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Asymmetric DNA Origami for Spatially Addressable and
Index-Free Solution-Phase DNA Chips
By Zhao Zhang, Ying Wang, Chunhai Fan,* Can Li, You Li, Lulu Qian,
Yanming Fu, Yongyong Shi, Jun Hu, and Lin He*
DNA nanotechnology has become an increasingly attractive field
of research since Seeman’s pioneering work on self-assembled
DNA nanostructures in the 1980s.[1] Because of the unparalleled
self-assembly ability of DNA molecules, it is possible to
bottom-up construct precise and uniform nanostructures which,
given their complexity, are usually difficult to realize with
conventional inorganic or organic nanomaterials.[2–8] DNA tiles
are regarded as the most important building block in the
bottom-up construction of nanoscale DNA structures, devices,
and machines. For example, by using a DNA double-crossover
(DX) tile, Winfree et al.[5] first designed two-dimensional (2D)
DNA crystals that could be imaged with atomic force microscope
(AFM). Ke et al. reported a 4 4 tile that could template the
formation of conductive nanowires or protein 2D arrays.[9] Mao,
LaBean and others[10–13] developed a series of motifs to create
self-assembled 2D lattices. A number of other projects have been
undertaken to control self-assembly with elaborate designs of
finite-sized lattices,[14,15] algorithmic self-assembled patterns,[3,16]
nanotubes,[17,18] or even 3D structures.[6,19–22] In this study, we
aim to develop a nanoscale chip for DNA detection using
self-assembled DNA structures.
In 2006, Rothemund reported seminal work on ‘‘DNA
origami,’’[4] which involves a long, single-stranded DNA that is
folded into designed shapes with the help of hundreds of short
‘‘helper’’ DNA strands (also called staple strands). In principle,
any nanoscale shape and pattern can be designed and fabricated
via DNA origami. For example, we recently constructed a Chinese
map in the form of an asymmetric DNA origami-based shape.[23]
Given that DNA origami is exceptionally well suited for designing
shapes of high complexity, it has been employed as a building
block for spatially addressable patterning nanoparticles and
proteins for potential applications in nanoelectronics and
nanosensors.[24–27] DNA-based biosening is an increasingly
[*] Prof. C. Fan, Prof. L. He, Prof. Y. Shi, Z. Zhang, C. Li, Y. Li, L. Qian
Y. Fu
Bio-X Center
Key Laboratory for the Genetics of Developmental and
Neuropsychiatric Disorders
(Ministry of Education)
Shanghai Jiao Tong University
Shanghai 200030 (PR China)
E-mail: [email protected]; [email protected]
Prof. C. Fan, Prof. J. Hu, Z. Zhang, Y. Wang
Laboratory of Physical Biology, Shanghai Institute of Applied Physics
Chinese Academy of Sciences
Shanghai 201800 (PR China)
DOI: 10.1002/adma.201000151
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important area with numerous applications.[28–30] More recently,
Yan and coworkers developed a DNA origami chip that allowed
AFM-based label-free detection of RNA targets with high
sensitivity and selectivity.[31,32] Their origami chip was of a
simple rectangular shape, which had to introduce built-in ‘‘index’’
oligonucleotides in order to break the symmetry of the tile. In the
present study we report the design of an index-free nanoscale
DNA chip using the asymmetric origami map developed in our
laboratory. Because of the asymmetric nature of the map, the
position of each DNA probe is fully spatially addressable,
obviating the need for index oligonucleotides.[23,33] To confirm
this, we set up a coordinate system on the origami map, based on
the folding path of scaffold strand. The x-coordinate is the
number of units of eight bases (or three quarter turns) counting
from the left side, while the y-coordinate is the number of folds of
scaffold strands counting from the lower side. Each staple
containing a probe can thus be located as a coordinate pair via its
complementary scaffold.
We first demonstrated that a protein label, streptavidin (STV),
is an appropriate contrast label for AFM imaging. In particular,
the biotin–STV binding is one of the strongest affinity pairs so far
identified, and the use of STV for pixel contrast enhancement has
been regularly employed in AFM imaging.[14,34–37] A staple strand
tailed with a 30 -biotinylated 17-mer probe was incorporated into
the tile (Fig. 1a), leading to a protruding probe positioning at the
(29, 19) site of the map. STV was then introduced and incubated
with the chip. AFM studies showed a clearly visible feature (a
white bulge) which is characteristic of STV binding (Fig. 1b),
suggesting that STV bound on protruding probes could serve as
an effective imaging label for AFM.
