Research Article
pubs.acs.org/synthbio

Nanopore Logic Operation with DNA to RNA Transcription
in a Droplet System
Masayuki Ohara,† Masahiro Takinoue,‡ 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, Japan‡
Department of Computer Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Yokohama, Kanagawa 226-8502, JapanS Supporting Information*

ABSTRACT: This paper describes an AND logic operation with ampliﬁcation andtranscription from DNA to RNA, using T7 RNA polymerase. All four operations,(0 0) to (1 1), with an enzyme reaction can be performed simultaneously, using fourdroplet devices that are directly connected to a patch-clamp ampliﬁer. The output RNA
molecule is detected using a biological nanopore with single-molecule translocation.
Channel current recordings can be obtained using the enzyme solution. The integration of DNA logic gates into electrochemical devices is necessary to obtain output
information in a human-recognizable form. Our method will be useful for rapid andconﬁned DNA computing applications, including the development of programmablediagnostic devices.

KEYWORDS: DNA computing, nanopore, lipid bilayer, droplet, microdevice

Nthat enables rapid detection (∼10 min).8 However, this process
has two issues. First, it is limited by the “one-to-one” reaction:
the DNA hybridization or chain displacement reaction involves
the same number of input and output molecules. The ampliﬁcation of output molecules is imperative to construct a highly
sensitive chip-based detection method. While PCR is broadly
used for DNA ampliﬁcation, it requires temperature cycling
and accordingly, is not well suited for small chip operations.
Another issue is that the input molecule and output molecule
are of the same type; when DNA molecules are used as inputs,
they are also the output in the system. The transcription function from DNA to RNA is useful for diagnosis or other medical
applications, but it cannot be achieved in the conventional
system.
To integrate ampliﬁcation and transcription functions into
the droplet system, we used a bacteriophage T7 RNA polymerase (T7RP) to construct a signal-ampliﬁed AND gate with
DNA as the input and RNA as the output.12 This ampliﬁcation
reaction can be performed in isothermal conditions and issuitable for integration into small chip operations. In this paper,
we report that T7RP can be synthesized from input DNA to
output RNA, using the AND operation, as shown in Figure 1.
In addition, we were able to amplify the input DNA, resulting
in a high concentration of output molecules; the output RNA
molecules can be rapidly electrically detected using αHL nanopores.

ucleic acids have become important materials for the construction of programmable nanomaterials and medical
systems in DNA computing.1,2 Initially, DNA computing basedon logic operations was expected to outstrip electrical computing in terms of parallel processing ability. Recently, DNA
computing has been applied to biological systems owing to its
biocompatibility and programmability.3 Extensive studies haveexamined its applications to life systems, such as cellular circuits4 and medical diagnostics.5 With respect to diagnostics,
mRNA and microRNA markers have been developed for the
diagnosis of various cancers.6,7 Unlike typical diagnostic devices
such as small glucose sensors, conventional DNA computing
is operated in a nonintegrated system. In conventional logic
operations, calculations are obtained by a three-step procedure,
as follows: (1) DNA or RNA molecules are used as inputs in asolution and handled by pipetting, (2) enzyme reactions or
chain displacement reactions occur in plastic tubes, and (3)output molecules and a human-recognizable readout are generated. To recognize output signals in conventional systems,
multistep procedures such as ﬂuorescent labeling, PCR (polymerase chain reaction), and gel electrophoresis are required.
These procedures are not suitable for practical diagnostic
applications.
We recently developed a method for the rapid and label-freedetection of output DNA, using α-hemolysin (αHL) nanoporesin microdroplet devices.8 DNA translocation through a nanopore can be detected electrically by the blocking of the channel
current signal at the single-molecule level.9−11 Using this system, we previously constructed a NAND gate in a droplet device© XXXX American Chemical Society

