a stimuli responsive dna walking...
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This journal is c The Royal Society of Chemistry 2010 Chem. Commun.
A stimuli responsive DNA walking devicew
Chunyan Wang,ab
Jingsong Ren*aand Xiaogang Qu
a
Received 5th October 2010, Accepted 3rd November 2010
DOI: 10.1039/c0cc04234j
A pH responsive DNA walker has been designed. The walker
can reversibly transport specific molecules along an assembled
track under environmental stimuli.
Kinesin, myosin and dynein are bipedal motor proteins. These
linear nanoscale motors move along complementary tracks
and perform a variety of functions such as cytokinesis, signal
transduction, intracellular trafficking, and locomotion of
cellular components. They operate with high efficiencies that
are not commonly encountered in artificial systems.1 Inspired
by this, researchers have paid increasing attention to the
construction of synthetic molecular motors over the past
years. Owing to the unique molecular recognition properties
and structural features, DNA has been recognized as an
attractive building material in nanotechnology and materials
sciences.2 Recently, significant efforts have been expended to
the fabrication of DNA walking nanomotors with foot-like
components that each can bind or detach from an array of
anchorage groups on the track, and transport an object from
one location to another on a nanometre scale.3–6 These kinds
of transportations fall into two categories. One is strand
displacement assay in which the walking system can be fueled
through adding a more energy favorable strand.7–10 For
example, a DNA biped system in which the walker moved
with precise bidirectional control via strand displacement was
first introduced.8 The walker was triggered to move along the
track in expected directions. Another is an enzyme powered
approach. Restriction endonuclease,11,12 DNAzyme13–15 and
polymerase5 had been reported individually to drive the
unidirectional DNA walker. The component sequences are
designed to incorporate the recognition sequences of
corresponding nucleases. The transfer of the walker is realized
by enzyme reactions, such as DNA ligation and cleavage, and
driven by hydrolysis of ATP. These nanodevices, although
promising, are offset by critical operation conditions
(optimized temperature and buffer solution), requiring specific
DNA sequences, and producing duplex waste products.
In addition, the stepping rate of strand displacement is
determined by the fuel hybridization rate, which is relatively
slow. Therefore, a new strategy is needed to overcome
these problems for the development of simple and easier to
manipulate DNA walking devices toward more sophisticated
functions. Recently, the construction of complex and adjustable
DNA devices in response to external stimuli has shown great
potential in the development of smart materials and become
one of the frontier challenges in DNA nanotechnology.
Despite great advances having been made in this field,16
stimuli responsive DNA walkers are still unexplored. Herein,
we demonstrate the first report of a pH responsive walking
system in which a walker strand could move along a track and
accomplish transportation of cargo under environmental
stimuli.
Our strategy is illustrated in Scheme 1. The pH-stimuli
walking device is based on two components: track and walker.
The track is prepared with three strands: T, S1 and S2
(detailed sequences are shown in the ESIw). Strand S1 and
S2 are partially complementary to scaffold strand T, each with
a 15-bp helix joining with the scaffold. Stoichiometric mixing
of strands T, S1 and S2 leads to a self-assembled track with
two protruding branches S1 and S2 that represent two
addresses A and B. Strands S1 and S2 are attached to the
track at the 50 end with about 5 nm spacing (15 bp) through a
3nt flexible hinge. Single-stranded hinges adjacent to either
end of these helices provide flexibility for adopting different
conformations. A single strand W that incorporates a
thrombin-binding aptamer was used as a model system to
perform as the walker. It is designed to be long enough to
switch between the A and B sites. The key part of the walker
described here is the protruding branch S1 which containing a
15 base i-motif DNA sequence.17,18 As can be seen in
Scheme 1, operation of the walker is powered by protons.
Under slightly acidic conditions, the cytosine residues are
partially protonated and the DNA folds into a closed i-motif
structure. Therefore, the designed walker could take one step
along the track each time when the environment is switched
between acidic and alkaline, and consequently it can move
back and forth between two destinations concomitant with pH
variations.
