DNA Origami Gatekeepers for Solid-State Nanopores**
Ruoshan Wei, Thomas G. Martin, Ulrich Rant,* and Hendrik Dietz*
Molecular self-assembly with DNA enables the construction
of soluble objects with nanometer to micrometer scale
absolute dimensions and custom chemical features,includ-
shapes,that can open novel paths to scientific discovery.
Herein, we report on DNA nanoplates for nanopore-based
sensing approaches. Nanopores in biological or solid-state
membranes offer great potential for label-free single-mole-
cule sensing applications.Biological nanopores, such as
alpha-hemolysin, can be customized within the limits of
protein engineering.Artificial nanopores in solid-state
membranes can be made with user-defined dimensions, but
chemical modifications require substantial effort.A chal-
lenge in the field is to gain control over both the geometrical
and chemical specifications of nanopores. We hypothesized
that using DNA-based nanoplates as covers for solid-state
nanopores could provide a route for meeting this challenge.
Our setup (Figure 1a) consists of two electrolyte reser-
voirs separatedby asilicon-supported free-standinginsulating
silicon nitride (SiN) membrane of thickness L=50 nm. The
membrane contains a single conical nanopore of diameter
D=18–25 nm (Figure 1b), which is fabricated by electron
beam lithography and reactive ion etching.When a voltage
is applied through the two electrodes, an ionic current flows
through the nanopore and is recorded with a current ampli-
fier. The dimensions and shape of the nanopore dominate the
resistance of the setup. The cis side of the nanopore is covered
with a rectangular nanoplate of width a, length b, and
thickness l. The plate includes a central aperture of width
x and length y (Figure 1a). The nanoplates are produced by
molecular self-assembly with scaffolded DNA origami (see
the Supporting Information, note S1) and consist of a double
layer of 46 tightly interlinked double-helical DNA domains in
a honeycomb-type packing lattice. We made nanoplate
a width and length of 50 nm, and a thickness of 6 nm. Correct
formation of the nanoplates was confirmed using negative-
stain transmission electron microscopy (Figure 1b; see also
the Supporting Information, notes S6–S9).
The nanoplates were electrically assembled onto the
nanopores by injection into the cis electrolyte compartment.
A sudden current drop and intensification in the current noise
was typically observed within a few seconds (Figure 1c) after
the bias voltage was applied (Supporting Information,
notes S2 and S3). The current blockades lasted for hours,
unless reverse voltages were applied or the membrane was
intensely rinsed. In both cases, the initial conductance level of
the nanopore was restored. In experiments using nanopores
with diameters exceeding the dimensions of the nanoplates,
we observed transient blockades (Supporting Information,
note S4), indicating that in those cases the nanoplates slipped
through the larger nanopores. In experiments with mem-
branes containing arrays of nanopores, we observed staircase-
like decreases in conductivity (Supporting Information, Fig-
ure S12), indicating the progressive capture of nanoplates by
individual nanopores in the array.
We find that, for nanoplates with apertures the conduc-
tance of nanoplate-on-nanopore hybrids decreases with
decreasing aperture size (Figure 1d). This finding can be
explained by a model in which the nanoplates cover the
nanopore in a flat orientation. Orthogonal or random
orientations should yield relative conductances that do not
depend on the size of the apertures. The nanopore conduc-
tance drops by approximately 17% when using nanoplates
lacking a central aperture. The DNA nanoplates are expected
to be permeable to ions, owing to a mesh of vertical and
horizontal channels. The vertical channels are formed by
cavities between neighboring double-helical DNA domains
between crosslinks along the helical axis,[4,5,12]while the
horizontal channels are intrinsic to the honeycomb-type
architecture of the DNA nanoplate. The corrugated lower
surface of a nanoplate may provide additional channels for
ion flow when it covers a nanopore.
The equivalent circuit model depicted in Figure 1e,f
accounts for the conductances of the bare nanopore (Gpore),
the nanoplate (Gplate), and the central aperture (Gaperture).
