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Cite This: Anal. Chem. 2018, 90, 8531−8537

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DNA Logic Operation with Nanopore Decoding To Recognize
MicroRNA Patterns in Small Cell Lung Cancer
Moe Hiratani and Ryuji Kawano*Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan

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S Supporting Information*

ABSTRACT: Although DNA computation has traditionally beendeveloped for parallel calculations in molecular analyses, this approachhas recently been considered for use in diagnostic or medical applications
in living systems. In this study, we propose that the DNA logic operation
may be a powerful tool for the recognition of microRNA patterns, which
may have applications for the early diagnosis of cancers. We developed arapid, label-free decoding method for output diagnostic molecules using
nanopore measurements. We designed diagnostic DNAs that autonomously recognized two microRNAs, miR-20a and miR-17-5p, and formed afour-way junction structure that was captured in the nanopore, showing
long blocking currents. We analyzed the blocking duration based on thecentral limit theorem and found that four diﬀerent operations, i.e., (0, 0),(0, 1), (1, 0), and (1, 1), could be discriminated. This pattern recognition
method has been diﬀerentiated from simple detection methods based on
DNA computing and nanopore technologies.

D

RNA and enzyme reactions using the droplet system; theoutput molecule (i.e., RNA) was detected rapidly, even whenthe enzyme reaction occurred in the droplet.5
Rapid, parallel operations using logic gates have recently
been applied to the medical and diagnostic ﬁelds.28 Benenson
et al. have proposed an autonomous diagnosis and treatment
approach with the logical control of gene expression.29
Moreover, we have also proposed a “theranostics” system forcancer therapy using microRNAs (miRNAs) as input
molecules; our diagnostic DNAs (dgDNAs) detected speciﬁc
miRNAs from small cell lung cancer (SCLC) and formed acomplex with a complementary sequence as a DNA drug
(antisense drug). The antisense oligonucleotides for SCLC
therapy were generated autonomously by isothermal ampliﬁcation.6 We then attempted to recognize the expression/
repression patterns of miRNAs in tumor cells using DNA
computing techniques.
miRNAs are potential early diagnostic markers for cancer;
certain cancers secrete speciﬁc miRNAs.30 If a single tumor
secreted only a single miRNA, diagnosis would be simple;
however, tumor cells show complicated miRNA expression or
repression patterns.31 For example, in a study of breast cancer,
10 miRNAs were overexpressed and 8 were downregulated,compared with expression in control tissues.30 For conventional
diagnosis using miRNAs, all miRNAs have to be analyzed
quantitatively. Gu and co-workers developed a method for

NA computing is a useful technology for molecular
calculations because it can be performed in living systems
owing to its inherent biocompatibility.1,2 Input and output
molecules contain intrinsic information, such as genetic codesfor proteins and recognition sites for enzymes, which can betransported under the logic operation. The decoding of
information from output molecules is conventionally performed
using ﬂuorescence-based methods, which require ﬂuorescentmolecules with speciﬁc or nonspeciﬁc binding and ampliﬁcation
by polymerase chain reaction (PCR) and gel electrophoresis,
which are time-consuming procedures.3
We have recently proposed methods for rapid electricaldecoding using nanopore measurement,4−7 a powerful tool forlabel-free, electrical detection at the single-molecule level.8−11
In particular, α-hemolysin (αHL), which forms a 1.4 nm
diameter pore in the lipid bilayer, can detect oligonucleotides
precisely based on size similarities between the pore andmolecules.12−14 To apply nanopore measurements to decoding,the durability of the lipid bilayer with nanopores must be
improved because the logic operation may require several
hours. The conventional lipid bilayer does not have suﬃcientstability over long periods; it is sometimes ruptured bymechanical vibration or the applied voltage.15,16 Several studies,including studies by our group, have described the developmentof stable lipid bilayer systems;17−20 consequently, we adopted adroplet system using microfabricated devices, referred to as thedroplet contact method (DCM).21−23 A lipid bilayer is formedat the interface of the lipid monolayer with the two droplets.
Based on the DCM, we have already reported parallel andstable nanopore or ion channel measurements.24−27 Moreover,
we have tested the decoding of logic operations using DNA/
© 2018 American Chemical Society

Received: April 9, 2018
Accepted: April 26, 2018
Published: June 28, 20188531

DOI: 10.1021/acs.analchem.8b01586
Anal. Chem. 2018, 90, 8531−8537

Article

Analytical Chemistry

Figure 1. Schematic illustration of the AND gate for the diagnosis of small cell lung cancer. When miR-20a and miR-17-5p are overexpressed, theybind to diagnostic DNAs and form a four-way junction (4WJ) structure. Then, the 4WJ structure is captured by α-hemolysin and causes current
signals with long blockades.

