Articles
https://doi.org/10.1038/s41565-021-01008-w

De novo design of a nanopore for single-molecule
detection that incorporates a β-hairpin peptide
Keisuke Shimizu1, Batsaikhan Mijiddorj2,3, Masataka Usami1, Ikuro Mizoguchi1, Shuhei Yoshida4,
Shiori Akayama4, Yoshio Hamada 4, Akifumi Ohyama5, Kenji Usui 4, Izuru Kawamura 2,5 andRyuji Kawano 1 ✉The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo
design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides
with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a β-hairpin
structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely
artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to
6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a
glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a β-hairpin peptide has the potential to create artificial nanopores,
which can be size adjusted to a target molecule.

T

he folded structure of proteins is determined by their linear
polypeptide sequence, as postulated in Anfinsen’s dogma1,
and gives rise to specific protein functionality. All proteins
have a unique structure and size. The folded structure relies on the
primary sequence of the amino acids, while this unique primarystructure is the result of structural evolution such as the mutationand selection of amino-acid residues over time. To reveal the relationship between this primary information and protein structure is
one of the ultimate goals of science.
The de novo design of the primary sequence of artificial proteins has been studied in the last four decades2–4, and recently has
also been described as design from scratch5–11. In early studies, thesecondary structures of proteins—α-helix and β-sheet structures—
were created synthetically by peptide chemistry12, with these secondary structures subsequently connected through a loop sequence
to construct the more complicated three-dimensional structure13.
Although the design strategy at the time was manual and basedon the physical model of proteins and peptides, computational
design has recently emerged. Baker and coworkers have proposed
extensive artificial proteins with computational design, such as afluorescence-activating protein with a β-barrel14 and transmembrane proteins with α-helical15 structures. The de novo design has
the potential not only to mimic natural proteins, but also to create artificial devices such as molecular machines. In the creationof manufactured devices for practical applications, pore-forming
transmembrane structures are meaningful targets because single
molecular detection and DNA sequencing have been achieved using
such pore-forming proteins and peptides16–19.
Nanopore sensing is a powerful tool for label-free single-molecule
detection20,21. Once a nanopore-forming membrane protein has
been reconstituted in a lipid bilayer to form a nano-sized pore,
the target molecule is able to electrophoretically pass through thenanopore under an applied voltage. A wide variety of applications
have been proposed such as DNA sequencing22, small molecule
detection using an adapter23 or DNA aptamer24, nanopore mass
spectroscopy25, decoding of DNA computations26–28 and protein or
peptide detection29,30. The choice of the applicable target molecule
is sometimes limited because the selectivity of nanopore sensing
mainly depends on the pore size and chemical properties, and thesize variation of natural pore-forming proteins is insufficient for thedetection of a range of molecules31. Bottom-up nanopore design
has great potential to expand target variation, owing to the possibility to tailor size compatibility between the nanopore and target
molecules. Moreover, this can offer improved accuracy of nanopore
detection, with a potentially substantial influence on amino-acidsequencing of proteins.
In designing an artificial protein nanopore, the process of pore
insertion into the lipid membrane must also be considered. In thecase of a natural system, membrane proteins are inserted into thecell membrane via chaperones or endoplasmic reticulum export.
One way to facilitate membrane insertion is to use short peptides. For example, an α-helical barrelled peptide (35 amino acids)
based on the Wza protein has previously been redesigned fromthe wild type and assembled to form monodisperse nanopores inlipid membranes18,19.
Here, we focus on the β-barrel structure of peptides because
the transmembrane region of most biological nanopores, including α-hemolysin (αHL), has a β-barrel structure. A transmembrane
peptide with a de novo design incorporating a β-hairpin structure
was chemically synthesized using an isoacyl dipeptide method32.
Our designed β-hairpin peptide, named SV28, assembled andformed a β-barrel structure with several different sizes of nanopore
ranging from 1.7 to 6.3 nm in diameter. To construct the monodispersely sized pore by designing the amino-acid sequence of the

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology (TUAT), Tokyo, Japan. 2Graduate School of Engineering,
Yokohama National University, Yokohama, Japan. 3School of Engineering and Applied Sciences, National University of Mongolia, Ulaanbaatar, Mongolia.
4
Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe, Japan. 5Graduate School of Engineering Science,
Yokohama National University, Yokohama, Japan. ✉e-mail: rjkawano@cc.tuat.ac.jp
1

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ArticlesaNature Nanotechnology
b

c

d

Water
Lipid

Water
Lipid

Water
Lipid

Water
Lipid

Water

Water

Water

+
Water

β-hairpinVβ-turn structureobphIon path–

Anchoring

Orientation
controlHO

Terminal structure+

2 a.a. ×2
28 a.a.

Snorkelling effectAmphiphilic arrangement

f

e
S D
G
G
D
Y
Y
S
S
S
S
Y
Y
V
V
S
S
S
S
V
V
V
V
S
S
S
S
V
V
V
V
S
S
S
S
V
V
Y
Y
S
S
S
S
Y
Y
G
G
G
G
R
R
R
R
Water

Water
Lipid

Anti parallel β-sheet

Assemble

β-hairpin motif

D

A

ro

ph

ilic

ro
10 a.a. ×2

D Negative charge
R Positive charge

Y Aromatic ringhydrogen bond donor

Hy
d

Hy
d

β-sheet structure+

Hydrophobic

S Hydrophilicic

4 a.a.