Gaperture can be estimated for a given aperture size by
a geometrical conductance model (Supporting Information,
note S5). The value of Gplatecan therefore be computed from
the measured hybrid conductance. We evaluated Gplateversus
Gporefor nanopores with diameters in the range of D=18–
25 nm and found that the conductance Gplateof the nanoplates
increases linearly with the conductance Gporeof the nanopore
[*] R. Wei,[+]U. Rant
Walter Schottky Institute, Technische Universit?t M?nchen
Am Coulombwall 4, 85748 Garching near Munich (Germany)
T. G. Martin,[+]H. Dietz
Center for Integrated Protein Science M?nchen & Institute for
Advanced Study, Physics Department, Technische Universit?t
Am Coulombwall 4a, 85748 Garching near Munich (Germany)
[+ +] These authors contributed equally to this work.
[**] This work was supported by the German Excellence Initiative
through grants from the Nano Initiative Munich and from the
Center for Integrated Protein Science Munich, by the Collaborative
Research Center SFB 863 of the German Research Society (DFG),
the TUM Institute for Advanced Study, and a European Research
Council Starting Grant to HD. R.W. was supported by the TUM
Graduate School’s Faculty Graduate Center of Physics. Discussions
with Friedrich Simmel, Michael Mayer, Daniel Branton, and George
Church are gratefully acknowledged.
Supporting information for this article is available on the WWW
? 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 4864–4867
(Figure 1g). Therefore, the
area of the nanopore con-
trols the area of the nano-
plate that is exposed to ion
flow. This finding agrees well
note S5) that further sup-
ports the notion that the
nanoplates cover the nano-
pore in the desired orienta-
ion fluxes that bypass the
nanoplates, we estimate the
transversal specific conduc-
tivity (splate ?) of the nano-
plates to be (7.8?0.4) Sm?1
in an aqueous solution of
custom apertures can serve
as size-selective molecular
gates for nanopores. To
demonstrate this option,
(52 kDa, hydrodynamic
diameter ca. 6 nm)to
the cis compartment of the
nanopore setup along with
width 9 nm and length
14 nm. Before insertion
of a nanoplate onto the
current blockades caused
by streptavidin molecules
translocating through the
ure 2b). After nanoplate
blockades caused by strep-
(Figure 2b,c; see also the
ments with a significantly
larger protein, immuno-
globulin G (IgG, 150 kDa,
diameter ca. 14 nm), we
found that with a nano-
plate in place, IgG trans-
Figure 1. DNA nanoplates on SiN nanopores. a) Schematic of the experimental setup. The free-standing SiN
membrane is shown in grey, the DNA nanoplate is shown in red. Inspired by previous work,[10d]we included
a ca. 1300 base long single-stranded or double-stranded DNA loop that protrudes near the central aperture (see
Figures S1-S5 for design details) to facilitate insertion into the nanopore. b) Top left: Transmission electron
micrograph (TEM) of a bare nanopore. Top right and bottom row: average of aligned negative-stain TEM
micrographs of nanoplates with varying aperture sizes (inset numbers indicate aperture width?length in nm)
imaged separately on thin carbon support layers (Figures S6–S9). Scale bars: 20 nm. c) Nanoplates were
injected at dilute effective concentrations of ca. 30 pmolL?1with bias voltages of up to +200 mV applied to the
trans electrode and captured electrically on nanopores. Horizontal scale bars: 2 s; Vertical scale bars: 10 nS.
Asterisk marks nanoplate capture event. d) Relative conductance of nanplate-on-nanopore hybrids compared to
conductance of bare nanopore before nanoplate insertion. Inset numbers indicate width?length in nm of
a central aperture in the nanoplates; 5?7’ indicates a nanoplate version with one single-stranded DNA
heptanucleotide overhang in the aperture (Figure S2). e,f) Schematic current paths and equivalent circuit model
for the conductance of the nanoplate-on-nanopore hybrid. g) Nanoplate conductance Gplatecorrelates linearly
with the conductance of the underlying nanopore Gpore(note S5); & no aperture, * 5 nm?7 nm aperture, and~
9 nm?14 nm aperture.