DNA Design and Free Energy Calculation. To conﬁrm
that DNA and miRNA hybridized as intended, a thermodynamic analysis was performed using NUPACK (California
Institute of Technology, http://www.nupack.org/)35 for each
operation pattern from (0, 0) to (1, 1). This analysis was
performed at 37 °C using a DNA or miRNA concentration of 3
μM in 1.0 M KCl-buﬀered electrolyte solution. Free energy was
also obtained from the NUPACK simulation. The details of
DNA design are provided below.
Design of Complementary Sequences of miR-17-5p andmiR-20a in Diagnostic DNA1 and Diagnostic DNA2. The 19mer from the 5′ terminus of diagnostic DNA1 (dgDNA1) andthe 5-mer from 3′ terminus of diagnostic DNA2 (dgDNA2)
formed a complementary structure with the 19-mer from the 3′
terminus and the 5-mer from the 5′ terminus of miR-17-5p,
respectively. The 15-mer from the 3′ terminus of dgDNA1 andthe 8-mer from the 5′ terminus of dgDNA2 formed acomplementary structure with the 15-mer from the 5′ terminus
and the 8-mer from 3′ terminus of miR-20a, respectively.
Thermodynamic Simulation Using NUPACK. The formation of a 4WJ structure was conﬁrmed using the sequence
designed above. The probability of 4WJ formation was 87%.
Redesign of the Sequences of dgDNA1 and dgDNA2. The
complementary sequences to miR-20a in dgDNA1 anddgDNA2 were ﬁxed. The complementary strand to miR-17-5p
in dgDNA1 was shortened by one base, and the shortened
sequence was added to dgDNA2.
Second Thermodynamic Simulation Using NUPACK. The
formation of 4WJ was conﬁrmed. The probability of 4WJ
formation when the two bases were changed, as describedabove, was 92%.
Addition of the Tail Structure to the 3′ Terminus in
dgDNA1. Thirty thymines were added to the 3′ terminus ofdgDNA1 because this structure enabled 4WJ to enter αHLeasily.
DgDNA/miRNA Hybridization. Diagnostic solutions were
solutions of each miRNA pattern from (0, 0) to (1, 1) with
dgDNA1 and dgDNA2 (30 μM each in 10 mM MOPS buﬀer;
pH 7.0, containing 1 M potassium chloride). The solutions
were heated to 95 °C for 5 min and then gradually cooled toroom temperature.
Nondenaturing Polyacrylamide Gel Electrophoresis.
4WJ structure formation was analyzed by nondenaturing
polyacrylamide gel electrophoresis (native PAGE, containing
19/1 acrylamide/bis (w/w)) on 15% gels in 1 × TBE buﬀer(89 mM Tris-borate, 2 mM ethylenediaminetetraacetic acid,

miRNA detection using nanopore technology with programmable polymer tags.32−34 They also reported that nanopore
measurements were superior to conventional quantitative
PCR.34In this study, as the ﬁrst step of pattern recognition using
DNA computing technology and nanopore measurement, we
attempted to construct a technology for the recognition of two
miRNAs secreted from SCLC using an AND operation: (0, 0),(0, 1), (1, 0), and (1, 1). Using our strategy, dgDNAs formed a
4WJ structure when miR-20a and miR-17-5p were presented, as
shown in Figure 1. The 4WJ could not be translocated throughthe αHL nanopore and showed a long blocking current
duration. In another case, dgDNA and miRNAs were singlestranded structures and passed through nanopores rapidly. To
analyze the current duration in a statistical framework, we usedthe central limit theorem, resulting in the recognition of not
only two miRNA patterns but also four diﬀerent operations.
Our proposed method can be used for the recognition of
miRNA patterns using DNA logic operations.