––

Terminal chargesg
SV14(half strand)

S D

SV28
(full strand)

N
N

C

N

C

C

N

C
0 ns

500 ns0 ns

500 nsFig. 1 | Design strategies and structural confirmation of the β-hairpin peptide. a, The design of β-hairpin peptide with 28 amino acids (a.a.) divided into
three sections: β-turn, β-sheet transmembrane and the terminals. b, Hydrophilic and hydrophobic amino acids are arranged in an alternating fashion.
c, Interaction of aromatic rings stabilizes the β-barrel pore. d, Designing specific charges at the terminus allows control of peptide orientation on an
applied voltage. e, Amino-acid sequence of the designed structure named SV28. The black lines indicate the hydrogen bonding. f,g, MD simulations of
the monomer structures. The 0 ns (left) and 500 ns (right) snapshots of the half-length (f) and full-length (g) SV28 in a lipid bilayer membrane.

SV28, we introduced a glycine mutation into the strand named
SVG28. The SVG28 forms a 1.7 nm pore monodispersely and can
detect a single polypeptide. The de novo design of β-barrel nanopores has great potential, with the ability to adjust size and shape toa target molecule, and with applications in the detection of DNA orvarious other molecules.

Design of the β-hairpin peptide

We designed the β-hairpin structure with three different regions ofamino-acid sequence: a β-strand backbone, a β-turn and two terminal structures (Figs. 1a–d). We first considered the length of thepeptide. The β-strands are necessary to have appropriate length in
the transmembrane region. We decided to use ten amino acids toprovide a length compatible with the thickness of a lipid bilayer,
because this is similar to the β-strand length of natural transmembrane β-barrel proteins33. The β-turn can form a bent structurewith four amino acids34. The terminal structures require two aminoacids with a random coil structure in the extra-membrane regions.
Overall, a length of 28 amino acids was selected for use in this study(Fig. 1a). The following three strategies were then used to determine
the type of amino-acid residue in each region.
(1) Construction of amphiphilic β-sheet structure as the β-strand
backbone. Alternation of hydrophilic and hydrophobic aminoacids was used to promote formation of the β-sheet structure(Fig. 1b)35. Separation of the hydrophilic and hydrophobic
surfaces to be inside and outside the nanopore facilitates construction of the β-barrel structure in the lipid bilayer. Ser was
selected as the hydrophilic residue because it is the smallest
hydrophilic amino acid and may prevent steric hindrance innanopore formation. Although Ala is the smallest hydrophobic
amino acid, it tends to form an α-helical structure and so Val
was selected instead as the hydrophobic residue.
(2) Stabilization of the membrane-spanning state using the snorkelling effect. In natural α-helix and β-barrel membrane proteins, Trp and Tyr are sometimes localized at the interface
between the aqueous and lipid phases, enhancing stability ofthe transmembrane structure36, also known as the snorkelling
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11-merSide view40

Top view

11-merPore diameter (Å)

a

5-mer

30
20
10
5-mer
0

d

e

20

δ (mdeg)

Residue

25
15
10
5

Residue25

fWavelength (nm)

2
1
0
–1
–2
–3
–4
–5
–6
–7

220

240

230

200

0

lipid
C=O

250

lipid
C=O

Not transferred
Transferred

190

20

180

400
600
Time (ns)800

Val10 13C’

Tyr12 13C’

170

160

13

C chemical shift (ppm)

g

15

h

15

N Val2210

13

Val10 ( C')

5
0

200
Coil
Bend

400
600
Time (ns)β-sheet
Turn

800

Val10

15

4.4 Å

N Val20β-bridge
3-helix

Val22
15

Val22 ( N)

160

140

120

100

80

15

N chemical shift (ppm)Fig. 2 | Prediction and confirmation of the assembling structure of the SV28 peptides in the lipid bilayer membrane. a, The last snapshot of 5-mer SV28
in a DOPC membrane in the MD simulation. Ribbons show the peptide structures, with the secondary structure indicated by the colour of the ribbon (red,β-sheet; yellow, turn and white, random coil structure). Ribbon arrows indicate the direction of the backbone from N to C terminal. Cyan lines indicate
water molecules, and the lipid molecules were omitted for clarity (excluding phosphorus atoms as orange spheres). Green and white spheres indicate
the potassium and chloride ions, respectively. b, MD simulation of the SV28 nanopore formation in the DOPC lipid membrane. The 11-mer nanopore wassimulated for 900 ns after 100 ns of initial simulation. Brown, lipid head; green, potassium ions and grey, chloride ion. c, Central diameters of 5-mer (black)and 11-mer (red) pores as a function of time were calculated using HOLE software at 1 ns intervals. d, Profile of the secondary structure of 5- and 11-merpores as a function of time. e, Circular dichroism spectra of non-transformed SV28 (red line) and transformed SV28 (blue line). f, 13C CP-MAS NMR
spectra of the triply isotope-labelled SV28 ([1-13C]Val10, [2-13C]Gly16, [15N]Val22-labelled and [1-13C]Tyr12, [2-13C]Gly16, [15N]Val20-labelled SV28) in
DOPC liposomes. The black lines indicate the spectra from DOPC liposomes, and the red lines indicate the spectra of isotope-labelled SV28 with DOPC
liposomes. g, 15N CP-MAS NMR signals of Val20 and Val22 of SV28. h, The β-barrel structure of SV28 nanopore. The interatomic distance between the
backbone amide nitrogen of Val22 and the carbonyl carbon of Val10 in the SV28 is estimated to be around 4.4 Å as measured by solid-state NMR.

