Electrochemistry
The Electrochemical Society of Japan
ArticleReceived: November 20, 2015
Accepted: February 4, 2016
Published: May 5, 2016
http://dx.doi.org/10.5796/electrochemistry.84.338

Electrochemistry, 84(5), 338–341 (2016)

Hairpin DNA Unzipping Analysis Using a Biological Nanopore Array
Masayuki OHARA, Yusuke SEKIYA, and Ryuji KAWANO*
Department of Life Science and Biotechnology, Tokyo University of Agriculture and Technology,
2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan* Corresponding author: rjkawano@cc.tuat.ac.jp
ABSTRACTThis paper describes a method of hairpin DNA (hpDNA) unzipping analysis using a biological nanopore array.
Various lengths of hpDNAs were captured and translocated through the nanopore and the individual duration times
were monitored. The correlation between the unzipping time and the simulated hybridization energy of theduplexes was able to be modeled as ﬁrst-ordered reaction. This method is one of the approach for understanding ofbasic biological reactions in DNA replication and RNA transcription.
© The Electrochemical Society of Japan, All rights reserved.

Keywords : Nanopore, DNA Unzipping, Lipid Bilayer
1. Introduction
Nanopore measurement is a powerful tool for single-molecule
analysis. A pore-forming protein, A-hemolysin (AHL) is a popular
biological nanopore, is reconstituted in a lipid bilayer, and thechannel current through the AHL nanopore can be measured usinga patch-clamp ampliﬁer.1,2 When a single molecule translocatesthrough the nanopore electrophoretically, the blocking current isclearly observed because the ion ﬂowing is prevented.3 Blocking
currents enable the electrical identiﬁcation of distinct molecularconﬁgurations and the elucidation of mechanisms responsible forsingle molecule behavior, such as single molecule transport,4 protein
unfolding,5 and DNA unzipping.6 Therefore, this method has been
attracting attention as a next-generation of DNA sequencing or asingle-molecule detection technique.7
The unzipping behavior of double-stranded DNA (dsDNA) is
important in many cellular processes, such as DNA replication and
RNA transcription. The forces and timescales associated with the
breakage of DNA/RNA strands have been studied at the singlemolecule level.8 The single DNA unzipping behavior has also beenstudied through nanopore measurement using the AHL pore.9–11 The

AHL pore, which consists of a vestibule (2.5 nm diameter) and acylindrical pore (1.5 nm diameter) as shown in Fig. 1, is suitable forthe dsDNA unzipping analysis. While ssDNA can pass through theentire AHL pore, dsDNA can get obstructed in the 2.5 nm vestibule
section (Fig. 1a). The clogged dsDNA is unzipped and translocatedwithin the nanopore, and the sequence-dependent duration time atsingle-molecule levels can be measured (Fig. 1b). In this experiment, a massive data set is needed for the stochastic analysis.
We recently proposed a multiple planar lipid bilayer system using
microdroplets, named as a “droplet contact method” (DCM). A
planar lipid bilayer can spontaneously form at the interface between
the contacting microdroplets in an oil/lipid mixture solution(Fig. 2a). In this method, membrane stability, which usually remains
for >300 h, is improved by reducing the contact area.12 Furthermore, we constructed a droplet array for high-throughput measurements.13 Using bilayer lipid membranes (BLMs), we measured
various membrane proteins and biological nanopore, for example,
single molecule detection using DNA aptamers,14 high-throughput
ion channel measurements at the single-protein level,13,15,16 solutionexchange techniques via the microdroplet system,17 and thedevelopment of portable nanopore sensors.18 In this study, weFigure 1. Schematic diagrams of hpDNA unzipping in the AHL nanopore. a) When hpDNA was clogged in the pore, the current signal
decreased with long duration times (bottom trace). b) When hpDNA was unzipped under applied voltage at 120 mV, it passed through the poreand the current signal returned to initial levels.
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Electrochemistry, 84(5), 338–341 (2016)
when the stem of the double-strand of the hpDNA was increased,
free energy also increased. Annealing of the folded strands was
conducted by heating hpDNA samples for 5 min at 100 µM in
ultrapure water to 95°C followed by rapid cooling to 4°C.
2.3 Preparation of multi-channel device
The multi-channel device was fabricated by machining a
6.0 mm thick, 10 © 10 mm polymethyl methacrylate (PMMA) plate(Mitsubishi Rayon, Tokyo, Japan) using a computer-aided design
and/computer-aided manufacturing three dimensional modelingmachine (MM-100, Modia Systems, Japan) as shown in Fig. 2b.
Two wells (2.0 mm diameter and 5.0 mm depth) and a chase
between the wells were manufactured on the PMMA plate. Eachwell had a through-hole in the bottom and Ag/AgCl electrodes set
into this hole. 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 photolithography method,12and then ﬁxed between PMMA ﬁlms (0.2 mm thick) using anadhesive bond (Super X, Cemedine Co., Ltd., Tokyo, Japan). The
ﬁlms, including the parylene ﬁlm, were inserted into the chase to
separate the wells. Ag/AgCl electrodes of each device were set toa solderless breadboard (E-Call Enterprise Co., Ltd., Taipei City,
Taiwan), which was connected to a Jet patch-clamp ampliﬁer(Tecella, Foothill Ranch, CA, USA) using jumper wire.