Received: March 28, 2017
Published: April 17, 2017
A

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DNAs containing part of the T7RP promoter region, as shownin Figure 2. For cases in which two input DNAs exist, deﬁned
as input (1 1), two input DNAs form a duplex and the duplex
hybridizes with the template DNA. The T7RP polymerase
binds to the promoter region and synthesizes a large amountof RNA as output 1 (Figure 2d). In other cases, inputs (0 0),(0 1), and (1 0), the input DNA cannot hybridize to the template DNA, resulting in output 0 (Figure 2a to 2c).
Design of the DNA for the AND Operation, Using a
Thermodynamic Simulation. We designed the sequence of
the input and template DNA molecules for the AND operation(Table 1) using a thermodynamic simulation. The design
requisites were as follows: (i) the template DNA had the T7RP
promoter region, which appeared only when both input DNAs
exist at the same time, and (ii) one of the input DNAs did notbind to the promoter region of the template DNA in the (1 0)and (0 1) systems. The template DNA had a partial doublestranded region, although most of the T7RP promoter region
remained single-stranded, as described in a previous report.12
To ensure hybridization between the template and two input
DNAs and a lack of hybridization for the single input DNA,
we designed Input B such that it formed a hairpin structure;
the double-stranded structure only formed in the presence of
Input A. This dsDNA was able to bind to the template DNA
and the enzyme reaction proceeded (Figure 2).
These DNAs were designed using thermodynamic simulations, and the base sequence and ΔG of each DNA system
are listed in Tables 1 and 2. The ΔG of the hybridized structureof Inputs A and B was −30.08 kJ mol−1, indicating thatthe double-stranded structure was much more stable than the
single-stranded structures (0 kJ mol−1). The hybridized DNA
could bind to the template DNAs to form the complete DNA
structure containing Input A, Input B, and template DNAs.
The calculated ΔG showed the largest stabilization energy, i.e.,−99.66 kJ mol−1, in this case. In contrast, when there was only
one input DNA, template DNA binding did not occur based on
the NUPACK simulation.
Veriﬁcation of the AND Operation by Gel Electrophoresis. To conﬁrm the AND operation using the designed

Figure 1. A schematic view of the AND gate with enzymatic reactions.
When two types of inputs exist, RNA is synthesized by the T7 RNApolymerase. After RNA synthesis, these output molecules pass throughα-hemolysin nanopores for detection.

The four individual calculations from (0 0) to (1 1) can be
operated simultaneously using four-microdroplet devices.

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RESULTS AND DISCUSSION
Principle of the AND Operation Using Biological
Nanopores. To construct the AND gate with the reverse transcription enzyme, we used two input DNAs and template

Figure 2. Reaction scheme for each input. (a−c) Output 0; Input DNA does not bind to the template DNA. Output RNA is not synthesized by
T7RP because the enzyme cannot recognize the template DNA. (d) Output 1; Input DNA A and B can hybridize to each other. Subsequently, theinput DNA binds to the template DNA. Finally, output RNA is synthesized by T7RP because the enzyme can recognize the input and template
DNA complex.
B

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Table 1. DNA Sequence for the AND Operationa
Input DNA A 5′-CGAAGCAGCAGAATCCGTAATA-3′

22 merInput DNA A 5′-CGAAGCAGCAGAATCCGTAATA-3′

Input DNA B

45 merInput DNA B

a5′-GTGAGTCGTATTACGGATTCTGCTGCTTCGTAATACGACTCACTA-3′

22 mer5′-GTGAGTCGTATT45 mer
ACGGATTCTGC
TGCTTCGTAATACGACTCACTA-3′

Input DNA A 5′-CGAAGCAGCAGAATCCGTAATA-3′
Input DNA B

5′-GTGAGTCGTATTACGGATTCTGCTGCTTCGTAATACGACTCACTA-3′

The four individual calculations from (0 0) to (1 1) can be operated simultaneously using four-microdroplet devices.