effect37. On the basis of this information, we selected Tyr, with
its location carefully chosen at positions 4, 12, 18 and 26 fromthe N terminus (Fig. 1c).
(3) Introduction of charged residues for controlling the orientationof SV28 using an applied voltage. Antiparallel β-sheets have morestrong interactions than parallel β-sheets. To control the peptide
orientation for forming the antiparallel β-sheets structure, two
negatively and positively charged residues were introduced atthe β-turn and the terminal regions, respectively (Fig. 1d). The
β-turn consists of four amino acids and the systematic study ofthe β-turn sequence has been previously reported34, so we decided on the sequence of –DSDG–. The N- and C-terminal regions
were designed as RG– and –GR, respectively. Gly is the linker between charged Arg residues connecting to the β-sheet backbone.
The final sequence of SV28 is shown in Fig. 1e. The β-hairpin
formation of the SV28 sequence was computationally confirmed
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using the MINNOU simulator, software that predicts transmembrane domains of membrane proteins and peptides38.
We verified the formation of the nanopore structure fromassembled half- and full-length of SV28 to confirm the stabilityof the β-hairpin structure in the membrane (Fig. 1f,g) by all-atom
molecular dynamics (MD) simulations. The full-length SV28
monomer kept the β-hairpin structure, while the half-length mostly
showed random coil secondary structures during these simulations(Supplementary Fig. 1). To confirm the β-barrel formation of SV28
peptides, we performed 1,000 ns MD simulations of 5- and 11-merpores in a dioleoylphosphatidylcholine (DOPC) membrane. Figure 2aand Supplementary Fig 2 display the final snapshots of thesesimulations, with the 11-mer structure also shown in Fig. 2b. Bothsimulations demonstrated stable nanopore structures. The 5-merpore was slightly tilted in the membrane in Fig. 2a, which may indicate the adaptation of the structure to the movement of surrounding lipids. The central diameter of the pores was calculated at 1 ns

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a

b

50 pA

10 pA

1s

0.5 s
Baseline

BaselinecdSquare-top

Step

100 pA

200 pA1s

200 pA

200 pA
0.5 s

e

0.5 s

1s

f
Unstable

100
90

70

Unstable pore
15

Count

60
50
40
30
20

Applied voltage (mV)
Incubation condition

+100

+150

+200

Cholesterol

0%

0%

0%

10
3

0%

4

5

n ≥ 100+200+200
24 h/37 °C 24 h/37 °C

n = 106

5

0
–5

0

5

10

15

20

25

30

Initial open conductance (nS)

20%

h

g 5

5
4

y = 0.014x 2 – 0.11x

Pore diameter (nm)Conductance (nS)2

Stable pore10

2

R = 0.87

3

1

20

StableSignal appearance ratio

80

4Erratic

Multi-level2
1

y = 0.20x – 1.0
R2 = 0.85

3
2
1

0
0

10

20
Number of strands

30

0
0

10

20
Number of strands

30

Fig. 3 | Evaluation of the stability and conductance of the SV28 nanopores using channel current measurements. a, The typical current and time tracesof SV28 at +100 mV with initial condition. b, The step signal was occasionally observed under the initial conditions. The traces were atypical before
optimization of the conditions. c, Step and square-top signals were defined as stable pore formation. d, Multi-level and erratic signals were defined as
unstable pore formation. e, The ratio of stable and unstable pore formation from the channel current analysis. f, The histogram of the current conductanceof the initial step from the 0 A increase of SV28 in the DOPC lipid bilayer. g,h, The relationships in β-barrel membrane proteins OmpA, OmpG, OmpF, VDAC
and FhuA between the channel conductance (g) and the number of β strands and the pore diameter and number of β strands (h).

intervals, as shown in Fig. 2c and Supplementary Fig 2. The average values are equal to 10.1 ± 0.4 and 28.5 ± 2.0 Å for 5- and 11-merpores, respectively.
Furthermore, the secondary structure profiles of two monomersof the pores as a function of time are shown in Fig. 2d. The tenth(Val10) and 22nd (Val22) positions form a β-sheet structure, andthe 16th position (Gly16) forms a turn structure while the turnsequence seems to be slightly shifted. To compare with the experimental results, we calculated the distance between the nitrogen of
Val10 and carbon of Val22 during the simulations (Supplementary
Fig. 3a). The average distances of the 5- and 11-mer peptides are

4.2 ± 0.1 and 4.1 ± 0.1 Å, respectively. These distances are consistent
with the usual value of the distance in the β barrels of outer membrane proteins. MD simulations of other oligomerized pores, 7- and16-mer, are also shown to give stable pore formation as shown in
Supplementary Fig. 3b,c.