■

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), potassium chloride(KCl; Nacalai Tesque, Kyoto, Japan), and 3-(N-morpholino)propanesulfonic acid (MOPS; Nacalai Tesque). Buﬀered
electrolyte solutions (1.0 M KCl, 10 mM MOPS, pH 7.0)
were prepared using ultrapure water. Wild-type αHL (SigmaAldrich, St. Louis, MO, USA and List Biological Laboratories,
Campbell, CA, USA) was obtained as a monomeric
polypeptide, isolated from Staphylococcus aureus in the formof a powder and dissolved at a concentration of 1 mg/mL in
ultrapure water. For use, samples were diluted to the designatedconcentration using a buﬀered electrolyte solution and stored at
4 °C. High-performance liquid chromatography grade DNA
oligonucleotides and miRNAs were synthesized by FASMAC
Co., Ltd. (Kanagawa, Japan) and stored at −20 and −80 °C,
respectively. TBE buﬀer (10× ) was obtained from Takara Bio
Inc. (Kusatsu, Japan) and was diluted 10-fold for gel
electrophoresis. The power supply and an LED transilluminator
were obtained from Bio Craft Co., Ltd. (Tokyo, Japan) andOptocode Corp. (Tokyo, Japan), respectively.
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Analytical ChemistryμL) on each side of the lipid bilayers contained 1 M KCl and 10
mM MOPS (pH 7.0). αHL was reconstituted in lipid bilayers
to form a nanopore from the ground side. The diagnostic
solution after dgDNA/miRNA (ﬁnal concentration, 3 μM)
hybridization was also added to the ground side. Within a few
minutes of the addition of the solutions, lipid bilayers wereformed, and αHL created nanopores within them. When thelipid bilayers ruptured during this process, they were recreatedby tracing with a hydrophobic stick at the interface of thedroplets.
Channel Current Measurements and Data Analysis.
The channel current was recorded using an Axopatch 200B
Ampliﬁer (Molecular Devices, San Jose, CA, USA) and ﬁlteredusing a low-pass Bessel ﬁlter at 10 kHz at a sampling rate of 50
kHz. A constant voltage of +150 mV was applied from therecording side, and the ground side was grounded. The
recorded data from Axopatch 200B were acquired using
Clampex 9.0 (Molecular Devices) with a Digidata 1440A
analog-to-digital converter (Molecular Devices). Data wereanalyzed using Clampﬁt 10.6 (Molecular Devices), Excel(Microsoft, Redmond, WA, USA), and R software (RFoundation). DNA or miRNA translocation and blocking
were detected when more than 85% of open αHL channel
currents were inhibited. Between 347 and 500 translocating or
blocking events were recorded. We examined the signiﬁcancetest between (1, 1) and the other systems after applying thecentral limit theorem: 10000 data points were extracted foreach of four miRNA patterns, and the mean value wascalculated by integrating 216 (65536) times. Subsequently,
histograms were generated from (0, 0) to (1, 1) using the data
which have enough normality, and then the signiﬁcance test
was examined. These processes are described in a ﬂowchart in
Supporting Information Figure S1. Normality was considered
statistically signiﬁcant at P < 0.05 using Kolmogorov−Smirnov
tests. Diﬀerences were considered statistically signiﬁcant at P <0.01 using Welch’s t-tests. Nanopore measurements wereconducted at 22 ± 2 °C.

pH 8.3) at a constant power of 2.3 W for 40 min at room
temperature. After electrophoresis, the gel was stained withdiluted SYBR Green II solution (Takara Bio Inc.) for 30 min
and visualized under blue LED irradiation; images wereobtained using a digital camera or a FAS-BG LED BOX(NIPPON Genetics Co., Ltd., Tokyo, Japan). The ﬂuorescenceintensity of each band was calculated within square regions withthe same area. Fluorescence intensity was measured ﬁve times
for each band.
Ultraviolet−Visible Light Spectrum Measurement.
The melting temperature for each miRNA pattern from (0,0) to (1, 1) was analyzed by measurements of the ultraviolet−
visible light (UV−vis) spectrum. The measurement solution foreach pattern contained DNA and miRNAs at 1 μM each in
MOPS buﬀer (10 mM, pH 7.0). UV−vis spectra were
measured using the V-660 instrument (JASCO Corp., Tokyo,
Japan) with the following settings: measurement wavelength,
260 nm; bandwidth, 2 nm; temperature, 30−80 °C for (1, 1)
and (1, 0) and 30−70 °C for (0, 1) and (0, 0).
Lipid Bilayer Preparation and Reconstitution of αHL.
Lipid bilayers were prepared using a device produced by
microfabrication (Figure 2a). Lipid bilayers can be simulta-■