Table 2. Gibbs Free Energy of DNA Combinations According to NUPACK Simulations
nameInput DNA A

Input DNA B

Input DNA A and B

temp. DNA 1

temp. DNA 2

temp. DNA 1 and 2(1 1)

ΔG (kJ/mol)

0−15.19−30.08−0.67

0−44.58−92.66system clearly exhibited the greatest RNA generation comparedwith other cases. In addition, this reaction diﬀered between aconventional plastic tube and the droplet with a surrounding
lipid layer (Figure S5). Therefore, we improved the droplet
device by minimizing width to enhance heat transfer. We concluded that this system was suﬃcient for the AND operation
by establishing an appropriate threshold (see Channel Current
Measurements and Data Analysis).
Asymmetric Solution Conditions for Nanopore Measurement in a Two-Droplet System. We fabricated fourdroplet contact devices to operate the four logic calculations
simultaneously (Figure 4a). The devices can be dismounted
from a breadboard, which was used as a platform for the electrical connection, and placed in a thermal cycler during the
enzyme reaction.13 To prevent droplet fusion and the mixtureof the solutions before nanopore measurements, we prepared aseparator that closes the aperture between each droplet chamber during the enzyme reaction (Figure 4b). In our two-droplet
devices, one of the droplets was used as the logic operation(enzyme reaction) droplet and the other was the electrolyte
solution, as shown in Figure 4c. Traditionally, nanopore measurements are performed using a symmetrical KCl solution to
obtain appropriate channel current conductance. However, theenzyme activity of T7RP decreases in solutions with high KCl
concentrations.14 Therefore, we used 0.2 M KCl for the enzyme
solution. This concentration has two problems: a decreased
the translocation event frequency15 and channel conductance.
To address these problems, we used asymmetric electrolyte
conditions (1 M KCl/0.2 M KCl).16 This asymmetric condition
resulted in an 8-fold higher translocation frequency and 1.7-foldhigher conductance than those of the symmetrical condition of(0.2 M KCl/0.2 M KCl).
AND Operation with Nanopore Measurements inFour Operation Devices. For the AND operation with nanopore measurements, the ﬂowchart of the calculation protocol
was conﬁgured as shown in Figure S2. (i) After nanopore
opening, the blocking of current signals shorter than 500 ms
and current blockage of less than 60% are measured for 5 s.
(ii) Procedure (i) is repeated 3 times and the mean event frequency is determined. (iii) If the mean event frequency is larger
than an established threshold value, the output is 1; otherwise,
the output is 0.
Using this protocol, the AND logic operation was performed
with the enzyme reaction and the output RNA was detectedusing nanopores. Figure 5a shows the typical current and time
traces for all inputs, (0 0) to (1 1). In the case of (0 0), theblocking of current signals greater than 60% was not observed,
but short current blocking events were detected. These shortcurrents might indicate partial blocking events17 of templateDNAs, we conducted the four calculations from input (0 0) to(1 1) in tubes, and the reaction results were examined by gelelectrophoresis, as shown in Figure 3a. In the case of (1 1), a

Figure 3. (a) Gel electrophoresis for the operation of RNA synthesis;
1 μM each DNA with the control was applied in lanes 2, 3, and 4.
(b) Relative ﬂuorescence of output RNA without DNase. Error bars
represent standard deviations (n = 3).

concentrated band was observed at around 35-mer DNA,
comparable to the length of synthesized RNA. To verify RNA
generation, we added DNase to degrade the DNAs. As a result,an unexpected band, consistent with RNA generation in the(0 1) system, was detected, suggesting that there were someerrors. Although Input B formed a hairpin structure to prevent
unexpected binding to the template DNA, this hairpin structure
may dissociate, enabling the binding of the T7RP promoter
region to the template DNA. As shown in Figure 3b, the (1 1)C

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Figure 4. (a) The measurement system for electrical measurement. This system can measure each input solution simultaneously. (b) Image of themeasurement multichannel device. Scale bar is 1 mm. (c) A schematic view of the experimental methods. Enzymatic reaction indicates activity for adroplet in the measurement device.