Figure 2. DNA nanoplates with custom apertures for size-selective macromolecular sensing with SiN
nanopores. a) Current–time traces observed for streptavidin (cis concentration=20 nm) translocation through
bare nanopores, b) Current–time traces after insertion of a nanoplate with a central aperture of width 9 nm
and length 14 nm (see Figure S5 for design details). c) Histogram of observed current levels. d–f) As in (a)–
(c), but with Immunoglobulin G (IgG; cis concentration=20 nm). g–i) As in (a)–(c), but with 6 kbp linear
double-helical DNA molecules (cis concentration=300 pm) and a nanoplate with aperture width 5 nm and
length 7 nm (Figure S2, version 0).
Angew. Chem. Int. Ed. 2012, 51, 4864–4867 ? 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
(Figure 2d–f). The DNA nanoplates can also be applied for
translocation experiments with double-stranded DNA mole-
cules, as seen in experiments with linear DNA fragments 6000
base pairs long and nanoplates that include a central 5 nm?
7 nm aperture (Figure 2g–i; see also the Supporting Informa-
tion, note S7). In experiments with a closed nanoplate, we did
not detect DNA translocation (Supporting Information,
DNA nanoplates can also provide chemical selectivity for
solid-state nanopores. To demonstrate this capability, we
included chemical bait modifications within the nanoplate
apertures to detect prey molecules that can selectively adhere
to the bait (Figure 3a). The nanoplates thus become gate-
keepers that can delay the passage of a target molecule
through the aperture. In our experiments, we used single-
stranded DNA motifs protruding from the double-helical
DNA domains that form the boundary of the nanoplate
aperture as bait (for design details, see Figures S2 and S3 in
the Supporting Information). It is important to note that
many different chemical bait moieties beyond single-stranded
DNA can be conjugated in a site-directed fashion to a DNA
We performed translocation experiments using single-
stranded DNA prey molecules with sequences [T]24CC[T]24
and [T]23CCGG[T]23(Figures 3b–e) that can format most two
or four DNA base pairs, respectively, with a bait motif of
sequence TTTCCGG located in the nanoplate aperture.
Immediately after a nanoplate was assembled on the nano-
pore, we observed a continuous switching between two
discrete current levels (Figure 3b,d). In the case of the
[T]24CC[T]24prey molecule, which can form at most two base
pairs with the bait motif, we found that the current switching
occurs much more quickly than in experiments with the
[T]23CCGG[T]23prey molecule, which can form four base
pairs with the bait. We analyzed the distribution of the current
blockade dwell times (Figures 3c,e) and found that the
distributions can each be fit to a single exponential distribu-
tion. In the case of the [T]24CC[T]24 prey molecule, the
average blockade dwell time was (41?3) ms, while for
[T]23CCGG[T]23we found a value of (487?28) ms. Differ-
ences in the binding energies, which in this case are controlled
by the number of base pairs formed between prey and bait
motifs, will be reflected in the bond lifetimes. We expected the
characteristic dissociation time for the two prey motifs to
differ by approximately a factor of e2=7.4, which is close to
what we observed. There-
fore, the short bait motif in
the nanoplate delays the
stranded DNA molecules
in a sequence-dependent
with bait motifs to more
complex patterns of prey
motifs yields current dwells
a multi-exponential fashion
with multiple characteristic
time constants. To demon-
strate this, we used the 7249
base long genome of the
which can adopt complex
secondary structures with
many loop-terminated hair-
pins, as our prey molecule.
One of many ground state
experimental conditions is
depicted in Figure 3 f.
with a 5 nm?7 nm central
motif were added to our
observed transient current
Figure 3. Sequence-specific prey DNA detection with functionalized DNA nanopores on SiN nanopores.
a) Molecular bait is displayed in the nanoplate aperture (see Figures S2 and S3 for detailed implementation).
b) Current–time trace observed in translocation experiments with an aperture-bound bait sequence TTTCCGG
and a single-stranded prey molecule of sequence [T]24CC[T]24(cis concentration=300 pm). c) Bars: logarithmi-
cally binned histogram (N=233) of current blockade dwell times. Solid line: single-exponential distribution
(note S11). d,e) Results as in (b),(c), but obtained with prey sequence [T]23CCGG[T]23. Histogram includes
N=193 dwells. f) Current–time traces observed in translocation experiments with M13mp18 genomic DNA
(cis concentration=100 pm) and a nanoplate with bait motif TTTAATT (Figure S2, version 1). Inset shows
a secondary structure map computed with Mfold.g) Bars: histogram of logarithmically binned current
blockade dwell times (N=6100). Solid line: fit to a sum of five single exponentials (note S11). Dashed lines:
individual single exponentials in the sum. h) * average dwell time tifrom (g) sorted according to magnitude.
