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: jren@ciac.jl.cn; 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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101, 238101.11 P. Yin, H. Yan, X. G. Daniell, A. J. Turberfield and J. H. Reif,

Angew. Chem., Int. Ed., 2004, 43, 4906.12 J. Bath, S. J. Green and A. J. Turberfield, Angew. Chem., Int. Ed.,

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Int. Ed., 2005, 44, 4355.16 J. Han, Science, 2000, 288, 1026; D. Stein, F. H. J. van der Heyden,

W. J. A. Koopmans and C. Dekker, Proc. Nat. Acad. Sci. USA,2006, 103, 15853; F. Xia, W. Guo, Y. D. Mao, X. Hou, J. M. Xue,H. W. Xia, L. Wang, Y. L. Song, H. Ji, O. Y. Qi, Y. G. Wang andL. Jiang, J. Am. Chem. Soc., 2008, 130, 8345.

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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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