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METHODS
Reagents and Chemicals. The following reagents wereused: 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC;
Avanti Polar Lipids, Alabaster, AL, USA), n-decane (Wako
Pure Chemical Industries, Ltd., Osaka, Japan), Tris(hydroxymethyl)aminomethane (Tris; Nacalai Tesque, Kyoto, Japan),magnesium chloride (MgCl2; Nacalai Tesque), potassium
chloride (KCl; Nacalai Tesque), urea (Wako Pure Chemical
Industries, Ltd.), Ribonucleoside Triphosphate mixture (NTP
mixture, F. Hoﬀmann-La Roche, Ltd., Basel, Switzerland), 10×Transcription Buﬀer (500 mM sodium chloride, 80 mM magnesium chloride, 50 mM dithiothreitol, and 400 mM Tris-HCl,
pH 8.0; TOYOBO Co., Ltd., Osaka, Japan), Thermo T7RP
(TOYOBO), Ribonuclease inhibitor (RNase inhibitor, TOYOBO
Co., Ltd.), deoxyribonuclease (DNase; Takara Bio, Inc., Shiga,
Japan), ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque),bromophenol blue (BPB, Wako), glycerol (Wako), Tris-BorateEDTA buﬀer (TBE buﬀer; Takara Bio), ammonium persulfate(Wako), and 40% (w/v) Acrylamide/Bis Mixed Solution(Nacalai Tesque). Buﬀered electrolyte solutions were preparedfrom ultrapure water, which was obtained from a Milli-Q
system (Millipore, Billerica, MA, USA). αHL (Sigma-Aldrich,
St. Louis, MO, USA) was obtained as a monomer protein
isolated from Staphylococcus aureus in the form of a powder,dissolved at a concentration of 1 mg/mL in ultrapure water,and stored at −80 °C. For use, samples were diluted to 0.3 μM
using a buﬀered electrolyte solution and stored at 4 °C. This
concentration was suitable for the single nanopore state, andthe single pore cannot be perfectly controlled. Each DNA(FASMAC Co., Ltd., Kanagawa, Japan) was obtained fromDNA synthesis in the form of a powder, dissolved at a concentration of 100 μM in ultrapure water, and stored at −20 °C.
Design and Formation of Input and Template DNA.
The free energy of Input DNA A, Input DNA B, and template
DNA was calculated by thermodynamic simulations using Nucleic
Acid Package (NUPACK) (http://www.nupack.org/).22 Annealing of the folded strands was conducted by heating each DNA
sample for 5 min at 100 μM in ultrapure water to 95 °C,
followed by rapid cooling to 4 °C.

DNAs. In the cases of (1 0) and (0 1), large blocking signals
were rarely observed, implying that unexpected RNA generation
occurred, as mentioned above. In the case of (1 1), frequent
blocking signals were observed. The mean event frequencies
for each system are shown in Figure 5b. Setting the thresholdto 0.702 s−1 based on the observed standard error andevent frequency (see Channel Current Measurements and Data
Analysis), we precisely deﬁned the output signals in our
droplet-nanopore system. All four-logic operations, (0 0) to(1 1), were carried out for 90 min, including 60 min of enzyme
reaction. In addition, DNase can eﬀectively reduce undesiredtranslocation by the template or input DNAs (Figure S6).
Combined with nanopore detection, the operation time can bemuch shorter than that of the conventional DNA logic operation (which can take several hours).18,19■

