for 30 min in the presence of miR-20a. Lanes of oblimersen
from 1 μM to 5 μM were used for making a calibration curve.

NUPACK cannot simulate the binding of DNA and RNA. The
results of these two diﬀerent simulations showed that the 3WJstructure was formed in both cases and that the Tm became thesame at 16 °C. The 3WJ structure would be formed because theΔG of DNA−RNA hybridization becomes an intermediate
value between those of DNA−DNA and RNA−RNA hybridization.36 Figure 4c shows the designed 3WJ sequences, which
formed a stable structure between the programmable DNA andmiR-20a. The speciﬁcity of this system was veriﬁed using
NUPACK simulation. The probability of 3WJ formation using
the sequence similar miRNA (miR-106) was 10 times lower
than that using miR-20a. Furthermore, it has been reported thathybridization cannot occur if there is a single-nucleotide
diﬀerence in the miRNA structure.26 The detailed procedure of
the design is presented in the SI. Next, we checked the functionof this design using gel electrophoresis.
Optimization of the isothermal reaction conditions.
Gel electrophoresis was ﬁrst performed to conﬁrm thefunctional capability of the primer and template DNAs.
Although oblimersen was not produced when miR-20a wasabsent (see the lane of miR-20a “−” at 30 min in Figure 4d), a
band corresponding to its molecular weight was observed in thepresence of miR-20a (see the lane of miR-20a “+” at 30 min in
Figure 4d), suggesting that oblimersen had been generated. In
this lane, the bands for the initially added miR-20a and primer
DNA were not observed because of their low concentrations

Figure 5. Results of nanopore measurement. (a, b) Typical current
and time traces of reaction solutions before and after the enzyme
reaction in the absence of miR-20a (a) and presence of miR-20a (b).
(c) Event frequency for each reaction solution. Error bars indicate thestandard error.

detection of output molecules before and after the enzyme
reaction without and with miR-20a, respectively. In thepresence of miR-20a, a number of spikelike signals wereobserved after the enzyme reactions, suggesting that oblimersen
had been generated and passed through the nanopore. Severaltranslocated signals were also observed in other cases because
ssDNA in the initial condition, including template DNA, primer
D

DOI: 10.1021/acs.analchem.6b03830
Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical ChemistryDNA, and miR-20a, can pass through the pore. To decrease
these background signals, the experimental conditions, such as
the voltage applied (+120 mV) and the use of a noise ﬁlter (10kHz), were regulated so that the translocation signal could
scarcely be observed in this concentration range (approximately
20 nM). In the current recordings, the short current blockages
were continually observed because of the collision (approximately 30% blockage) or the transient partial entry(approximately 60% blockage) of ssDNA into the vestibule ofthe αHL pore.38 Next, the event frequencies of each condition
were compared to estimate the concentration of oblimersen
generated, as shown in Figure 5c (see also the histograms in
Figure S3). The translocation frequency of DNA/RNA directly
reﬂects the concentration in the solution.23,26 As shown in
Figure 5c, the event frequency of oblimersen generated whenmiR-20a was present was clearly higher than that when miR20a was absent. Quantiﬁcation of the generated oblimersen is
discussed below.
Quantitative polymerase chain reaction (qPCR) and gelelectrophoresis are the conventional methods for thequantiﬁcation of polynucleotides. It has been previously
reported that the accuracy of qPCR for oligonucleotide
quantiﬁcation is lower than that of nanopore measurement.26
Therefore, we examined the capability of nanopore measurement compared with that of gel electrophoresis. Although gelelectrophoresis has been used as a simple quantiﬁcation
method in oligonucleotide quantiﬁcation, its accuracy has
been questioned due to the low reliability in using ﬂuorescenceintensity to determine oligonucleotide quantity. Speciﬁcally,
this intensity is not consistent in each experiment, even at thesame concentration; instead, it depends on factors such as thephotophysical conditions. Thus, a calibration curve was createdin each gel experiment by loading ﬁve diﬀerent calibration
samples (Figure 6a; the other calibration curves are presented
in Figure S4). In the nanopore measurement, the calibration
curve was established using the translocation event frequency,
as mentioned above (Figure 6b). The correlation factor (R2) inthe nanopore measurement (n > 3) was higher than that in thegel experiment (mean R2 value of 0.82, n = 3). In addition, theaccuracy of each method was checked using a standard solution
containing 3.0 μM oblimersen. As presented in Figure 6c, theaccuracy of the determined value from the nanopore measurements (3.0 ± 0.26 μM, n = 3) was higher than that from gelelectrophoresis (2.4 ± 2.0 μM, n = 3). Moreover, the durationof a single experiment in the nanopore measurement wasapproximately 30 min, which is much shorter than that for gel
electrophoresis (ca. 90 min).
Based on the results of nanopore quantiﬁcation, theoblimersen concentration values before and after the enzyme
reaction were determined, as depicted in Figure 6d. For thequantiﬁcation of initial oligonucleotides (primer DNA,
template DNA, and miR-20a) using nanopore measurement,
the calibration curve from before the reaction was established(Figure S6) because the event frequency in the initial condition
was varied in the case of oblimersen. After the enzyme reaction,
the concentration of translocated DNA/RNA was estimated at
550 ± 35 nM. The solution before the enzyme reaction
contained 86 ± 14 nM of the initial DNAs and miRNA.
Therefore, 460 ± 20 nM oblimersen was generated from 20
nM miR-20a (ca. 23-fold) in 30 min under 37 °C. The
isothermal reaction with the 3WJ structure allowed us toautonomously amplify from the target miRNA to the antisense
oligonucleotide.