We then employed protruding probes without a biotin label to
detect their biotinylated complementary targets. Given that STV
can provide sufficient contrast for AFM imaging, we employed
linear probes rather than the ‘‘V-shaped’’ probes employed in the
previous report.[31,32] We designed eight probes split into two
groups, i.e., one group (four probes) which had their 30 -end (red
dots in Fig. 1c) and one group which had their 50 -end (green dots
in Fig. 1c) exposed to solution. As a result, when the
30 -biotinylated target hybridized with the probes, the biotin label
and subsequent STV binding was placed either proximal (red
dots) or distal (green dots) to the nanochip surface. Our AFM
studies showed that the probes could bind to the targets via
specific DNA hybridization, leading to visible STV features in the
case of proximal biotin (Fig. 1d). We found that not all positions
with probes led to consistent hybridization and STV binding.
There are at least three possible explanation for this: i) DNA
hybridization efficiency, ii) biotin–STV binding efficiency, and iii)
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the influence of the force of AFM tips on samples. While all these
effects may influence the signal readout, we note that the
tip-induced effect should be taken into careful consideration. It is
worthwhile pointing out that we had to optimize tip–surface
interactions in order not to either damage the tip or scratch bound
proteins. Indeed, we found that STV features might disappear
under non-optimized imaging conditions, and a similar
phenomenon has been reported in other studies.[24,38,39] The
recently developed super-resolution microscopy might provide an
alternative way to study these effects.[33]
Interestingly, we found that the group with proximal biotin led
to significantly clearer STV bulges than the group with distal
biotin (Fig. S3 of the Supporting Information), which is in fact
contrary to our intuition. Since the biotin label is relative small,
the hybridization efficiency is similar in both cases, while the STV
binding is more difficult in the former case due to the steric effect.
We also ascribe this counter-intuition phenomenon to the
tip–surface interaction. While double-stranded DNA is basically
stiff, it may dangle at the surface, particularly when a large STV
protein is sitting at the top of the duplex. Such dangling is likely to
significantly affect the imaging process.[4,40] Consequently, we
always employed proximal biotin positions in subsequent
experiments.
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Figure 1. a) Scheme showing that streptavidin (STV) serves as the AFM
label for a staple strand tailed with a 17-mer 30 biotinylated ssDNA probe.
The AFM image in (b) shows that the biotin–STV binding is visible.
c) Scheme for DNA hybridization of biotinylated targets at the DNA origami
chip. Green or red dots show the biotin tags are at either 30 - or 50 -end,
leading to biotin–STV binding at different positions. d) AFM images show
the successful hybridization in pre-designed positions. Three representative zoom-in images are shown at the bottom.
The significant probe position effect in the previously reported
origami chip was ascribed to the different electrostatic effect at
different positions of the tile.[41] We therefore designed two types
of probes in order to test such a position effect, namely four
probes located near the edge of the map with the four other
probes close to the middle of the map (Fig. S4a of the Supporting
Information), thus the former was expected to have less
electrostatic repulsion than the latter. However, it was interesting
that we did not find such significant position effect in our
map-based nanoscale DNA chip (Fig. S4b). Among 110 tiles with
middle probes, there were 104 tiles on which at least one white
bulge can be seen, while 116 of the 122 tiles with edge probes had
at least one STV bulge. No significant difference was found
(Fisher’s exact test, one-sided, p ¼ 0.43). We also put the same
probes at the two columns simultaneously (left column: X ¼ 31;
right column: X ¼ 43), and again no significant difference in
hybridization efficiency was found (Fig. S4c). We reason that the
linear probe employed in this work led to smaller electrostatic
repulsion or steric hindrance than the V-shaped probe.[41] This
improvement means that a large portion of staple strands in the
middle can be used in our chip design, which is important for
realizing high-density DNA detection with the nanoscale chip.
However, we note that the distribution of binding numbers (out
of the four sites) could still be dependent on the position, and on
the target sequence and length as well.
Given that the origami chip could specifically detect DNA
hybridization, we further explored its ability to perform multiplex
detection. Two groups of probes with two different sequences
were placed at the surface of the chip forming two columns (left
column: X ¼ 21; right column: X ¼ 31). Importantly, we found
that the bulge feature of STV appeared only in the left column in
the presence of the target 10 specific for probe 1, while the target 20
only led to the appearance of STV in the right column (Fig. 2).
This high specificity demonstrated that the origami DNA chip
was suitable for multiplex DNA detection with minimal sequence
perturbation.
It is important to detect unlabeled rather than biotinylated
targets in practical DNA assays as it reduces the time/cost of
target modification. In order to achieve this goal, we designed a
‘‘sandwich-type’’ hybridization detection strategy that is usually
employed in conventional DNA chips to detect DNA targets.