CONCLUSIONSIn summary, the signal-ampliﬁed AND operation with DNA-toRNA transcription can be achieved using bacteriophage T7RP.
Output RNA molecules were detected by αHL nanopores withthe single-molecule translocation, and the system was labelfree. The four diﬀerent operations from (0 0) to (1 1) weresuccessfully conducted simultaneously using the microdroplet
devices. Using this method, the DNA/RNA transcription AND
operation was performed over a short time period, using a timeconsuming enzyme reaction. The enzyme reaction requiredca. 60 min and was the rate-limiting process in the operation.
To construct a rapid operation, other methods, such as toehold
reactions, should be considered.
Highly complex operations, such as NAND or XOR withenzyme reactions, can be readily performed using our system.
In addition, the integration of DNA logic gates into electrochemical devices is important to ensure that molecules containing output information, such as diagnostic results, can beprocessed as human-recognizable information.20 In our system,
the output molecules can be observed as nanopore current
signals in microdroplet devices. This property is imperative forthe practical usage in DNA computing, e.g., microRNA21 ormRNA detection for cancer diagnosis.
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each device were attached to a solderless breadboard (E-CallEnterprise Co., Ltd., Taipei City, Taiwan), which was connected
to a Jet patch-clamp ampliﬁer (Tecella, Foothill Ranch, CA,
USA) using a jumper wire.
Gel Electrophoresis and Analysis of Fluorescence
Intensity. Products of output RNA ampliﬁcation were analyzed by 10% 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 7.5 W constant power for 45 min at 22 ± 2 °C. The gel was made in ourlaboratory. After electrophoresis, the gel was stained withdiluted SYBR Gold (Thermo Fisher Scientiﬁc, Waltham, MA,
USA) solution for 30 min. Images were obtained under blue
LED radiation using a digital camera. The ﬂuorescent intensity
was analyzed using ImageJ 1.50 (National Institutes of Health,Bethesda, MD, USA).
Enzymatic Reaction in Droplets, Preparation of Bilayer
Lipid Membranes, and Reconstitution of α-Hemolysin.
Bilayer lipid membranes (BLMs) were prepared using anarrayed device with four chambers on the breadboard. Fourindividual BLMs can be simultaneously formed in this device.
First, the DPhPC (lipids/n-decane, 10 mg/mL) solution(2.3 μL) was added to all chambers. Next, the buﬀer solution(4.0 μL) without αHL, DNA, or proteins was poured into eachrecording chamber. The enzyme solution (4.7 μL) with αHL(ﬁnal concentration 1 to 5 nM) and each DNA (ﬁnal concentration 0.1 μM) was added to each ground chamber. In thisstudy, the enzyme solution (200 mM KCl, 20 units of RNase
inhibitor, 50 units of T7RP, 2 mM NTP mixture, 1× Transcription Buﬀer pH 8.0) was used for the ground chamber, and the
buﬀer solution (1 M KCl, 1× Transcription Buﬀer pH 8.0) wasused for the recording chamber. The devices were set in athermal cycler during the enzyme reaction for 60 min at 37 °C.
They were then mounted on a breadboard platform, and theseparator was opened (Figure 4b). A few minutes after applying
the sliding separator, the two lipid monolayers connected andformed BLMs, and αHL formed nanopores by reconstitution in
the BLMs.
Channel Current Measurements and Data Analysis.
Channel current was monitored using a Jet patch-clamp ampliﬁer connected to each chamber. Ag/AgCl electrodes werealready present in each droplet when the solution was added tothe chambers. A constant voltage of +120 mV was applied to
the recording chamber, and the ground chamber was grounded.
Reconstituted αHL in BLMs allowed ions to pass through ananopore under the voltage gradient, and channel current
signals were obtained. When output RNA was present in the
ground chamber, the RNA passed through the nanopore andchannel current blockage was observed. Spike signals of RNAtranslocation were obtained, and the processes of electric
current change were deﬁned as a translocating event. When the
BLMs ruptured, BLMs were reformed with the separator gateusing tweezers. The signals were detected using a 4 kHz lowpass ﬁlter at a sampling frequency of 20 kHz. Analyses ofchannel current signals and duration were performed using
pCLAMP ver. 10.6 (Molecular Devices, Sunnyvale, CA, USA)and Excel2013 (Microsoft, Redmond, WA, USA). RNAtranslocation events were obtained from more than 60% of
the current blockades from an open current level in a single
nanopore. Channel current measurements were conducted at
22 ± 2 °C. The detailed protocol of the AND operation
described in Figure S2 as the ﬂowchart. All operations wereconducted from 5 to 16 times (5 < n < 16) in the nanopore

Figure 5. (a) Typical current traces of each input at +120 mV for 0.2
M KCl on the trans side, 1 M KCl on the cis side. (b) The event
frequency for each input. Error bars indicate three times the standard
error (3σ).