Synthesis of a hydrophobic β-hairpin peptides using
isoacyl dipeptide

Conventional solid-phase synthesis and subsequent purification of SV28 is challenging because the β-sheet structure is prone
to aggregation. A method using isoacyl dipeptides32 gives the
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aqueous-soluble SV28 preform; the final chemical structure
can be derived from this preform after transition from the side
to the main chain in the isoacyl peptide (Supplementary Fig. 4).
In this way, it was possible to synthesize and purify SV28 using
the conventional peptide synthesis method (Supplementary
Fig. 5). The ester bond was transferred to a peptide bond byincubation in a basic solution (pH 13) for 5 min before using
the peptides in the experiments. Acyl migration was confirmed
using reversed-phase high-performance liquid chromatography(Supplementary Fig. 6).
The secondary structure of the transferred SV28 precursors
with DOPC liposomes was confirmed by circular dichroism spectroscopy under similar conditions to the following channel current
measurements. The SV28 preform did not show any large negative
Cotton effect at 216 nm (Fig. 2e and Supplementary Fig 7). After thetransference of isoacyl dipeptide, a large negative Cotton effect at
216 nm was observed (Fig. 2e and Supplementary Fig 7), indicatingthat the transformation of isoacyl dipeptide facilitated the formation of a β-sheet structure in SV28. Moreover, after 24 h incubation
in a buffer solution, the peptides were seen to maintain the β-sheet
structure (Supplementary Fig. 7).
The solid-state nuclear magnetic resonance (NMR) measurements also supported the proposed conformation of SV28 in DOPC
liposomes. 13C and 15N NMR signals of [13C]Val10, [13C]Gly16(Fig. 2f and Supplementary Fig 8), and [15N]Val22 were observed
at 174.2, 45.3 and 124.1 ppm (Fig. 2g), corresponding to the β sheet
and the random coil structure at the transmembrane (Val10 and
Val22) and the β-turn (Gly16) region39. Next, we confirmed theformation of hydrogen bonding between two β strands using Val10
and Val22 (Fig. 2h), with the distance between them also checked by
MD simulation as mentioned above. Rotational echo-double resonance was used to estimate the interatomic distance between the
carbonyl carbon of Val10 and amide nitrogen of Val22 in SV28 to
be around 4.4 Å (Supplementary Fig. 9), indicating the formation ofhydrogen bonds between the β strands.
The NMR-deduced structure approximately corresponds to thestructure predicted by the MD simulation. Overall, the information
indicates the formation of the β-hairpin structure of SV28.

Confirmation of pore formation using channel current
measurements

The pore-forming properties of SV28 were examined by the channel current recording in our lipid bilayer system (Supplementary
Fig. 10)40. Several pore-opening states in which the current was
raised, but did not plateau, were observed in the DOPC lipid
bilayer under +100 mV (Fig. 3a). Sometimes step-like signals wereobserved (Fig. 3b). Other different shapes of current signals werealso observed. We have previously proposed current signal classification for several different signal shapes, and assigned these to various pore-forming models of α-helical peptides41–43. In this study, we
also classified these signals into four types of current signal: step,square-top (Fig. 3c), multi-level and erratic (Fig. 3d). The definition of signal classification is described in Supplementary Fig. 11.
To estimate the pore-forming behaviour of SV28, we here definethat step and square-top signals reflect stable pore formations, andmulti-level and erratic signals reflect unstable pore formations. In
the initial measurement of SV28, the stable signals were observed
only 3% of the time (Fig. 3e, left bar), which led us to investigate
how to improve stable pore formation. To optimize formation of
stable pores, we investigated three different conditions as follows(Fig. 3e and details in Methods):(1) Optimization of applied voltage to align the SV28 monomers.
(2) Incubation of SV28 monomers to form oligomer structureswith the lipid monolayer surface.
(3) Adding cholesterol to the lipid bilayer.
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Table 1 | Table of conductance values showing the numberof monomers, strands and the pore diameters of the SV28
nanopores
Peak no.

Conductance (nS)

Monomer no.

Strand no.

Diameter (nm)

1

1.1

6.9

13.8

1.7

2

3.3

9.9

19.8

3.0

3

7.0

13.3

26.6

4.4

4

10.5

15.8

31.6

5.4

5

14.4

18.2

36.4

6.3

Combining the three optimized conditions led to a further
increase in the ratio of stable signals to 67% (Fig. 3e, right). Thisratio is close to the ratio we previously reported for pore-forming
proteins31. The other typical current signals and step signals areshown in Supplementary Figs. 12 and 13.
The pore diameter of the SV28 nanopore was calculated using
the conductance of the open channel state. The open channel conductance was determined as the initial step signal from the baseline(≅0 A). The histogram of the pore conductance of SV28 is shownin Fig. 3f. Several peaks are observed in this histogram, with fiveidentified peaks picked by the second deviation method. The peakconductances at 1, 3, 7, 11 and 14 nS are identified. We estimatedthe pore size using the experimental results instead of the theoretical Hille model. We assessed the relationship between the current
conductance and the pore diameter of β-barrel proteins, measurements that were taken by electrophysiology and by crystallographyusing microscopes. OmpA, OmpF, OmpG, VDAC and FhuA wereused in this estimation as the β-barrel transmembrane proteins. On
the basis of the relationship between the current conductance andthe pore diameter of the natural proteins (Fig. 3g,h), the five different pore sizes and the number of monomers in the case of SV28
are approximately estimated as 1.7, 3.0, 4.4, 5.4 and 6.3 nm with 7,
10, 13, 16 and 18 monomers of SV28, respectively (Table 1). The
experimentally estimated pore sizes are larger than those from thetheoretical Hille model.