RESULTS AND DISCUSSION
Design of Diagnostic DNAs. dgDNAs were required toform a 4WJ structure with miR-20a and miR-17-5p only whenboth miRNAs were present. To satisfy this requirement, we
designed two dgDNAs using a thermal dynamic simulation(NUPACK) considering the following qualiﬁcations: (1) both
dgDNA1 and dgDNA2 must have sequences complementary toboth miR-20a and miR-17-5-p and form the 4WJ structure
simultaneously with the four oligonucleotides (dgDNA1,
dgDNA2, miR-20a, and miR-17-5p), and (2) 4WJ must enteran αHL nanopore after formation of the structure. Therefore,
30 nucleotides composed of polydeoxythymine (poly(dT30))
were added to the 3′ terminus of dgDNA1. The length ofpoly(dT30) was suﬃcient to reach the trans side of the αHLpore when 4WJ was captured in the nanopore. The rate of theblocking current was previously reported to be 85%.37,38 A

Figure 2. (a) Photograph of the devices for measuring channel
currents. (b) Schematic illustration of lipid bilayer preparation.

neously formed using this device by the DCM (Figure 2b).21,36
In this method, the two lipid monolayers contact each otherand form lipid bilayers on a parylene C ﬁlm, which separates
the two chambers. Lipid bilayers were formed as follows. First,
the wells of the device were ﬁlled with n-decane (2.5 μL)
containing DPhPC (10 mg/mL). The recording solutions (4.7
Table 1. DNA and miRNA Sequences
DNA, miRNAbase sequence

dgDNA1dgDNA2
miR-20amiR-17-5p5′ACTACCTGCACTGTAAGACTATAAGCACTTTA(T)303′
5′CTACCTGCCACTTTG3′5′UAAAGUGCUUAUAGUGCAGGUAG3′5′CAAAGUGCUUACAGUGCAGGUAGU3′
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Analytical Chemistryeach) with various concentrations of the target miRNAs (0−3.0μM). For 4WJ conditions, the strength of the bands increased
as the concentration of miRNAs increased, as shown in thewhite frame in Figure 3b. In addition, the location of the band
diﬀered from that of the (0, 1) and (1, 0) band (Figure S3).
The intensities of the bands for 4WJ, dgDNA1, and dgDNA2 in
Figure 3b are also shown in Figures S4 and S5. There wereweak bands of unhybridized oligonucleotides, even under equal
molar conditions (3.0 μM), probably due to the occurrence ofnonhybridization by heat generation during gel electrophoresis.
The ratio of unhybridized to hybridized molecules wasestimated to be 0.5:1 (miR-17-5p:4WJ) based on the intensityof each band.
Detection of Output Molecules Using αHL Nanopores. Nanopore measurements can detect the output
molecule of DNA logic operations rapidly and electrically, as
we have previously shown. Therefore, in this study, we usedαHL nanopores to recognize the four AND operations: (0, 0),(0, 1), (1, 0), and (1, 1). Figure 4 shows the channel current

detailed protocol of this design is described in the Experimental
Section. The most suitable sequence and the simulated
structures are shown in Table 1 and Figure 3a. In addition,

Figure 3. (a) Secondary structure of 4WJ predicted by NUPACK
simulation. (b) Native PAGE image for conﬁrmation of 4WJ
formation. The 4WJ structure decreased as the miRNA concentration
decreased.

all other possible binding patterns using four oligonucleotides(dgDNA1, dgDNA2, miR-20a, and miR-17-5p) were simulated(Table 2 and Figure S2). The designed 4WJ structure showed
the most stable state among the possible structures.
Next, we experimentally conﬁrmed the formation of the 4WJstructure using native PAGE. Figure 3b shows the results foreach of four oligonucleotides and the two dgDNAs (3.0 μM

DNA, miRNA constructs

free energy (kJ/mol)Figure 4. (a−d) Typical current and time traces of (0, 0) (a), (0, 1)(b), (1, 0) (c), and (1, 1) (d) using single αHL nanopore under 150
mV voltage application (cis side positive). The concentrations of
dgDNA and miRNA were 3 μM each. The molar ratios of [dgDNA1]:[dgDNA2]:[miR-20a]:[miR-17-5p] were 1:1:0:0 in (0, 0), 1:1:1:0 in(0, 1), 1:1:0:1 in (1, 0), and 1:1:1:1 in (1, 1).

dgDNA2dgDNA1
miR-20amiR-17-5p
dgDNA2 + miR-20a
dgDNA2 + miR-17-5p
dgDNA1 + miR-20a
dgDNA1 + miR-17-5p
sgDNA1 + dgDNA2 + miR-20a + miR-17-5p