Preparation of the Multichannel Device. The multichannel device23,24 was fabricated by machining a 6.0 mm-thick,
10 × 10 mm poly(methyl methacrylate) (PMMA) plate(Mitsubishi Rayon, Tokyo, Japan), using computer-aided
design and a computer-aided manufacturing three-dimensionalmodeling machine (MM-100; Modia Systems, Koshigaya,
Japan), as shown in Figure 4a. Two wells (2.0 mm diameterand 5.0 mm depth) and a ditch between the wells were
manufactured on the PMMA plate. Each well had a throughhole in the bottom and Ag/AgCl electrodes were set in thehole. A polymeric ﬁlm made of parylene C (polychloro-pxylylene) with a thickness of 5 μm was patterned with single
pores (100 μm diameter) using conventional photolithographymethods and then ﬁxed between PMMA ﬁlms (0.2 mm thick)using an adhesive bond (Super X; Cemedine Co., Ltd., Tokyo,
Japan). The ﬁlms, including the parylene ﬁlm, were inserted
into the ditch to separate the wells. Ag/AgCl electrodes of
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management via single nanopore-based sensors. Chem. Rev. 112,6431−6451.
(10) Deamer, D. W., and Branton, D. (2002) Characterization of
nucleic acids by nanopore analysis. Acc. Chem. Res. 35, 817−825.
(11) Branton, D., Deamer, D. W., Marziali, A., Bayley, H., Benner, S.
A., Butler, T., Di Ventra, M., Garaj, S., Hibbs, A., Huang, X. H.,
Jovanovich, S. B., Krstic, P. S., Lindsay, S., Ling, X. S. S., Mastrangelo,
C. H., Meller, A., Oliver, J. S., Pershin, Y. V., Ramsey, J. M., Riehn, R.,
Soni, G. V., Tabard-Cossa, V., Wanunu, M., Wiggin, M., and Schloss, J.
A. (2008) The potential and challenges of nanopore sequencing. Nat.
Biotechnol. 26, 1146−1153.
(12) Ayukawa, S., Takinoue, M., and Kiga, D. (2011) RTRACS: A
Modularized RNA-dependent RNA transcription system with highprogrammability. Acc. Chem. Res. 44, 1369−1379.
(13) Ohara, M., Sekiya, Y., and Kawano, R. (2016) Hairpin DNA
unzipping analysis using a biological nanopore array. Electrochemistry84, 338−341.
(14) Matsukage, A. (1972) Effects of KCl concentration ontranscription by E.coli RNA-polymerase 1. Specific effect of
combination of nucleoside triphosphates. Mol. Gen. Genet. 118, 11−22.
(15) Jeon, B. J., and Muthukumar, M. (2014) Polymer capture byalpha-hemolysin pore upon salt concentration gradient. J. Chem. Phys.
140, 015101.
(16) Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y., andMeller, A. (2010) Electrostatic focusing of unlabelled DNA into
nanoscale pores using a salt gradient. Nat. Nanotechnol. 5, 160−165.
(17) Akeson, M., Branton, D., Kasianowicz, J. J., Brandin, E., andDeamer, D. W. (1999) Microsecond time-scale discrimination amongpolycytidylic acid, polyadenylic acid, and polyuridylic acid ashomopolymers or as segments within single RNA molecules. Biophys.
J. 77, 3227−3233.
(18) Li, W., Zhang, F., Yan, H., and Liu, Y. (2016) DNA basedarithmetic function: a half adder based on DNA strand displacement.
Nanoscale 8, 3775−3784.
(19) Seelig, G., Soloveichik, D., Zhang, D. Y., and Winfree, E. (2006)
Enzyme-free nucleic acid logic circuits. Science 314, 1585−1588.
(20) Feng, L. Y., Lyu, Z. Z., Offenhausser, A., and Mayer, D. (2015)
Multi-level logic gate operation based on amplified aptasensor
performance. Angew. Chem., Int. Ed. 54, 7693−7697.
(21) Hiratani, M., Ohara, M., and Kawano, R. (2017) Amplificationand quantification of an antisense oligonucleotide from target
microRNA using programmable DNA and a biological nanopore.
Anal. Chem. 89 (4), 2312−2317.
(22) Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M.
B., Khan, A. R., Dirks, R. M., and Pierce, N. A. (2011) NUPACK:
analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170−173.
(23) Kawano, R., Tsuji, Y., Sato, K., Osaki, T., Kamiya, K., Hirano,M., Ide, T., Miki, N., and Takeuchi, S. (2013) Automated parallel
recordings of topologically identified single ion channels. Sci. Rep. 3,
1995.
(24) Watanabe, H., and Kawano, R. (2016) Channel current analysis
for pore-forming properties of an antimicrobial peptide, magainin 1,
using the droplet contact method. Anal. Sci. 32, 57−60.