Detection of double-stranded DNA and G4 structure of
DNA using the SV28 nanopore

We used SV28 with a pore diameter of around 5.4 nm and attempted
to detect double-stranded DNA (dsDNA) with lengths of 50 basepairs (bp) and 1 kilobasepairs (kbp). Although the translocation signal was not discernible by the inherent blockage of the pore in thecase of 50-bp dsDNA measurements at 1 and 10 μM concentrations(Supplementary Fig. 14), the blocking currents were consistently
observed using 1-kbp dsDNA ranging from 50 to 200 nM (Fig. 4a andSupplementary Fig 15). The scatter plot of the blocking rate and duration time of translocation at 100 nM under application of 100 mV isshown in Fig. 4b, with peak values of 195 pA and 1.8 ms, respectively(scatter plots without bootstrapping are presented in Supplementary
Fig. 16). Additionally, the translocation of multiple dsDNA throughthe pore was occasionally observed (Supplementary Fig. 17). We next
examined the dependency of the translocation frequency of dsDNA
on the concentration and applied voltage. The event frequency versus concentration showed a linear dependency ranging from 50
to 200 nM under the application of 80 mV (Fig. 4c). Moreover, theevent also depended exponentially on the voltage application from40 to 100 mV at concentrations of both 100 and 200 nM (Fig. 4d).
These linear and exponential dependencies on the concentration
and applied voltage are consistent with previously reported resultsof DNA translocation using the αHL nanopore44. The voltage dependency of the duration time did not show a simple decrease with voltage application, but had a peak voltage (Fig. 4e). A similar result has

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b 260

100 pA

Blocking current (pA)

a

220
+
180

20 s

n = 55140
0.5

dn > 100

y = 0.05x + 0.56
R 2 = 0.99

12
8

80 mV
4
0
0

50

100

150

200

250

e
50
40
30

y = 0.27 × 10
R 2 = 0.98

20

y = 0.04 × 10
R 2 = 0.99

10
0
20

40

G

G
G

+
G M
+
G M

80

100

4

50 nM

3
100 nM2

200 nM

1
0

120

30

40

50

60

70

80

90

Applied voltage (mV)h

A38TTAGGG20TT

G
G
G
G
G
G

n > 33y = 0.012x + 7.39
R 2 = 1.00

8
Duration time (ms)

3.0 nmG

60

0.064 xn > 100

Applied voltage (mV)g

2.7 nm

200 nM

0.046 x

100 nM

dsDNA concentration (nM)f5

n > 50Duration time (ms)16

Event frequency (events (s–1))

Event frequency (events (s–1))

c

1.5
2.5
Duration time (ms)Poly A tail

7
6
5
4
3

G-quadruplex hybrid fold

100

150

200

Applied voltage (mV)

0.2 s

300 pAj

0.2 s

Blocking current (pA)

300 pA

1,000

w/ G4 100 mV
w/ G4 150 mV
w/ G4 200 mV
w/o G4 100 mV

+ ++

100
+
n > 33
10
2

3

4

5

6

Duration time (ms)78Event frequency (events (s–1))

l

k

i

120

n > 33100
80
60
y = 7.45 × 100.012 x
R 2 = 0.92

40
20
0

100

150

200

Applied voltage (mV)Fig. 4 | The dsDNA (1 kbp) translocation through the SV28 nanopore with diameter of around 5 nm. a, The current and time trace of the SV28 nanopore
with 1 kbp dsDNA (100 nM) under application of 40 mV. The dashed lines indicate the threshold for the dsDNA translocation events. b, The scatter plotof the translocation data of 100 nM and 100 mV after bootstrapping. c, The event frequency of the translocation as a function of the concentration of
dsDNA. d, The event frequency of the dsDNA translocation as a function of the applied voltage. Blue and orange lines indicate the dsDNA concentration
at 100 and 200 nM to guide the eye. e, Duration time of dsDNA with 50, 100 and 200 nM dependence on the applying voltages. Blue, orange and green
lines indicate each concentration to guide the eye. f,g, Schematic structure of G4 and (f) five G4s (g) in the hybrid hold in a series. h, Duration time of
the G4 dependence on the applying voltages at 100, 150 and 200 mV. i,j, The current and time trace of the SV28 nanopore with (i) and without (j) G4.
k, Scatter plots of the blocking current and duration time after bootstrapping of DNA with (w/) G4 and without (w/o) G4 structure. The peak tops of eachcondition with G4 are (duration, blocking current of 4.24 ms, 669 pA) at 100 mV (4.07 ms, 797 pA), 150 mV, (3.72 ms, 678 pA) 200 mV and without G4(4.05 ms, 62 pA) at 100 mV. f, The duration time of G4 (2 μM) as a function of the applying voltages at 100, 150 and 200 mV. l, The event frequency of thetranslocation of G4 (2 μM) as a function of the applied voltages. The lines in c–h,l show the results of fitting by liner or exponential models.

previously been reported, and may be due to rejection of the target
molecule from the pore under low voltage application45. The scatterplots from all data are shown in Supplementary Fig. 18.