^ average dwell time observed in translocation experiments with M13mp18 without bait motif (note S9,
Figure S17).~,!average dwell times t found in (c;~) and (e;!).
? 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 4864–4867
characteristic time constant around 500 ms (Supporting Infor- Download full-text
mation, note S9).When a
TTTAATTwas included in the nanoplate aperture (Support-
ing Information, Figure S2), we observed current switching
(Figure 3 f) between two defined levels. When this experi-
ment was repeated with six bait motifs protruding into the
nanoplate aperture, we observed multi-level current block-
ades (Supporting Information, Figure S3 and note S10).
These observations suggest that the blockades are caused by
excursions of segments of M13mp18 molecules, which may
temporarily adhere to the bait motif, into the aperture. In the
case of multiple bait motifs, several M13mp18 segments can
dwell in the aperture simultaneously, thus explaining the
multilevel blockades. In the absence of bait, the segments
rapidly either retrocede or translocate.
We recorded several minute-long current–time traces and
analyzed the statistical distribution of blockade dwells for the
case of the M13mp18 genome as prey and a single bait with
sequence TTTAATT in the nanoplate aperture. We found
a dwell time distribution that is an additive superposition of
five single exponential distributions (Figure 3g; see also the
Supporting Information, note S11). One of the five distribu-
tions has a time constant that matches the one found for the
translocation of M13mp18 through a nanoplate lacking a bait
sequence. Two of the five single exponential distributions
have time constants that match those seen in Figure 3c,e
within a factor of two. Taken together, these findings suggest
that the five characteristic dwell time constants reflect the
dissociation of different types of duplexes formed between
the bait and prey molecules (Figure 3h). The time constants
increase exponentially, suggesting that the duplexes formed
differ by their length in base pairs. This theory is based on the
assumption that duplex lifetimes grow exponentially with the
number of base pairs formed.
In conclusion, we have presented DNA nanoplates that
function with solid-state nanopores, which can be fabricated
through standard electron beam lithography. The nanoplates
are permeable to small ions, but the passage of macro-
molecules can be controlled by including custom apertures.
The chemical addressability of the DNA nanoplates enables
bait–prey single-molecule sensing experiments, as highlighted
here by the sequence-specific detection of DNA snippets and
genomic phage DNA. Applications in biomolecular interac-
tion screens and for detecting DNA sequences by hybrid-
ization are readily conceivable. High-resolution sensing
applications, such as electrical DNA sequencing will require
reducing both the leakage current and current fluctuations.
bait motifwith sequence
Received: January 25, 2012
Published online: April 4, 2012
nanotechnology · single-molecule studies
Keywords: DNA self-assembly · DNA structures · nanopores ·
 N. C. Seeman, Annu. Rev. Biochem. 2010, 79, 65–87.
 a) E. Winfree, F. Liu, L. A. Wenzler, N. C. Seeman, Nature 1998,
394, 539–544; b) J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R.
Sha, P. E. Constantinou, S. L. Ginell, C. Mao, N. C. Seeman,
Nature 2009, 461, 74–77.
 P. W. K. Rothemund, Nature 2006, 440, 297–302.
 S. M. Douglas, H. Dietz, T. Liedl, B. Hogberg, F. Graf, W. M.
Shih, Nature 2009, 459, 414–418.
 E. S. Andersen, M. Dong,M. M. Nielsen, K. Jahn, R. Subramani,
W. Mamdouh, W. W. Golas, B. Sander, H. Stark, C. L. Oliveira,
J. S. Pedersen, V. Birkedal, F. Besenbacher, K. V. Gothelf, J.
Kjems, Nature 2009, 459, 73–76.
 a) H. Dietz, S. M. Douglas, W. M. Shih, Science 2009, 325, 725–
730; b) D. Han, S. Pal, J. Nangreave, Z. Deng, Y. Liu, H. Yan,
Science 2011, 332, 342–346.
 a) S. M. Douglas, J. J. Chou, W. M. Shih, Proc. Natl. Acad. Sci.