experiments. The threshold output for our AND gate wasdeﬁned as follows:y = max{x(0,0)+ 3σ(0,0) , x(0,1)+ 3σ(0,1) , x(1,0)+ 3σ(1,0)}
̅
̅
̅
(1)

z=

− 3σ(1,1) + yx(1,1)
̅2(2)

where x indicates the event frequency, y is a maximum of threevalues, and z is the threshold value.
(i) The mean event frequency of three inputs (0 0), (1 0),
and (1 1) and three times the standard error (3σ) were
calculated. Each mean value and 3σ was added. The largest
value of the three input patterns was determined. (ii) The mean
event frequency of input (1 1) was determined by 3σ. (iii) The
mean value of (i) plus (ii) was deﬁned as the threshold.

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ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssynbio.7b00101.
Experimental details of the enzyme reaction and theprotocol of logic operation (PDF)■

AUTHOR INFORMATION

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

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

R. K. and M. T. conceived the original idea. M. O. conducted
the experiments and M. O. and R. K. wrote the paper.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
This work was partially supported by KAKENHI (Molecularrobotics: Nos. 24104002, 15H00803, and 16H06043) from
MEXT Japan.

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REFERENCES(1) Seeman, N. C. (2010) Nanomaterials based on DNA. Annu. Rev.
Biochem. 79, 65−87.
(2) Simmel, F. C., and Dittmer, W. U. (2005) DNA nanodevices.
Small 1, 284−299.
(3) Benenson, Y. (2009) Biocomputers: from test tubes to live cells.
Mol. BioSyst. 5, 675−685.
(4) Benenson, Y. (2012) Biomolecular computing systems:
principles, progress and potential. Nat. Rev. Genet. 13, 455−468.
(5) Jung, C., and Ellington, A. D. (2014) Diagnostic applications of
nucleic acid circuits. Acc. Chem. Res. 47, 1825−1835.
(6) Benenson, Y., Paz-Elizur, T., Adar, R., Keinan, E., Livneh, Z., andShapiro, E. (2001) Programmable and autonomous computing
machine made of biomolecules. Nature 414, 430−434.
(7) Zhang, X. Y., Wang, Y., Fricke, B. L., and Gu, L. Q. (2014)
Programming nanopore ion flow for encoded multiplex microRNAdetection. ACS Nano 8, 3444−3450.
(8) Yasuga, H., Kawano, R., Takinoue, M., Tsuji, Y., Osaki, T.,
Kamiya, K., Miki, N., and Takeuchi, S. (2016) Logic gate operation by
DNA translocation through biological nanopores. PLoS One 11,
e0149667.
(9) Reiner, J. E., Balijepalli, A., Robertson, J. W. F., Campbell, J.,
Suehle, J., and Kasianowicz, J. J. (2012) Disease detection andF

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