Since the SV28 forms several different sizes of nanopore, here we
detect the G4 structure of DNA using a large SV28 nanopore (6.4 nm).
The G4 DNA forms a hybrid hold with a 3.0 × 2.7 nm2 box-like
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c

d

100

Unstable

a

90
20

S D

D
G
Y
S
S
Y
V
S
S
V
V
S
S
G
S
V
V
S
G
S
V
Y
S
S
Y
G
G
R
R

80
70
60
50
40

10

30

Top view

20
5n = 88n = 100

0–4 –2 02

4 6 8 10 12 14 16 18
Conductance (nS)5s

1s

Blocking conductance (nS)

Duration time (ms)

i
40
30

y = 42.9 × 10–0.007 x
R 2 = 0.996

20n > 29600

50 100 150 200
Applied voltage (mV)

250

0.8
0.6
0.4
0.2

50 mV
100 mV
200 mVn > 200

0
20

40
60
Duration time (ms)6Signal to noise ratio

Blocking conductance (nS)

1

l
0.8
0.6
0.4

L-PLL
S-PLL

0.2

n > 100

0
0

5
10
15
Duration time (ms)

SVG28
+200

SVG28
+50

24 h/37 °C 24 h/37 °C
20%
20%

30
25

20

5
4
3
2
1
0

n=5
αHL

SVG28

80

y = 2.22 × 100.011 x
R 2 = 0.971

20
15
10
5

n > 296

0
0

j

0

k

24 h/37 °C
20%Event frequency (events (s–1))

100 pAh

Incubation condition
Cholesterol

50

100 150 200 250

Applied voltage (mV)

Event frequency (events (s–1))

0.5 nS

SV28
+200

g

f

e

10

PeptideApplied voltage (mV)1612
y = 0.016x + 7.28
R 2 = 0.982

8
4n > 14800m

Event frequency (events (s–1))

G Glycine-kink

10

Stable

Count

15

200
400
600
L-PLL concentration (nM)

12
10
8
6
4
2
0

n > 27
αHL

SVG28Fig. 5 | The SVG28 structure and sensing capability for a poly-arginine. a, Sequence of the SVG28 that has G-kink into the transmembrane region.
b, The structure of the SVG28 in a DOPC membrane in the MD simulation from side and top views. c, The histogram of the current conductance of theinitial step from the baseline level (0 A) increase of SVG28 in the DOPC lipid bilayer. d, The ratio of stable and unstable pore formation from the channel
current analysis with comparing to the SV28 and the SVG28. e,f, Typical current and time trace of the SVG28 nanopore with L-PLL (f) and without L-PLL(e). g, The event frequency of the L-PLL detection as a function of the applied voltages. h, Duration time of the L-PLL detection dependence on the applying
voltages at 50, 100 and 200 mV. i, The scatter plot of the L-PLL detection in 100 nM: the blocking rate and the duration after the bootstrapping. j, The
event frequency as a function of the concentration of the L-PLL. k, The scatter plot of the L-PLL and shorter length of poly-l-lysine (molecular weightroughly 10,000) detection in 250 nM: the blocking rate and the duration after the bootstrapping. l, Comparison of the signal to noise ratio on the L-PLL
detection between αHL and SVG28 nanopore. m, Comparison of the event frequency on the L-PLL detection between αHL and SVG28 nanopores.

structure and the G4 structure is strung out as five boxes in this saltcondition (Fig. 4f,g)46,47. In the case of the G4 structure, the blocking signals are observed, although the same length of DNA without
G4 structure is not recognized as shown in Fig. 4i,j. The scatter plots
with and without the G4 structure are markedly different (Fig. 4k).
The peak top of the blockade of the G4 structure is 669 pA at 100 mVof applied voltage. The ratio of the blocking amplitudes between
dsDNA and G4 is IdsDNA/IG4 = 0.3 (195/669 pA). This value roughly
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consists of the ratio of their molecular volume: VdsDNA/VG4 = 0.4(12.5 nm3/29.2 nm3 and 4 nm long). The dwell time and translocation
frequency of G4 depend on the applied voltages (Fig. 4h,l). The areaof the scatter plots at different voltages is seen to occur at in a different location in the graph in Fig. 4k. These scatter areas are also
located at different positions to the inherent current blocking by the
SV28 nanopore (Supplementary Fig. 19). The SV28 nanopore candiscriminate the human telomeric DNA at the single-molecule level.