Protruding probes on the origami tile with specially designed
sequences serve as the capture probe with biotinylated probes as
the reporter probe, both flanking the DNA target that has two
different regions complementary to the probes (Fig. 3a). In the
presence of the target, the reporter probe, along with the target, is
brought to the proximity of the tile via the capture probe/target/
reporter probe sandwich hybridization. STV is then introduced to
provide a contrast label for AFM imaging. As shown in Figure 3,
this sandwich detection also led to clearly visible STV features in
the presence of the target, while non-complementary sequences
did not result in any observable features.
In summary, we have demonstrated a solution-phase, spatially
addressable DNA origami chip for DNA detection. This nanoscale
DNA chip offers several advantages. First, it is an asymmetric tile
that allows index-free detection without having to involve position
index labels. Second, we employed linear probes rather than
‘‘V-shaped’’ probes, which led to a much smaller position effect.
Third, the sandwich detection strategy is able to detect unlabeled
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Chinese map is not indispensable, and in principle any
asymmetric origami shapes can be use for index-free DNA
detection. The relationship between the origami shape and
optimal distribution of probes is worth studying in future. Our
origami chip still possesses some disadvantages. For example,
hybridization efficiency is still not satisfactory, thus a column of
identical probes have to be employed in order to assure sufficient
binding. AFM imaging still has limitations as it does not allow
rapid monitoring of DNA hybridization. Despite that, given the
rapidly increasing ability to construct complicated DNA selfassembled structures[6,20] and hybrid nanobio complexes,[42–48]
we expect that this origami chip will find important applications
in nanotechnology-based DNA detection.[49–53]
Experimental
Figure 2. Schematic demonstration (left) and AFM images (right) for
multiplex detection. Four probes in (a) are complementary to target 1
and four probes in (b) are complementary to target 2. Each probe is
represented by a pair of coordinates. All eight probes were placed on the tile
simultaneously. Upon the addition of the specific target, STV bulges are
only observed at specific positions in AFM images. Scale bar: 250 nm.
long-piece DNA targets, without having to use two pieces of
protruding capture probes (as in the ‘‘V-shaped’’ probes), which
potentially means that the origami chip with the sandwich
strategy can accommodate twice as many capture probes as the
‘‘V-shaped’’ probes. Since biotinylated oligonucleotides (reporter
probes) are commercially available at reasonably low cost and
have been widely used in DNA assays, the added cost and design
labor are also relatively small. In addition, the sandwich detection
strategy is fairly generic and can be easily extended to virtually any
DNA detection. However, we note that the use of the shape of
Materials: All staple strands were purchased from Generay, Inc.[54] and
were used without further purification. M13mp18 viral DNA was purchased
from New England Biolabs, Inc. (Catalog number: #N4040S) and no
digestion by restriction enzyme was performed. The biotinylated ssDNA
strands were purchased from Takara, Inc. and STV was purchased from
AMRESCO, Inc. (Catalog number: E497).
Assembly and Hybridization: M13mp18 DNA was mixed with over 200
short DNA oligonucleotides with a molar ratio of 1:10 (1.6 nmol L1
M13mp18 DNA, 16 nmol L1 of each short strand) in a 100 mL buffer
(1 TAE-Mg2þ). Anneal the mixture from 94 to 4 8C in a PCR machine (ABI
9700) at a rate of 0.1 8C/10 s. Then the biotinylated target strands of the
same amount as the probes were added, and incubated for 0.5 h at 37 8C.
After hybridization, STV of twofold concentration of the biotinylated target
strand was added to the mixture. The mixture was incubated for another 1 h
at room temperature before imaging.
AFM Imaging: Samples were prepared by deposition of 3 mL onto
freshly cleaved mica. Imaging was performed in Tapping Mode under
30 mL 1 TAE/Mg2þ buffer on a Digital Instrument Nanoscope III a
Multimode AFM (Veeco) with J scanner, using an NP-S oxide-sharpened
silicon nitride tip (Veeco). The tip–surface interaction was minimized by
optimizing the scan set-point.
Acknowledgements
Z.Z., Y.W., C.F., and C.L. contributed equally to this work. We thank the
financial support from National Natural Science Foundation (20725516,
90913014). C.F. and L.H. also acknowledge support from Ministry of
Science and Technology (2007CB936000, 2006AA02A407, 2006CB910601,
2006BAI05A05, 2007CB947300, and 07DZ22917), Ministry of Health
(2009ZX10004-301), the Shanghai Leading Academic Discipline Project
(B205), the Shanghai Municipality Science & Technology Commission
(05JC14090, 0952nm04600) Supporting Information is available online
from Wiley InterScience or from the authors.
Received: January 14, 2010
Revised: February 1, 2010
Published online: May 3, 2010
Figure 3. Schematic demonstration (a) and AFM images (b) for the
sandwich strategy for target detection. Specific white bulges are found
in the AFM image, suggesting the sequence specific hybridization at the
nanoscale DNA chip. Scale bar: 250 nm.
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