Science and Technology of Advanced Materials

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Identifying DNA methylation in a nanochannel
Xiaoyin Sun, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Sakon Rahong,Masaki Kanai, Kazuki Nagashima, Tomoji Kawai & Yoshinobu Baba
To cite this article: Xiaoyin Sun, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Sakon Rahong,Masaki Kanai, Kazuki Nagashima, Tomoji Kawai & Yoshinobu Baba (2016) Identifying DNA
methylation in a nanochannel, Science and Technology of Advanced Materials, 17:1, 644-649,
DOI: 10.1080/14686996.2016.1223516
To link to this article: http://dx.doi.org/10.1080/14686996.2016.1223516© 2016 The Author(s). Published by National
Institute for Materials Science in partnershipwith Taylor & Francis
Accepted author version posted online: 24
Aug 2016.
Published online: 11 Oct 2016.
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Date: 12 October 2016, At: 18:01

Science and Technology of Advanced Materials, 2016VOL. 17, NO. 1, 644–649
http://dx.doi.org/10.1080/14686996.2016.1223516

OPEN ACCESS

FOCUS ISSUE ARTICLE

Identifying DNA methylation in a nanochannel
Xiaoyin Suna,b, Takao Yasuia,b,c, Takeshi Yanagidad,e, Noritada Kajia,b, Sakon Rahonga,b, Masaki Kanaid,
Kazuki Nagashimad, Tomoji Kawaie and Yoshinobu Babaa,b,f
a

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan;
ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Nagoya, Japan;cJapan Science and Technology Agency (JST), PRESTO, Saitama, Japan;dInstitute of Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan;eInstitute of Scientific and Industrial Research, Osaka University, Osaka, Japan;fHealth Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japanb

ABSTRACT

DNA methylation is a stable epigenetic modification, which is well known to be involved ingene expression regulation. In general, however, analyzing DNA methylation requires rather
time consuming processes (24–96 h) via DNA replication and protein modification. Here
we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level
without any protein modifications by measuring the contracted length and relaxation time of
DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA
molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower
contraction rate). The present methodology offers a promising way to identify DNA methylation
without any protein modification at a single DNA molecule level within 2 h.

1. Introduction
DNA methylation is a stable epigenetic modification that it is known to be involved in the regulationof gene expression. Researchers have reported DNA
methylation in eukaryotic cells might lead to silencingof important genes, such as tumor suppressor genes.
[1] This gene silencing for tumor suppressor will affectrelated transcriptional pathways, and ultimately leadto cancer progression.[2] To date, methylation for thepromoter regions of tumor suppressor genes have been
detected in patients with lung, hepatic, breast, cervical,
colorectal and genitourinary cancers.[3] In addition, the­methylated number at single DNA molecules has beenfound to increase abnormally in breast and prostate cancers.[4] In order to detect and predict various cancers intheir early stage, simple and rapid detection of methylated DNA molecules at a single molecule level is desired.

CONTACT Takao Yasuiapchem.nagoya-u.ac

yasui@apchem.nagoya-u.ac.jp; Xiaoyin Sun

ARTICLE HISTORY

Received 5 March 2016
Revised 9 August 2016
Accepted 9 August 2016
KEYWORDS

DNA methylation; single DNA
molecule; nanochannel; DNA
contraction
CLASSIFICATION

30 Bio-inspired andbiomedical materials; 102
Porous / Nanoporous /Nanostructured materials

The best-known detection methods for DNA­ ethylation are bisulfite conversion followed by sequencm
ing [5] and chromatin immunoprecipitation followed
by sequencing.[6] These strategies can obtain details of
the epigenetic modification; however, they have serious
disadvantages of consuming a relatively large sample
and taking much time. Micro- and nano-devices are
powerful tools that have been applied to analyze DNA
methylation. They allowed researchers to analyze DNA
methylation through optical and electrical approaches
by detecting DNA molecules with methylation on the
methyl-CpG-binding domain (MBD).[7,8] However,DNA methylation detection in previous studies isbased on modification of methylation sites using MBD
proteins, which has a risk of non-specific absorption
to DNA molecules. Such a non-specific absorption is
crucial for the detection of DNA methylation, and therefore a simple DNA methylation detection without any
sun.xiaoyin@h.mbox.nagoya-u.ac.jp; Yoshinobu Babababaymtt@© 2016 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sci. Technol. Adv. Mater. 17 (2016) 645X. SUN et al.