Articles
Re-design of SV28 with a glycine-kink and poly-lysine
detection using the SVG28 nanopore

Although the SV28 nanopore forms five different-sized pores andcan detect differently sized target molecules, the construction of amonodisperse sized pore is a significant challenge in the de novo
design. To realize monodisperse pore formation, we redesigned theβ-hairpin peptide by introducing a Gly-kink into the transmembrane region of the SV28, named SVG28 (Fig. 5a and Supplementary
Fig 4b). A Gly-kink is an intrinsic residue in both natural and de novo
proteins with a β-barrel structure14,48 that increases the local β-sheet
curvature, resulting in stabilization of the β-barrel conformation14.
We initially confirmed the pore structure of the SVG28 using MD
simulation (Fig. 5b and Supplementary Fig 20) and synthesized itusing the isoacyl dipeptide method (Supplemental Informationand Supplementary Figs. 4f, 5d and 6c). After the transference of
isoacyl dipeptide, a large negative Cotton effects at 216 nm was also
observed in circular dichroism (Supplementary Fig. 7c), indicating
the formation of a β-sheet structure in SVG28.
In the channel current measurements, the SVG28 showeda step signal with a single-channel conductance (Fig. 5c,e andSupplementary Fig 21), indicating more monodisperse pore formation while large pores were rarely observed at the lower voltage application. The main channel conductance of the SVG28
pore (g = 1.0 nS) almost corresponds to the smallest pore of the
SV28 (peak no. 1 g = 1.1 nS, pore diameter, 1.7 nm in Fig. 3f and
Supplementary Fig 21f), and the size of the pore can be approximately estimated as 1.7 nm with the same assessment using naturalprotein pores that are predicted to form a 7-mer pore. Moreover, theratio of the stable pore formation of SVG28 was higher than that of
the SV28 nanopore (Fig. 5d); the ratio reached around 90%, whichis almost the same as the alpha-hemolysin (αHL) nanopore31. The
results support the hypothesis that a G-kink makes a flexure in theβ strand and stabilizes the β-barrel structure. In addition, the sizeof the 7-mer SVG28 nanopore was estimated to be around 1.3 nm
using MD simulations (Supplementary Fig. 20).
We next attempted to detect a polypeptide chain using thesize-compatible SVG28 nanopore, because the next main targetof nanopore sensing is in the amino-acid sequencing of proteins49.
The translocation-like blocking events of a poly-l-lysine (L-PLL,
molecular weight 30,000–70,000) at 100 nM were observed under
an applied voltage of 100 mV (Fig. 5f and Supplementary Fig 22).
The blocking event frequency (Fig. 5g) and the duration time
(Fig. 5h) depended on the applied voltage as well as the SV28 nanopore measurements. The scatter plots of the current blocking andduration also show the voltage dependency of the L-PLL measurement (Fig. 5i), and the event frequency also depended on the concentration (Fig. 5j). These results indicate that the SVG28 nanopore
can detect L-PLL as a nanopore sensor. Furthermore, the shorter
length of poly-l-lysine (molecular weight roughly 10,000) can also
be detected using the SVG28 nanopore (Fig. 5k and SupplementaryFig 22h). Next, we assessed the capability of polypeptide detection
compared with the αHL nanopore, which is the most popular nanopore protein for single-molecule detection. Figure 5l,m presents thesignal to noise ratio of the blocking events and the event frequency
in the L-PLL measurement. Both detection parameters of SVG28
are much better than that of the αHL nanopore for polypeptide
detection. Specifically, the event frequency in the SVG28 pore washigh, probably due to the electrostatic effect between the L-PLL andthe entrance of the nanopore. The αHL has positive charges at theentrance of the pore, which inhibit the entrance of polycations. In
contrast, the SVG28 has a negative charge at the entrance, resulting
in a high event frequency.

Conclusions

We designed a pore-forming peptide with a β-sheet structure that
forms five differently sized or monodispersely sized nanopores

Nature Nanotechnology
in the lipid membrane. A particularly pleasing result was that weachieved success with our initially designed sequence of SV28; we
did not need to adjust this initial sequence via trial-and-error experiments, and we did not need to design many different sequencesof peptides as we had initially anticipated. We consider the reason
that the reduction of the information hierarchy is one of the keys toour design; in other words, the key is a reduction of the amino-acidsequence space. Our nanopore structure was designed with several limitations and requirements: a β-sheet structure, amphiphilic
properties and strong interaction between monomers. Moreover,the length of our peptide was decided as less than 30 amino acids
so as to be suitable for chemical synthesis. These requirements limited the sequence and variety of amino acids incorporated into thetransmembrane nanopore design.
Design of the peptide nanopore provides insight into the application of nanopore sensing of DNA secondary structures and polypeptides, and also of hurdles encountered in nanopore technology.
Our design strategy could also be integrated in the creation ofmolecular machines with use as part of a molecular robot50.

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of
author contributions and competing interests; and statements ofdata and code availability are available at https://doi.org/10.1038/
s41565-021-01008-w.
Received: 6 January 2021; Accepted: 13 September 2021;
Published: xx xx xxxx