−0.00
−5.10
−16.3
−16.3
−67.3
−73.6
−129.6−142.2−276.3

recordings for the four operations. Deep blocking currents overthe 85% threshold level were observed in all four operating
systems. For (0, 0), the oligonucleotides (dgDNA1 anddgDNA2) did not form hybridized structures, resulting in
spike-like translocation signals of single-stranded DNA (Figure4a). For (0, 1) and (1, 0), one of the diagnostic DNAs binds tothe target miRNA and forms a weak complex. This complex
initially blocks the pore and should then translocate throughTable 2. Gibbs Free Energy of DNA and miRNA
Combination According to NUPACK Simulations

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histograms, normality was not detected (p < 0.05), indicatingthat t-tests could not be used to analyze durations. Therefore,
we used another statistical method to evaluate durations.
Evaluation of Durations Using the Central Limit
Theorem. The central limit theorem (CLT) states that thesum of a large number of random variables is approximately
normal, even if the original variables themselves are notnormally distributed. Based on this theorem, we generated anormal distribution from the duration data and evaluateddiﬀerences using t-tests. The histograms of the normal
distribution for the four operations are presented in Figure 6aand Figure S6.
Interestingly, each distribution gave diﬀerent peak values(Xc), suggesting that all four systems had intrinsic durations.
We then evaluated the intrinsic duration for each system, as
follows. For (0, 0), Xc was 2.03 ± 0.0605 ms; this blocking
event should reﬂect the translocation of dgDNA1 anddgDNA2, which formed a single-stranded state. For (0, 1)
and (1, 0), the Xc values were 25.9 ± 0.707 and 728 ± 49.0 ms,
respectively; the thermal dynamic simulation predicted thatthese formed a weakly hybridized structure comprised of themiR-20a/dgDNA1 (0, 1) and miR-17-5p/dgDNA1 (1, 0)
duplexes. The hybridized free energies of the dgDNA1/miR-175p duplex were much higher than those of the dgDNA1/miR20a duplex. These duplexes should translocate through thenanopore with an unzipping mechanism or eject spontaneously
from the pore against the applied voltage. Finally, for (1, 1), Xc
was the longest (3440 ± 292 ms), and all four oligonucleotides
formed a stable 4WJ structure, resulting in the long blocking
event. Additionally, Xc increased as the concentration of thetarget miRNAs decreased (Figure S7).
To discriminate each operation statistically, we next
performed signiﬁcance tests. After applying the CLT, allhistograms exhibited normality. Thus, we used Welch’s t-test toevaluate each operating system. Notably, all four systems
showed signiﬁcant diﬀerences (p < 0.01, as shown in Figure6b). This indicated that we could use this approach todiscriminate not only the two miRNAs but also all four patterns
for each miRNA. Moreover, we checked the concentration
dependency in this system. We set the speciﬁc duration of 4WJ
to over 3148 ms based on the CLT analysis for the (1, 1)
system and generated a calibration curve (Figure S8). The
speciﬁc duration exhibited a linear correlation with theconcentration of miRNAs.
Relationships between Duration and the Hybridized
Structure. In the nanopores, the hybridized DNA blocks thepores and translocates with the unzipping of strands. The freeenergy of hybridized DNA is strongly related to the unzipping
time in nanopore measurements. Here, we discuss thisrelationship for our four operation systems. The unzipping
kinetics can be presented as a ﬁrst-order reaction as follows:36,40

the pore by unzipping the duplex (Figure 4b,c). This unzipping
mechanism has been previously studied using αHL nanopores.36,39,40 In the case of (1, 1), the four oligonucleotides
formed the 4WJ structure and should block the pore, yielding
long blocking duration signals (Figure 4d).
Conventionally, analyses of durations have focused on thepeak of the histogram of durations or the median value because
the points are not normally distributed and usually show a wide
range, e.g., from approximately a millisecond to approximately asecond.40 In our case, the histogram of durations also showed
an asymmetrical distribution and ranged from sub-microsecond
to several tens of seconds, as shown in Figure 5 on a

Figure 5. Histograms of the duration for each miRNA pattern. Solidlines are ﬁts of the histograms to Gaussian curves, which were used toobtain peaks. All samples of dgDNAs and miRNAs were simple
mixtures subjected to annealing treatment before obtaining measurements.

logarithmic scale. To discriminate between (1, 1) and the othersystem, we initially used t-tests with Gaussian ﬁtting because
the distribution seemed nearly normal. The peak durations arelisted in Table 3. The peak duration increased from (0, 0) to (1,1). We then evaluated normality using Kolmogorov−Smirnov
tests to determine whether the assumptions of t-tests are met.
Table 3 shows the results of normality tests. For all four