Figure 1. The multi-step fabrication process of the nanochannel device. (a) Cleaning quartz substrates; (b) depositing a Cr layer;(c) coating photolithography resists; (d) drawing microchannel patterns; (e) etching the Cr layer; (f) etching the microchannels;(g) etching the remaining Cr layer; (h) deposition of another Cr layer; (i) coating EB resist; (j) drawing nanochannels; (k) etching thesecond Cr layer; (l) etching the nanochannels; (m) etching the remaining second Cr layer; (n) bonding with a cover glass.

modification is desired for DNA methylation detection
with no site specificity. Here we fabricated a nanochannel device to detect the DNA methylation without site
specificity at a single DNA molecule level by measuring acontraction process (Figure 1). The nanochannel device
was fabricated on fused silica substrates by photolithography, reactive ion etching and electron beam lithography techniques. To measure the contraction process, we
introduced methylated and non-methylated single DNA
molecules into the nanochannel to elongate and contract
them (Figure 2(a)). Both ends of the nanochannel wereconnected to microchannels, which have reservoirs to
put electrodes for introduction of the DNA molecules(Figure 2(b–e)). The nanochannel was 300 nm, 300 nm,
and 250 μm in width, height, and length, respectively.
The microchannels were 2 μm in height and 5 mm inlength, and the widths of the microchannels were gradually changed from 8.25 μm to 7 mm. As we applied
electrical potential difference of 3 V between two reservoirs, negatively charged DNA molecules were driven
from a reservoir to the other one via the microchannels
and the nanochannel, electrophoretically. If the nanochannel geometry is smaller than the gyration radius of

the DNA molecules, the DNA molecules were forced
to deform from random-coil to linear conformation
inside the nanochannel, and finally, the DNA molecules had elongated conformation. After elongation of
DNA molecules, we turned off the electrical potential toevaluate the contraction process of DNA molecules withand without methylation inside the nanochannel. This
simple analysis method helps to detect DNA moleculeswith methylation.

2. Experimental details
2.1. Device fabrication
At first, in order to pattern the microchannel, a Cr layer
as a metal mask was deposited on a quartz glass substrateusing a sputtering apparatus (model SVC-700LRF,
Sanyu Electron Co., Tokyo, Japan). Then, a positive
photoresist (TSMR-V50 EL, Tokyo Ohka Kogyo Co.
Ltd, Tokyo, Japan) was spin-coated on the Cr layer. After
that, the microchannel pattern was developed using
AZ 300 MIF developer (AZ Electronic Materials Plt,Tokyo, Japan). Next, the Cr layer in the microchannel
pattern was removed by immersing the substrate inSci. Technol. Adv. Mater. 17 (2016) 646X. SUN et al.

Figure 2. Methylation detection system using the nanochannel device. (a) Schematic illustration of the experimental setup.
Negatively charged DNA molecules were transported into a nanochannel by applying an electric field. The red arrow shows thetranslocation direction of the DNA molecules in the nanochannel. Contracted length analysis of DNA molecules is shown on theright. The contracted length of a single DNA molecule was observed with an inverted fluorescence microscope after switching off
the electric field. (b) Schematic illustration of the fabricated device. The device has a nanochannel connected to two microchannels.
(c) Scanning electron microscope (SEM) image of the nanochannel with a 300 nm width, 300 nm depth and 250 μm length; scalebar, 10 μm. (d) SEM image of the nanochannel entrance; scale bar, 10 μm. (e) SEM image (30° tilt angle) of the nanochannel entrance;scale bar, 10 μm.

Cr etchant solution. The microchannel was etched toa 2 μm depth by reactive ion etching (RIE; RIE-10NR,
Samco Co., Kyoto, Japan). The remaining Cr layer was
lifted off using Cr etchant solution. Then, another Cr
layer of 20 nm thickness was deposited on the quartz
glass substrate by rf sputtering for the nanochannel
fabrication. The positive electron beam (EB) resist(ZEP520A-7, Zeon Corp., Tokyo, Japan) was spin-coatedon the sputter-deposited Cr layer. The nanochannel
pattern was formed by electron beam lithography(EBL; SPG-724, Sanyu Electron Co.), and developed
using electron beam developer solution (ZED-N50,
Zeon Corp.). The second Cr layer in the nanochannel
pattern was lifted off using Cr etchant solution. After
that, the 300 nm nanochannel depth was etched by RIE.
The remaining Cr layer was removed using Cr etchantsolution. Finally, the nanochannel device was bonded
using a cover glass (Crystal Base Co., Osaka, Japan) by
a chemical bonding method.