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Methods

MD simulation. The MD simulations of β-barrel structures of SV28 and
SVG28 were performed in a DOPC membrane using GROMACS-5.1.4 and
GROMACS-2021.1 (ref. 51) and Charmm36 force field52. The structural modellingand simulation details are included in the Supplementary Information.
Preparation of the bilayer lipid membrane (BLM) and peptide pretreatments.
BLMs were prepared by the droplet contact method using a microdevice53,54. First,
the DOPC (lipids/n-decane, 10 mg ml−1) solution (2.3 μl) was poured into eachchamber. Next, the buffer solution (4.7 μl) without any peptide was poured into
the recording chamber. The buffer solution (4.7 μl) with SV28 and SVG28 (final
concentration 1 μM) was poured into the ground chamber. In this study, a buffer
solution (1 M KCl, 10 mM MOPS, pH 7.0) was used. Before the measurement, theSV28 and SVG28 were added to 100 mM KOH and incubated for 5 min to allow
transfer of isoacyl dipeptide to the native dipeptide of Val and Ser. Then, HCl wasadded to make the buffer pH 7. A few minutes after adding the buffer solution,
the two lipid monolayers connected to form BLMs. When the BLMs ruptured,
they were reconstituted as BLMs by tracing with a hydrophobic stick between thetwo droplets. The solutions were prepared comprising 2 μM transformed SV28
and SVG28, 1 M KCl, 10 mM MOPS and 10 mg ml−1 DOPC or DOPC:cholesterol,
4:1 (w/w) in n-decane at pH 7. The solution was agitated in a vortex for 30 s andincubated for 24 h at 37 °C. The lipid and buffer solutions were added to the groundchamber. The details are included in the Supplementary Information.
Channel current measurements and data analysis. The channel current wasmonitored using a JET patch-clamp and PICO2 amplifier (Tecella) connectedto the chambers. Ag/AgCl electrodes were already present in droplets when
the solution was added to the chambers55. A constant voltage ranging from 40
to 200 mV was applied to the recording chamber, and the other chamber was
grounded. Pore formation in BLMs allowed ions to pass through the nanopore
under the voltage gradient, giving the channel current signal. The signals weredetected using a 4-kHz low-pass filter at a sampling frequency of 20 kHz. Analysisof channel current signals and duration time was performed using pCLAMP
v.11.0.3 (Molecular Devices), Excel (Microsoft) software and in-house programs
coded using Python. Channel current measurements were conducted at 22 ± 2 °C.
Peaks of conductance histograms were identified by the second derivative method(threshold 15%) and fitted by nonlinear and Gaussian curve fitting methods
using OriginPro8.5j (Light Stone). The scatter plots of the blocking current andduration of the DNA translocation and PLL translocation are described after
bootstrapping26. The bootstrap method is based on the resampling of the original
random sample drawn from a population with an unknown distribution. We usedthe exact bootstrap method, which used the entire space of resamples. In the exact
bootstrap method, the accuracy is verified with a sample number over 30. In thisstudy, our bootstrap procedure took the same amount of data randomly from thetranslocation data, and the mean values for these samples were calculated 65,536
times. The details are included in the Supplementary Information.
The optimization of the SV28 pore formation.
(1)

(2)

Optimization of applied voltage to align the SV28 monomers. It is proposedthat the orientation of SV28 can be controlled by applying a voltage, since wedesigned the positively and negatively charged amino acids to be positioned
at the turn and terminal regions. As predicted, the ratio of stable signals increased with increasing the applied voltage from +100 to +200 mV (Fig. 3e).
Incubation of SV28 monomers to form oligomer structures with the lipid
monolayer surface. To give an energetically stable structure of the assembled
SV28, we attempted to incubate the SV28 monomers in a mixed ‘aqueous’‘lipid/n-decane’ solution. We expected the SV28 monomers to assemble at theinterface between aqueous and lipid/n-decane solution and form a stable pore
during incubation. After incubating for 24 h at 37 °C, the ratio of the stable signals dramatically increased from 27 to 61% (Fig. 3e). A similar phenomenon(3)

was previously reported wherein amyloid β(1–42), channel-forming β-sheet
peptide, forms stable pores after incubation with lipid micelles56.
Adding cholesterol to the lipid bilayer. Cholesterol addition enhanced theformation of stable pores (Fig. 3e). This result may be explained in thatcholesterol addition results in reduced fluidity of the DOPC bilayer, andsubsequently there is reduced disturbance and pore dissociation.

Data availability

The data that support the plots in this paper and other findings of this study areavailable from the corresponding author on reasonable request.

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Acknowledgements

We thank K. Ueda at Yokohama National University for the technical comments of thecalculations. The calculations were performed by the clusters of supercomputers of the
Research Centre for Computational Science, Okazaki, Japan. We also thank N. Koga at
Institute for Molecular Science and Y. Sekiya at TUAT for the discussion of the peptide
design and A. Cooney for the language editing. This work is partially supported by the
KAKENHI: grant nos. 19H05382 and 21H00390 for the ‘Molecular Engine’ projectand grant no. 19H00901 from MEXT. B.M. thanks the Mongolian-Japan Engineering
Education Development Program (grant no. J11B16) for support.

Author contributions

K.S. and R.K. conceived the original idea and K.S. designed the pore-forming peptides.
K.S., S.Y., S.A., Y.H. and K.U. synthesized the peptides and performed the circulardichroism measurements. B.M. and M.U. performed MD simulations. A.O. and I.K.
performed solid-state NMR measurements. K.S., M.U. and I.M. perform the channel
current measurements. K.S. and R.K. wrote the entire paper. M.U., I.M., Y.H., B.M., K.U.
and I.K. wrote the experimental details and results.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41565-021-01008-w.
Correspondence and requests for materials should be addressed to Ryuji Kawano.
Peer review information Nature Nanotechnology thanks the anonymous reviewers fortheir contribution to the peer review of this work.
Reprints and permissions information is available at www.nature.com/reprints.

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