Scheme 1 Schematic illustration of the walker locomotion. Green
sphere represents fluorescent dye (ROX) and brown sphere represents
quencher (BHQ-2). The diagrams depict (a) unbound walker;
(b) walker anchors to site A; (c) walker anchors to site B.
a Laboratory of Chemical Biology and State Key laboratory of RareEarth Resources Utilization, Changchun Institute of AppliedChemistry, Chinese Academy of Sciences, Changchun, 130022,China. E-mail: [email protected]; Fax: +86 0431-85262625
bGraduate School of the Chinese Academy of Sciences,Chinese Academy of Sciences, Beijing, 100190, China
w Electronic supplementary information (ESI) available: Experimentaldetails, electrophoresis and fluorescence time course measurment. SeeDOI: 10.1039/c0cc04234j
COMMUNICATION www.rsc.org/chemcomm | ChemComm
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Chem. Commun. This journal is c The Royal Society of Chemistry 2010
Under alkaline conditions, the cytosine residues in the
protruding S1 region are deprotonated and hybridized with
the walker to form a rigid duplex. Meanwhile, thrombin is not
capable of binding to its aptamer upon duplex formation. At
low pH, the C-rich region of S1 would adopt a stable compact
i-motif structure with a 6nt hanging tail. Subsequently, the
originally formed duplex W�S1 will be destabilized and the
walker has a great tendency to detach from site A and
hybrid with the protruding S2 through branch migration.
Consequently, the target protein was captured at site B. When
the solution pH was switched back to alkaline, the i-motif
structure collapsed. The 30 end of S1 will then displace S2
through the 6nt toehold, and hybrid with the walker to form
thermodynamically more favored duplex S1�W and the target
protein is released. Therefore, the walker can transport the
protein between two destinations repeatedly in response to
environmental cues. More importantly, the switch is simple to
manipulate and does not accumulate duplex waste products to
poison the system. Furthermore, the walker can be designed to
incorporate specific sequences making it an ideal transporter
for loading and releasing corresponding targets in a robust,
programmable and controllable fashion.
Native polyacrylamide gel electrophoresis and a fluorescent
resonance energy transfer technique were used to confirm the
assembly and operation of the pH responsive DNA walker.
Gel-electrophoretic experiments were performed first to
demonstrate the proper associations between the walker and
track. The experiments were carried out in a cold room at
10 V cm�1. The DNA structures were assembled in a stepwise
fashion and then analyzed by native polyacrylamide gel
electrophoresis. As can be seen from Fig. S1w, each complex
migrated as a clear sharp band with expected gel mobility,
suggesting that proper combination of DNA strands led to the
formation of stable complexes under native conditions. The
assembled structure (a combination of strands T, S1, S2 and
W) migrated much slower than the bands corresponding to
walker and track. The migration shift was due to the fact that
address branches protruding from the double strand scaffold
dramatically decrease the mobility of the device. The observed
mobility change under pH 8.0 and 5.5 illustrates a stepwise
association of complementary segments and also confirms that
no dissociation was observed at each pH. The walker strand
could switch between two destinations by alternate addition
of HCl and NaOH. The movement was accompanied by
formation and destabilization of the quadruplex, as well as
the capturing and releasing of thrombin. To further confirm
this observation, the pH responsive system was conducted in
the presence of thrombin as well. The complex incubated with
thrombin was allowed to stay for about three hours during
each switch at room temperature and then pipetted out for gel
analysis. The holding and releasing processes at two different
states were demonstrated in Fig. 1 (lane 3). At pH 8.0, the
band corresponding to the complex incubated with thrombin
was slightly influenced. At pH 5.5, the band corresponding to
complex in the presence of thrombin was obviously retarded,
signalling the creation of species with higher molecular weight
corresponding to the loading state. The slower mobility shift
indicated the formation of a higher molecular complex which
was too large to migrate and was unable to penetrate the
gel.19,20 The dye stained gel confirmed that correct structures
were formed and the thrombin could be held by the walker
at pH 5.5 and released at pH 8.0. Therefore, the results
indicate the single strand walker could transport specific
molecules between two destinations repeatedly in response to
environmental cues.