Figure 6. (a) Calibration curve for the estimation of oblimersen madefrom the ﬂuorescence intensity in gel electrophoresis. (b) Calibration
curve for the estimation of oblimersen made from the event frequency(using Axopatch 200B). (c) Estimation accuracy for 3.0 μM
oblimersen with nanopore measurement and gel electrophoresis.
The calibration curve for this estimation is depicted in Figure S4(using Pico2). Error bars indicate the standard deviation. (d)Concentrations of miR-20a and oblimersen. Error bars indicate thestandard error.

■

CONCLUSIONSIn summary, we developed an autonomous ampliﬁcation
system using DNA computing techniques. Two programmable
DNA molecules, a primer and a template DNA, bound to the
input miR-20a molecule and formed a 3WJ structure. After that,
an isothermal reaction with enzymes was repeatedly conducted
to generate a number of output oblimersen molecules. The
generated oblimersen was quantiﬁed by the translocation
frequency in nanopore measurement; through this experiment,
we found that the quantitative capability of the nanopore
measurement was greater than that using ﬂuorescent measurement in gel electrophoresis experiments. It should thus benoted that the nanopore measurement is a more useful tool forthe quantiﬁcation of oligonucleotides than conventional gel
electrophoresis. The results of nanopore quantiﬁcation showedthat oblimersen was ampliﬁed more than 20-fold from miR-20a,
which meets the dosage requirement for SCLC therapy. This
autonomous ampliﬁcation strategy is a potent candidate for abroad range of theranostics with antisense oligonucleotides.

■

ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.analchem.6b03830.
Design protocol of template and primer DNA, gelelectrophoresis data for the optimization of enzyme
reaction, histograms of the event frequency of oblimersen
in nanopore measurements, and calibration curves of thequantiﬁcation of oligonucleotides (PDF)
E

DOI: 10.1021/acs.analchem.6b03830
Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry■(23) Kawano, R.; Osaki, T.; Sasaki, H.; Takinoue, M.; Yoshizawa, S.;
Takeuchi, S. J. Am. Chem. Soc. 2011, 133 (22), 8474−8477.
(24) Ohara, M.; Sekiya, Y.; Kawano, R. Electrochemistry 2016, 84 (5),338−341.
(25) Zhang, X. Y.; Wang, Y.; Fricke, B. L.; Gu, L. Q. ACS Nano 2014,
8 (4), 3444−3450.
(26) Wang, Y.; Zheng, D. L.; Tan, Q. L.; Wang, M. X.; Gu, L. Q. Nat.
Nanotechnol. 2011, 6 (10), 668−674.
(27) Zadeh, J. N.; Steenberg, C. D.; Bois, J. S.; Wolfe, B. R.; Pierce,
M. B.; Khan, A. R.; Dirks, R. M.; Pierce, N. A. J. Comput. Chem. 2011,32 (1), 170−173.
(28) Kibbe, W. A. Nucleic Acids Res. 2007, 35, W43−W46.
(29) Watanabe, H.; Kawano, R. Anal. Sci. 2016, 32 (1), 57−60.
(30) Kawano, R.; Tsuji, Y.; Kamiya, K.; Kodama, T.; Osaki, T.; Miki,N.; Takeuchi, S. PLoS One 2014, 9 (7), e102427.
(31) Tsuji, Y.; Kawano, R.; Osaki, T.; Kamiya, K.; Miki, N.; Takeuchi,
S. Anal. Chem. 2013, 85 (22), 10913−10919.
(32) Tsuji, Y.; Kawano, R.; Osaki, T.; Kamiya, K.; Miki, N.; Takeuchi,
S. Lab Chip 2013, 13 (8), 1476−1481.
(33) Kawano, R.; Tsuji, Y.; Sato, K.; Osaki, T.; Kamiya, K.; Hirano,
M.; Ide, T.; Miki, N.; Takeuchi, S. Sci. Rep. 2013, 3, 1995.
(34) Kawano, R.; Osaki, T.; Sasaki, H.; Takeuchi, S.; Polymer-Based
Nanopore-Integrated, A. Small 2010, 6 (19), 2100−2104.
(35) Zhang, Q.; Chen, F.; Xu, F.; Zhao, Y. X.; Fan, C. H. Anal. Chem.
2014, 86 (16), 8098−8105.
(36) Nakano, S.; Fujimoto, M.; Hara, H.; Sugimoto, N. Nucleic Acids
Res. 1999, 27 (14), 2957−2965.
(37) Lv, W. F.; Zhao, J. M.; Situ, B.; Li, B.; Ma, W.; Liu, J. M.; Wu, Z.
X.; Wang, W.; Yan, X. H.; Zheng, L. Biosens. Bioelectron. 2016, 83,
250−255.
(38) Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.;
Deamer, D. W. Biophys. J. 1999, 77 (6), 3227−3233.