Figure 2. (Color online) a) A schematic illustration of the droplet
contact method used for preparing a lipid bilayer. b) A photographof the lipid bilayer array. This device has six chambers and isconnected to multiple patch-clamp ampliﬁers.

designed the variable array device using commercially available
breadboard in an attempt to reveal the relationship between the
hybridization energy (¦G) and the unzipping kinetics of hairpin
DNA (hpDNA) using this device.
2. Experimental
2.1 Reagents and chemicals
In this study, we used the following reagents: 1,2-diphytanoyl-snglycero-3-phosphocholine (DPhPC; Avanti. Polar Lipids, AL, USA);n-decane (Wako Pure Chemical Industries, Ltd., Osaka, Japan); 3morpholinopropane-1-sulfonic acid (MOPS, Nacalai Tesque, Kyoto,
Japan); and potassium chloride (KCl; Nacalai Tesque). Buffered
electrolyte solutions were prepared from ultrapure water, which was
obtained from a Milli-Q system (Millipore, Billerica, MA, USA).
AHL (Sigma–Aldrich, St. Louis, MO, USA) was obtained as amonomer protein isolated from Staphylococcus aureus in the form of
a powder, and dissolved at a concentration of 1 mg/mL in ultrapure
water and stored at ¹80°C. For use, samples were diluted to 0.5 µM
using a buffered electrolyte solution and stored at 4°C. Hairpin DNA(FASMAC Co., Ltd., Kanagawa, Japan) was obtained from DNA
synthesis in the form of a powder, and dissolved at a concentrationof 100 µM in ultrapure water and stored at ¹20°C.
2.2 Design and formation of hairpin DNA
The free energy of the hpDNA was calculated from thethermodynamic simulation using the Nucleic Acid Package(NUPACK) software (http://www.nupack.org/).19 In this simulation,

2.4 Bilayer lipid membrane preparation and reconstitution of
α-hemolysin
Bilayer lipid membranes (BLMs) were prepared using an arrayeddevice, which had six chambers on the breadboard. Six individual
BLMs can be simultaneously formed in this device, which allowed
for a higher-throughput measurement compared to the conventional
system. First, the DPhPC (lipids/n-decane, 10 mg/mL) solution(2.4 µL) was poured into all chambers. Next, the buffer solution(4.7 µL) without AHL and hpDNA was poured into each recording
chamber. The buffer solution (4.7 µL) with AHL (ﬁnal concentration
0.005 µM) and hpDNA (ﬁnal concentration 10 µM) was poured into
each ground chamber. In this study, the buffer solution (1 M KCl,
10 mM MOPS, pH 7.0) was used for all droplets. A few minutes
after adding the buffer solution the two lipid monolayers connected
and formed BLMs, and AHL formed nanopores by reconstitution in
the BLMs. When the BLMs ruptured, we reformed the BLMs by
tracing with a hydrophobic stick between two droplets.18
2.5 Channel current measurements and data analysis
Channel current was monitored using a Jet patch-clamp ampliﬁer
connected to each chamber. Ag/AgCl electrodes were alreadypresent in each droplet when we added the solution into thechambers. A constant voltage of +120 mV was applied to therecording chamber, and the ground chamber was grounded.
Reconstituted AHL in BLMs allowed ions to pass through ananopore under the voltage gradient, and we obtained the channel
current signals. When hpDNA was present in the ground chamber
the nanopore containing the AHL was blocked by this DNA andthe channel current decreased. When hpDNA passed through thenanopore, the channel current increased. We deﬁned those processesof electric current change as the duration time. The signals weredetected using a 4 kHz low-pass ﬁlter at a sampling frequency of
20 kHz. Analysis of channel current signals and duration time was
performed using pCLAMP ver. 10.5 (Molecular Devices, CA, USA)and Excel (Microsoft, Washington, USA) software. Signature eventsof hpDNA were obtained from more than 10% current blockades
from an open current level in a single nanopore. Between 40 and1300 unzipping events were recorded in this study. All data were
presented as mean values « SE, where each duration time was takenusing the natural logarithm. Channel current measurements wereconducted at 22 « 2°C.
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Electrochemistry, 84(5), 338–341 (2016)
Table 1.
HP4
HP6
HP8
HP10
HP12
HP14
HP16

The hairpin DNA sequences.