2.2. Sample preparation
T4 DNA molecules (166 kbp, T4GT7 DNA, Nippon
Gene Co. Ltd, Tokyo, Japan) were used for identifying
occurrence of DNA methylation in the nanochannel.
The non-methylated and methylated T4 DNA molecules were stained with an intercalating fluorescencedye YOYO-1 at a dye-to base pair ratio of 1:5. For asingle DNA molecule observation, the T4 DNA solution was diluted to 5 ng ml–1. In order to prepare themethylated T4 DNA molecules, a mixture of phage T4
DNA molecules (166 kbp, T4GT7 DNA, Nippon Gene
Co., Ltd) and CpG methyltransferase (M.SssI) (NewEngland Biolabs Inc., Tokyo, Japan) was incubated at
37°C for 1 h, and then heated at 65°C for 20 min to inactivate the methyltransferase. The CpG methyltransferase(M.SssI) methylates all cytosine residues (C5) within the
double-stranded dinucleotide recognition sequence 5′...
CG...3′. The M.SssI could methylate all 4816 CpGs in theSci. Technol. Adv. Mater. 17 (2016) 647T4 DNA molecules, which are distributed throughout
the entire molecule. After that, the methylation state
was confirmed using the restriction enzyme SalI (NewEngland Biolabs Inc.), which can digest DNA moleculesin the methylation region. The methylated T4 DNA molecules were stained by YOYO-1 overnight at 4°C beforeuse.
2.3. Measurement setup
T4 DNA molecules were introduced into the nanochannel under an applied DC electric field provided by ahigh voltage source-measure unit (model 236, Keithley,
Cleveland, USA). Stretching of the T4 DNA molecules
was observed and was attributed to the electric force
pulling them into the nanochannel against the entropic
barrier at the entrance of the nanochannel. Since electrical potential difference less than 3 V between two
reservoirs could not transport DNA molecules frommicrochannel to nanochannel due to the entropy barrier at the entrance of nanochannel, we used 3 V aselectrical potential difference, which took 20 min totransport the DNA molecules from microchannel tonanochannel. When the T4 DNA molecules were fully
inside the nanochannel, the electric field was switched
off immediately. Then, the contraction process of theT4 DNA molecules was observed. We measured nine
DNA molecules, four of which showed relatively short
DNA length due to photocleavage of DNA molecules;
therefore we used only five DNA molecules data (successful rate: 55%). The fluorescence images of T4 DNA
molecules were obtained with an inverted fluorescencemicroscope (ECLIPSE TE300, Nikon, Tokyo, Japan)
coupled with a CCD camera (C7190-43, Hamamatsu
Photonics K.K.) through a 100 × / 1.40 NA objective
lens. A 488 nm laser (FLS-448-20, Sigma Koki Co., Ltd.)was used as a light source to observe the fluorescently
stained T4 DNA molecules. The fluorescence images
were recorded on a DV tape (Sony DV 180 ME DigitalVideo Cassette, Sony Corp.).

3. Results and discussion
Methylation of DNA molecules causes a difference intheir contraction process in the nanochannel (Figure 3).
The presence of methylated DNA molecules was confirmed by enzymatic digestion; the methylated DNA
molecules had one band, while the non-methylated
DNA molecules had several bands (Figure 3(a)). After
single DNA molecules were introduced into and elongated in the nanochannel by applying the electric field(Figure 3(b)), the time-course contraction process of asingle methylated and a single non-methylated DNA
molecule in the nanochannel was obtained as shown in
Figure 3(c). We found that the length of the ­methylated
DNA molecule after a 7-s contraction was longer thanthe length of the non-methylated one. And the data

X. SUN et al.

points of DNA length showed the contraction rate of
the methylated DNA molecule was slower than that of
the non-methylated one (Figure 3(c)). Since the exact
contraction mechanism of methylated DNA moleculesin the nanochannel must be rather complex, such DNA
contraction behaviors are an interesting subject for theoreticians and experimentalists alike.
To interpret the difference in the contraction process, we focused on the rigidity increase by methylation. It has been reported that the rigidity of DNA
molecules increases in response to DNA methylation.
[9–11] Conformational spaces restricted by steric hindrance of the bulky methyl groups trigger an increased
persistence length of methylated DNA molecules ofabout 30% with a methylation rate of 20% compared to
non-methylated DNA molecules.[12–14] In the present
study, we ­methylated all cytosine residues (C5) on 4816
CpG positions within the DNA molecules, which have
31,034 cytosine in total, using CpG methyltransferase;
the methylation rate of DNA molecules was about 15%,
and we could calculate a 23% increase of persistence
length to get the theoretical value.[10] The DNA lengths
after the 7-s contraction of non-methylated and methylated DNA molecules were 11.06 μm and 13.58 μm,
respectively (Figure 3(c)); this was a 23% increase in thecontracted length, which was the same per cent increaseas the theoretical increase of the persistence length witha methylation rate of 15%. The contracted length of DNA
molecules has been reported to have a relationship withthe persistence length,[15] and therefore our explanationof the contracted length increasing using persistence
length makes sense.
Seven-second contraction of non-methylated and
15% cytosine-methylated (100% CpG-methylated)
DNA molecules showed a significant difference of thecontracted length around 2.5 μm. Since the spatial resolution of optical microscopy is limited to c.200 nm,[16]we might find a significant difference of the contracted
length between non-methylated and 8% CpG-methylated
DNA molecules. And even if we use stimulated emission depletion condition with optical resolution of
70 nm,[17] we might distinguish the contracted lengthof non-methylated from 2% CpG-methylated DNA molecules. The report for methylation frequencies in humanmalignancies shows that a possibility for 8% methylation
discrimination is good enough for various cancers prediction in their early stage.[5] This kind of prediction has
enormous significance in using DNA molecules with atleast 100 kbp due to an importance of ensuring genome
information integrity.[18,19]
In order to explain the difference in the contraction
process of methylated and non-methylated single DNA
molecules, it can be plotted using the following equation
[20]:
)
(
5kB TD1∕3tl(t) = le + (l0 − le ) exp −(1)
8𝜋𝜂L2 (pw)2∕3