To gain further support of the proposed design and
construction, fluorescent measurements was used to monitor
the real time motion of the walker. In our system, branch S1
and the walker are end-labeled with the quencher and fluorescent
dye to allow monitoring of the fluorescence intensity changes.
Specifically, fluorescent labels rhodamine derivative (ROX)
and black hole quencher (BHQ-2) were covalently bound
at 50 end of the walker strand W and 30 end of strand S1,
respectively. The fluorescence of BHQ-2 and ROX is insensitive
to pH change between 4.0 and 9.0. Under alkaline conditions,
walker strand hybrids with S1 and the two fluorescent dyes
were brought adjacent to each other. Consequently, the
fluorescence of ROX would be efficiently quenched by
BHQ-2. Under acidic conditions, the single strand tail at the
50 end of strand W will fold back to form a quadruplex. The
fluorescent label located at its 50 end is well separated from
the quencher. The energy transfer efficiency is low and the
fluorescence intensity was strong. As can be seen from Fig. 2A,
the fluorescence intensity of the walker strand was strong at
pH 8.0. Upon mixing with the track solution, the fluorescence
of ROX decreased. The intensity change resulted from the
binding of the walker to the track at site A. Similarly, upon the
addition of HCl into the preassembled sample, the fluorescence
intensity increased (Fig. 2A, blue line). The walker locomotion
was further confirmed by fluorescence time course measurements.
The walker strand can continuously switch between site A and
B when the solution pH oscillates between 5.5 and 8.0. Fig. 2B
demonstrated the cyclical fluorescent changes at 603 nm. The
fluorescent intensity changes result from the mechanically
switching duplex and quadruplex states of the pH responsive
system. The switching process was also performed in the
presence of thrombin21 (Fig. S2w). Since the waste products
are water and NaCl, which would not interfere with the
system, the pH stimuli walker still performs efficiently
after three full operation cycles. The decreased fluorescence
intensity should attribute to the photobleaching of fluorescent
dye and dilution of the solution.
In conclusion, we have shown a novel strategy to construct a
stimuli responsive DNA walker system powered by protons.
The DNA walker has important properties that make it useful
Fig. 1 Electrophoretic analysis of the device at two solution
conditions: pH 8.0 and pH 5.5. Lane 1: strand (T + S1 + S2); lane 2:
strand (T + S1 + S2) +W; lane 3: strand (T + S1 + S2) + W +
thrombin.
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This journal is c The Royal Society of Chemistry 2010 Chem. Commun.
for further development. First, the incorporation of a
G-quadruplex and i-motif sequence into the system allowed
the device to accomplish movements such as repeatedly
capture or release target protein in response to external
stimuli. This kind of DNA walker is robust and reversible
without the need of injecting external energy. Second and
more importantly, many other sequences that widely exist in
living systems and play key roles in many biological processes
could be introduced into the system through rational design
and DNA devices with more intricate functions could be
constructed. Different nanosized objects/functional groups,
such as nanoparticles, fluorophores and drugs could be
incorporated into this system with precise control. Our work
demonstrated the first report of a DNA walker system that
could move along a track and accomplish transportation
under pH stimuli. Therefore, this work is an important step
forward in obtaining artificial nanomotors with precise motion
control and will be highly beneficial for future applications
and complex operations in diverse areas ranging from drug
delivery to nanoscale assembly or patterning.
The work was supported by the National Basic Research
Program of China (Grant 2011CB936004) and the National
Natural Science Foundation of China (Grants 20831003,
90813001, 20833006, 90913007).
Notes and references
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Fig. 2 Fluorescence spectra change of ROX while attached to the
track under different pH. (A) Spectra of walker before (black line) and
after attached to the track (red line) under pH 8.0, spectra of the
walker at pH 5.5 (blue line). The spectra was recorded from 590 nm to
650 nm at an excitation wavelength length of 580 nm, both slits were
set to 5 nm. (B) Cycling the DNA walker in the absence of thrombin.
The intensity change at 603 nm was recorded.
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