5B-AACGTTTTCGTTAAAAAAAAAAAAAAAAAAAA-3B
5B-CGAACGTTTTCGTTCGAAAAAAAAAAAAAAAAAAAA-3B
5B-GTCGAACGTTTTCGTTCGACAAAAAAAAAAAAAAAAAAAA-3B
5B-CAGTCGAACGTTTTCGTTCGACTGAAAAAAAAAAAAAAAAAAAA-3B
5B-CCCAGTCGAACGTTTTCGTTCGACTGGGAAAAAAAAAAAAAAAAAAAA-3B
5B-AACCCAGTCGAACGTTTTCGTTCGACTGGGTTAAAAAAAAAAAAAAAAAAAA-3B
5B-CGAACCCAGTCGAACGTTTTCGTTCGACTGGGTTCGAAAAAAAAAAAAAAAAAAAA-3B

32
36
40
44
48
52
56

mer
mer
mer
mer
mer
mer
mer

Table 2. The melting energy of each hairpin DNA calculatedusing NUPACK. (at 22°C)
Stem of hairpin(base-pair)¦Gsim(kJ mol¹1)

4
6
8
10
12
14
16

¹18.2
¹38.5
¹53.2
¹66.9
¹84.9
¹95.2
¹115.5

3. Results and Discussion
3.1 Channel current recordings of hairpin DNA by lipid bilayerarray
We ﬁrst designed the hpDNA sequence using thermodynamic
simulation. Table 1 shows the hpDNA, which has 4 to 16 base pairs
with a 20 adenine tail designed by NUPACK, and their melting freeenergy (¹¦Gsim) is listed in Table 2. The absolute-value of freeenergy was gradually increased with increasing the lengths of thehairpin duplex. We next measured the unzipping time of the
hpDNA in the AHL nanopore. The typical current and time traces ofeach hpDNA (HP6 to HP12) are shown in Fig. 3. We consideredin this paper that there are two consecutive steps in the nanopore
unzipping process: i) the hairpin region of dsDNA are clogged in
the vestibule following the entry and sliding of the ssDNA tail inthe pore; ii) the hairpin region is unzipped and the unzipped singlestranded DNA translocates through the nanopore. These two steps
can be observed in the channel current signals. We deﬁned theblocking time of hpDNA as the duration time (t) in the subsequent
experiments.

Figure 3. The typical current and time traces of hairpin DNA
unzipping in the nanopore.

3.2 The length dependency of the complementary strand of
hairpin DNA on the unzipping behavior
Figure 4a shows the duration time, t, as a function of ¦Gsim
calculated from thermodynamic simulation. Although we expected
the duration time to initially scale linearly with the free energy, tincreased with increasing ¦Gsim logarithmically in this experiment.
It has been previously argued that the unzipping kinetics of
dsDNA can be exhibited in ﬁrst-order reactions.6 It is well known
that the ﬁrst-ordered dissociation of enzyme reaction is described
as Michaelis-Menten model. This reaction can be represented
schematically as below.
½E þ ½S  ½ES ! ½E þ ½P
where E is enzyme, S is substrate, ES is complex with enzyme andsubstrate, and P is product. Thus, in this paper, we considered this340

Figure 4. a) Duration time of hpDNA as a function of hybridization energy calculated using NUPACK. b) The relationshipbetween the ﬁrst order rate constant and the hybridization energy.
The plots were ﬁtted using the Eq. (1).

Electrochemistry, 84(5), 338–341 (2016)
unzipping behavior as the ﬁrst-ordered dissociation reaction, and it
can be described as following.
½hpDNA þ HL ! ½ssDNA þ ½ HL
The rate constant of this reaction can be described as below.
k ¼ k0 expðGsim =NA kB TÞ

ð1Þ

k ¼ 1=twhere k is the dissociation rate constant, k0 is the initial rate
constant, NA is the Avogadro constant, kB is Boltzmann constantused for this single molecule experiment instead of a gas constant,and T is temperature. The k (s¹1) should be equal to the inverse of t
because 1/t is the reaction frequency per unit of time at the single
molecule reaction. Figure 4b shows the plot of k as a function of¦Gsim. The plots were well ﬁtted using the Eq. (1), indicating theinverse value of the duration time is as the ﬁrst-order rate constant.
This result can be used for the prediction of unzipping time using thesimulated free energy. The unzipping time of dsDNA is occasionallyrequired in the nanopore/DNA experiments. Therefore, thisprediction will be useful for DNA or RNA deign in the extended
nanopore study and also be useful for understanding of DNA related
biological reactions.
4. Conclusion
In summary, we examined hpDNA unzipping time using an AHL
nanopore by changing the length of the complementary region of
the hpDNA from 4 to 16 bases. The hpDNA was initially obstructedin the nanopore, and thus it was unzipped and electrophoreticallypassed through the nanopore. The duration time of captured
hpDNA had a logarithmic relation with the melting free energy(¹¦Gsim) of hpDNA estimated from thermodynamic simulation.
Therefore, we modeled it as a ﬁrst-order dissociation reaction, andﬁtted the data using the kinetic equation. Our study will be usefulfor the design of dsDNA, to be able to determine their blocking
time in the nanopore. Moreover, the high-throughput nanopore
measurement described here will be applied to analysis of extensive
biological reactions, such as the binding of transcription factors to
dsDNA and the movement of enzymes along the length of a DNA
chain.

Acknowledgment
This work was partly supported by a Grant-in-Aid for ScientiﬁcResearch on Innovative Areas (No. 24104002) and ScientiﬁcResearch No. 26540160 and 25708024 of The Ministry of
Education, Culture, Sports, Science and Technology, and Global
Innovation Research Organization in TUAT.
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