[DNA/miRNA + α HL]→ [single‐stranded nucleic acids] + [α HL]

The dissociation rate constant can also be presented as follows:

Table 3. Analysis of the Histograms Shown in Figure 5(0, 0)(0, 1)(1, 0)(1, 1)

value of peak (ms)normality (p < 0.05)

0.085
0.69
0.81
1.4, 575

no
no
no
no

k = k 0 exp(−ΔGsim /NAkBT )(1)

k = 1/twhere k is the dissociation rate constant, k0 is the initial rate
constant, NA is the Avogadro constant, kB is the Boltzmann
constant used for this single molecule experiment, instead of
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Figure 6. (a) Histograms of the duration for each miRNA pattern after data ampliﬁcation based on the law of large numbers. (b) Mean value of theduration for each miRNA pattern. Error bars indicate the three-time standard deviations. (c) Correlations between the ﬁrst-order rate constant andthe hybridization energy (dotted line) and between the ﬁrst-order rate constant and Tm (solid line).

the gas constant, T is the temperature, and t is the duration.
Note that k (s−1) should be equal to the inverse of t because 1/t
is the reaction frequency per unit time in the single-molecule
reaction. Figure 6c shows the relationship between theexperimentally determined k and ΔGsim from the thermodynamic simulation (NUPACK). The ﬁtting result was given as ln
k = 1.66 × 1019 (−ΔGsim/NAkBT) + 6.06 (R2 = 0.85). Figure S9shows the relationship between t and ΔGsim for reference.
Although the ﬁtting parameters were similar to those previously
reported from unzipping experiments of hairpin DNAs,36 thecorrelation coeﬃcient did not indicate suﬃcient linearity
between lnk and ΔGsim. Therefore, we next measured themelting temperatures (Tm values) of the four operating
systems. This value was experimentally obtained and is plottedin Figure 6c. For (0, 0), the Tm value could not be obtained
because there was no double-stranded structure. In the othersystems, (0, 1), (1, 0), and (1, 1) gave melting temperatures of
61.9, 66.4, and 70.5 °C, respectively. We plotted these values
and ﬁtted them using eq 1. The ﬁtting results yielded theequation ln k = −0.489Tm + 32.8, with a correlation coeﬃcientof R2 = 0.99. This result indicated that there was a linear
correlation between Tm or ΔGsim and ln k. The unzipping time
could be predicted by the duplex structure using the kinetic
model of the unzipping events.

these durations could be predicted by the hybridized strength
estimated by the Gibbs free energy or the melting temperature.
The selectivity of DNA hybridization with miRNA34 and thesensitivity of nanopore detection (∼1 fM)27 would be suﬃcientfor practical applications. Typically, in methods for miRNAdetection from bioﬂuids, such as RT-PCR or RNA arrays, thetarget miRNAs are extracted from real samples during
pretreatment. Our method can also be combined with general
pretreatment procedures before nanopore measurements. Our
proposed method, based on DNA computing and nanopore
technologies, is useful for miRNA pattern recognition. In futurestudies, we will evaluate the complex patterns of miRNAs inother cancers, e.g., bile duct cancer.
Moreover, our strategy will be integrated into theconstruction of a molecular robot,41,42 i.e., a next-generation
biochemical machine made of biomaterials, such as DNA,
proteins, and lipids. Three prerequisites, namely, sensors,
intelligence, and actuators, have been proposed for theconstruction of such a robot. Pattern recognition with
nanopores will be a strong tool for the sensor and intelligenceparts of this robot.

■■

ASSOCIATED CONTENT* Supporting InformationS

CONCLUSIONS
Our designed dgDNA could recognize two diﬀerent miRNAs,
which were found to form the 4WJ structure, resulting in longblocking signals in nanopore measurements. Statistical analysis
with CLT precisely discriminated four AND operation systems:(0, 0), (0, 1), (1, 0), and (1, 1). In addition, we showed that

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.analchem.8b01586.
Thermal dynamic simulation of DNAs/RNAs andﬂuorescent intensity of native PAGE (PDF)
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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. conducted the experiments. All authors have given
approval to the ﬁnal version of the manuscript.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
We thank M. Takinoue (Tokyo Institute of Technology) for a
useful discussion on the design of the 4WJ structure. This work
was partially supported by KAKENHI (Molecular Robotics,
Grant Nos. 15H00803 and 16H06043) from MEXT Japan.

■

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DOI: 10.1021/acs.analchem.8b01586
Anal. Chem. 2018, 90, 8531−8537