AUTHOR INFORMATION

Corresponding Author*E-mail: rjkawano@cc.tuat.ac.jp.
ORCID

Ryuji Kawano: 0000-0001-6523-0649
Author Contributions

M.H. and R.K. conceived the original idea and wrote the paper.
M.H. and M.O. conducted the experiments. All authors havegiven approval to the ﬁnal version of the manuscript.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
We thank M. Takinoue (Tokyo Tech.) for a useful discussion
on the design of the 3WJ structure. This work was partially
supported by KAKENHI (Molecular Robotics: No. 15H00803
and No. 16H06043) from MEXT Japan.

■

REFERENCES(1) Kahan-Hanum, M.; Douek, Y.; Adar, R.; Shapiro, E., A library of
programmable DNAzymes that operate in a cellular environment. Sci.
Rep. 2013, 3.10.1038/srep01535(2) Culler, S. J.; Hoff, K. G.; Smolke, C. D. Science 2010, 330 (6008),
1251−1255.
(3) Jung, C.; Ellington, A. D. Acc. Chem. Res. 2014, 47 (6), 1825−
1835.
(4) Benenson, Y.; Gil, B.; Ben-Dor, U.; Adar, R.; Shapiro, E. Nature
2004, 429 (6990), 423−429.
(5) Shapiro, E.; Gil, B. Science 2008, 322 (5900), 387−388.
(6) Benenson, Y.; Paz-Elizur, T.; Adar, R.; Keinan, E.; Livneh, Z.;
Shapiro, E. Nature 2001, 414 (6862), 430−434.
(7) Schwarzenbach, H.; Hoon, D. S. B.; Pantel, K. Nat. Rev. Cancer
2011, 11 (6), 426−437.
(8) Komiya, K.; Yamamura, M. New Generation Computing 2015, 33(3), 213.
(9) Rabinowits, G.; Gercel-Taylor, C.; Day, J. M.; Taylor, D. D.;
Kloecker, G. H. Clin. Lung Cancer 2009, 10 (1), 42−46.
(10) Ebi, H.; Sato, T.; Sugito, N.; Hosono, Y.; Yatabe, Y.;
Matsuyama, Y.; Yamaguchi, T.; Osada, H.; Suzuki, M.; Takahashi, T.
Oncogene 2009, 28 (38), 3371−3379.
(11) Rudin, C. M.; Kozloff, M.; Hoffman, P. C.; Edelman, M. J.;
Karnauskas, R.; Tomek, R.; Szeto, L.; Vokes, E. E. J. Clin. Oncol. 2004,
22 (6), 1110−1117.
(12) Rudin, C. M.; Otterson, G. A.; Mauer, A. M.; Villalona-Calero,
M. A.; Tomek, R.; Prange, B.; George, C. M.; Szeto, L.; Vokes, E. E.
Annals of Oncology 2002, 13 (4), 539−545.
(13) Bayley, H.; Cronin, B.; Heron, A.; Holden, M. A.; Hwang, W. L.;
Syeda, R.; Thompson, J.; Wallace, M. Mol. BioSyst. 2008, 4 (12),1191−208.
(14) Taniguchi, M. Anal. Chem. 2015, 87 (1), 188−199.
(15) Ying, Y.-L.; Zhang, J.; Gao, R.; Long, Y.-T. Angew. Chem., Int. Ed.
2013, 52 (50), 13154−13161.
(16) Stoloff, D. H.; Wanunu, M. Curr. Opin. Biotechnol. 2013, 24 (4),699−704.
(17) Reiner, J. E.; Balijepalli, A.; Robertson, J. W. F.; Campbell, J.;
Suehle, J.; Kasianowicz, J. J. Chem. Rev. 2012, 112 (12), 6431−6451.
(18) Deamer, D. Annu. Rev. Biophys. 2010, 39, 79−90.
(19) Yasuga, H.; Kawano, R.; Takinoue, M.; Tsuji, Y.; Osaki, T.;
Kamiya, K.; Miki, N.; Takeuchi, S. PLoS One 2016, 11 (2), e0149667.
(20) Deamer, D. W.; Branton, D. Acc. Chem. Res. 2002, 35 (10),817−825.
(21) Meller, A.; Nivon, L.; Brandin, E.; Golovchenko, J.; Branton, D.
Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (3), 1079−1084.
(22) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc.
Natl. Acad. Sci. U. S. A. 1996, 93 (24), 13770−13773.
F

DOI: 10.1021/acs.analchem.6b03830
Anal. Chem. XXXX, XXX, XXX−XXX