Sci. Technol. Adv. Mater. 17 (2016) 648X. SUN et al.

Figure 3. Contracted length analysis of T4 DNA molecules in the nanochannel. (a) Non-methylated and methylated T4 DNA molecules
were digested by using the restriction enzyme Sall. Methylated DNA molecules showed a resistance to enzymatic digestion.
(b) The contraction process of a single DNA molecule was observed with the inverted fluorescence microscope after switching off
the electric field. The florescence images of the contraction process for a non-methylated and a methylated single T4 DNA molecule
inside the nanochannel. Scale bars are 10 μm. (c) Data points for average length of non-methylated (blue) and methylated (red)single T4 DNA molecules inside the nanochannel plotted as a function of time. Red and blue dotted lines showed fitting curves from
Equation (1). Error bars show the standard deviation for a series of measurements (n = 5).

where le and l0 are the equilibrium length and the initiallength in the nanochannel, respectively, η is the viscosityof the solvent, L is DNA contour length, p is the DNA
persistence length, w is the DNA molecule width, kB
is the Boltzmann constant, and D is channel diameter.
Using fitting curves based on the above equation, we
calculated the coefficients in the exponential expression
as 0.303 and 0.348 s−1 for methylated and nonmethylated DNA molecules, respectively. An inverse of
the coefficients in the exponential expression showed
relaxation times for methylated and non-methylated
DNA molecules of 3.300 and 2.874 s, respectively.
From the fitted and calculated relaxation time, we also
calculated a 23% increase for persistence length, andthese results highlighted that the contraction rate ofmethylated DNA molecules was slower than that ofnon-methylated DNA molecules in the nanochannel;
and therefore, our methodology could distinguishmethylated and non-methylated DNA molecules
based on a difference of the relaxation time related to adifference of the persistence length.
Since methylation of DNA molecules changes thecontracted length and the contraction process, the fluorescence intensity profile of DNA molecules may beable to show methylation positions. Considering methylation causes relatively long contracted length andslow contraction process, we suppose that fluorescenceintensity profiles reveal methylation location of DNA
molecules, approximately. This kind of measurement
allows researchers to identify approximate methylation
location and distinguish methylated or non-methylated
DNA molecules. As for cancer prediction in early stages,
detecting methylated rate increase abnormally is more
important than reading out sequence information, andtherefore our proposed measurement would contributeto first screening of DNA methylation analysis.

Sci. Technol. Adv. Mater. 17 (2016) 6494. Conclusions
In summary, we demonstrated that our nanochannel
device is a powerful tool to detect DNA methylation without any protein modification at the level of a single DNA
molecule. It assists in detecting DNA methylation witha small amount of sample and in a relatively short time
including pretreatment and analysis (approximately 2 h).
We use the contracted length and contraction rate ofa single DNA molecule to determine whether or not a
DNA molecule is methylated, and identify methylation
location of DNA molecules, approximately. Our methodology allows users to analyze intact DNA moleculesin nanochannels. We expect that our device will become
an important part of biomolecule analysis systems.

Competing financial interests
The authors declare no competing financial interests.

Funding
This research was supported by the Cabinet Office,
Government of Japan and the Japan Society for the Promotionof Science (JSPS) through the Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST
Program), the ImPACT Program of the Council for Science,
Technology and Innovation (Cabinet Office, Government ofJapan), Nanotechnology Platform Program (Molecule andMaterial Synthesis) of the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), Japan, the JSPS
Grant-in-Aid for Scientific Research (A) [16H02091], and
the Grant-in-Aid for Scientific Research on Innovative Areas‘Nanomedicine Molecular Science’ [number 26107709] from
the MEXT and PRESTO, Japan Science and Technology
Agency (JST).

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