USA 2007, 104, 6644–6648; b) M. J. Berardi, W. M. Shih, S. C.
Harrison, J. J. Chou, Nature 2011, 476, 109–113; c) Y. Ke, S.
Lindsay, Y. Chang, Y. Liu, H. Yan, Science 2008, 319, 180–183;
d) R. J. Kershner, L. D. Bozano, C. M. Micheel, A. M. Hung,
A. R. Fornof, J. N. Cha, C. T. Rettner, M. Bersani, J. Frommer,
P. W. K. Rothemund, G. M. Wallraff, Nat. Nanotechnol. 2009, 4,
557–561; e) N. A. Bell, C. R. Engst, M. Ablay, G. Divitini, C.
Ducati, T. Liedl, U. F. Keyser, Nano Lett. 20112, 12, 512–517.
 a) G. M. Church, D. W. Deamer, D. Branton, R. Baldarelli, J.
Kasianowicz, US patent 5,795,782, 1995; b) J. J. Kasianowicz, E.
Brandin, D. Branton, D. W. Deamer, Proc. Natl. Acad. Sci. USA
1996, 93, 13770–13773; c) S. Howorka, Z. Siwy, Chem. Soc. Rev.
2009, 38, 2360–2384; d) B. M. Venkatesan, R. Bashir, Nat.
Nanotechnol. 2011, 6, 615–624.
 a) L. Q. Gu, O. Braha, S. Conlan, S. Cheley, H. Bayley, Nature
1999, 398, 686–690; b) L. Movileanu, S. Howorka, O. Braha, H.
Bayley, Nat. Biotechnol. 2000, 18, 1091–1095; c) S. Howorka, S.
Cheley, H. Bayley, Nat. Biotechnol. 2001, 19, 636–639; d) J.
Clarke, H. C. Wu, L. Jayasinge, A. Patel, S. Reid, H. Bayley, Nat.
Nanotechnol. 2009, 4, 265–270; e) F. Olasagasti, K. R. Lieber-
man, S. Benner, G. M. Cherf, J. M. Dahl, D. W. Deamer, M.
Akeson, Nat. Nanotechnol. 2010, 5, 798–806.
 a) Z. Siwy, L. Trofin, P. Kohli, L. A. Baker, C. Trautmann, C. R.
Martin, J. Am. Chem. Soc. 2005, 127, 5000–5001; b) S. M. Iqbal,
D. Akin, R. Bashir, Nat. Nanotechnol. 2007, 2, 243–248; c) S.
Ding, C. Gao, L. Q. Gu, Anal. Chem. 2009, 81, 6649–6655;
d) A. R. Hall, A. Scott, D. Rotem, K. K. Mehta, H. Bayley, C.
Dekker, Nat. Nanotechnol. 2010, 5, 874–877; e) E. C. Yusko,
J. M. Johnson, S. Majd, P. Prangkio, R. C. Rollings, J. Li, J. Yang,
M. Mayer, Nat. Nanotechnol. 2011, 6, 253–260; f) S. W. Kowalc-
zyk, L. Kapinos, T. R. Blosser, T. Magalhaes, P. van Nies, R. Y.
Lim, C. Dekker, Nat. Nanotechnol. 2011, 6, 433–438; g) R. Wei,
V. Gatterdam, R. Wieneke, R. Tamp?, U. Rant, Nat. Nano-
technol. 2012, DOI: 10.1038/NNANO.2012.24.
 R. Wei, D. Pedone, A. Z?rner, M. Dçblinger, U. Rant, Small
2010, 6, 1406–1414.
 C. E. Castro, F. Kilchherr, D. N. Kim, E. L. Shiao, T. Wauer, P.
Wortmann, M. Bathe, H. Dietz, Nat. Methods 2011, 8, 221–229.
 M.Firnkes, D. Pedone,J. Knezevic,M.Dçblinger,U.Rant,Nano
Lett. 2010, 10, 2162–2167.
 M. Zuker, Nucleic Acids Res. 2003, 31, 3406–3415.
Angew. Chem. Int. Ed. 2012, 51, 4864–4867? 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim