Speeding Up a Bidirectional DNA Walking Device - Langmuir (ACS

Sep 22, 2012 - A strategy to speed up DNA walking devices through the use of DNA catalysts has been developed. The DNA walker is designed to move on a...
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Speeding Up a Bidirectional DNA Walking Device Chunyan Wang,†,‡ Yu Tao,†,‡ Guangtao Song,†,‡ Jinsong Ren,*,† and Xiaogang Qu† †

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China S Supporting Information *

ABSTRACT: A strategy to speed up DNA walking devices through the use of DNA catalysts has been developed. The DNA walker is designed to move on a three-foothold molecular track with the assistance of fuel strands. The movement can be accelerated in the presence of catalysts. The motor could be halted at a desired location by a simple control, and the locomotion is about 1 order of magnitude faster than previous hybridization-based walker. Additionally, one branch of the walker can be designed to capture and transfer protein or some other inorganic molecules along the designed track with easy control, which makes our engineered DNA system more versatile.

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INTRODUCTION Nanoscale motors that operate with high efficiencies are not commonly encountered in artificial systems but ubiquitous in biology. A few notable examples are the bipedal motor proteins: kinesins, myosins, and dyneins. 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.1−4 Inspired by this, researchers have paid increasing attention to the construction of artificial molecular motors over the past years. The controlled linear motion of molecular motors in a defined direction is one of the most fascinating features of nanobiotechnology. The motors are supposed to move and function at a molecular level. DNA walkers represent initial steps toward a new and exciting direction of technology. Initial attempts to produce DNA walking nanomotors involved the addition of DNA fuel strands at each step of walking. A DNA biped system and a synthetic DNA walker were first reported,5,6 which could move toward both directions with precise control. The walker moved along the track in response to added input. The more energy favorable strand migrated through sticky ends and displaced original strand via strand migration. Fluorescent dyes were incorporated to illustrate the state of the walker. Various enzymes were introduced afterward to facilitate the walking process. Endonuclease,7,8 DNAzyme,9,10 and polymerase11 were reported individually to drive the autonomous and unidirectional DNA walker. The component sequences were designed to incorporate the recognition sequences of the corresponding nucleases. Hence, transfer of the walker was realized by enzyme reactions, such as DNA ligation, cleavage, and elongation, driven by the energy of ATP hydrolysis. Autonomous directional motion was achieved by making cleavage of the anchorage conditional in the presence of the cargo. After further endeavors, walkers that could take complicated route and actions were also reported.12,13 The © 2012 American Chemical Society

transportation of cargos could be precisely controlled at each time. Lately, walking systems constructed by organic molecules using covalent chemistry were also reported.14 These walking devices could transport objects from one location to another on a nanometer scale. Most of the DNA nanodevices, although promising, are offset by the time-consuming operational cycles. Considering that the stepping rate of strand displacement determined by the fuel hybridization rate15,16 is relatively slow. Therefore, a strategy is needed to overcome this problem and develop faster, efficient, and easy to manipulate DNA walking devices toward more sophisticated functions. Recently, a new concept of DNA catalyst was put forward. Turberfield et al.17 demonstrated that is possible to decrease the reaction time for DNA hybridization by 2−4 orders of magnitude in an artificial molecular machine. This was realized by the use of geometrical and topological constraints induced by loop structures. The research group also used helper DNA to make the DNA hairpin accessible for hybridization to its complement.18 Their walker catalyzed the reaction between two complementary hairpin fuel molecules. This concept was also demonstrated by a research group to accelerate a simple circulation.19,20 The mechanism behind their strategy consisted in that a specified input oligonucleotide catalyzed the release of a specified output oligonucleotide, which in turn could serve as a catalyst for other reactions. This reaction provided an amplifying circuit element that is simple, fast, modular, and robust. After reviewing the ongoing efforts, we address this issue by meticulous improvement to the previous design. Herein, we demonstrate the acceleration of the DNA walking device through the approach of DNA catalyst. The advantage of using strand-displacement-based catalysts over enzymes is that Received: August 16, 2012 Revised: September 21, 2012 Published: September 22, 2012 14829

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Scheme 1. Schematic Illustration of the Walking Processa

a

The track was constructed from strands T1, T2, T3, T4, and T5. The walker was constructed from W1, W2, and W3. Arrowheads indicate the 3′ends of strands. FAM, ROX, and HEX labeled on 3′ end of T1, T3, and T4 are represented as red, green, and purple spheres, respectively. BHQ labeled on the 5′ end of W1 and W2 are represented as gray and cyan spheres. Six steps that needed to complete locomotion in one direction are marked in the scheme. which contained 50% glycerol and 0.2% each of Bromophenol Blue and Xylene Cyanol FF, was added to each sample. Gels were stained with Stains-All. Fluorescence Spectra. Fluorescence measurements were carried out on Jasco-FP-6500 spectrofluorometer (Jasco International Co. Ltd. Tokyo, Japan) with a quartz cell of 1 cm path length. Using excitation wavelengths of 497, 585, and 538 nm, fluorescence emission spectra were monitored from 507 to 650 nm, 595 to 650 nm, and 548 to 650 nm at room temperature, respectively. The slits for the excitation and emission monochromators were both set to 5 nm. The sensitivity and dynamic response of fluorescence spectroscopy allows monitoring the motion of walker. Fluorescence Time Course Measurements. The kinetics of fluorescence intensity changing with time of T1 was measured at excitation wavelength of 497 nm, monitored at wavelength of 520 nm. The intensity change of T3 was monitored at wavelength of 602 nm with excitation wavelength of 585 nm. The intensity change of T4 was monitored at wavelength of 555 nm with excitation wavelength of 538 nm. The slit widths were both set to 5 nm. After the addition of attachment strands or detachment strands, solution was mixed within 4 s by rapidly drawing up with a pipet and releasing it. All measurements were performed at room temperature.

the former generally have fewer sequence constraints and are less likely to be influenced by environmental conditions, such as pH, temperature, and salt concentrations. Additionally, we designed a branched structure as a walker, which could carry diverse cargoes through rational design and move along the track at the same time. More importantly, it is a helpful attempt to put the concept of DNA catalyst into application.

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EXPERIMENTAL SECTION

Materials. Tris(hydroxymethyl)aminomethane (Tris) was purchased from USB. MgCl2, NaOH, and Stains-All were purchased from Sigma. DNA oligomers were purchased from Sangon (ShangHai, China) without further purification. The concentration of each strand was estimated by absorption at 260 nm. Molar extinction coefficients were estimated by the nearest-neighbor method. Sequences labeled with fluorophores were purchased from Sangon. All DNA oligonucleotide used in this paper are listed in the Supporting Information. Electrophoresis. The native PAGE experiments were carried out on a 7.5 cm × 7.5 cm plate. 14% polyacrylamide (19:1 acrylamide:bis(acrylamide) ratio) on nondenaturing gel at 10 V/cm for 3.0 h at 4 °C. The running buffer consisted of 50 mM Tris HCl, pH 8.0, 20 mM acetic acid, and 2 mM EDTA (TAE). 1 μL of tracking dye solution, 14830

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Formation of Track. The track was formed by mixing strands T1, T2, T3, T4, and T5 in equal molar ratio. The walker was formed by mixing strands W1, W2, and W3. A stoichiometric quantity of each strand designed in the complex was fixed at a concentration of 5 μM, in TAE/Mg buffer (40 mM Tris·HCl, pH 8.0, 20 mM acetic acid, 2 mM EDTA, and 12.5 mM magnesium acetate). The solution was cooled slowly from 95 to 20 °C over 48 h. Operation of the Walking System. An equal molar ratio of walker was added to the track solution and incubated for an hour. The walker was able to move back and forth between three destinations by alternately addition of attachment stands and detachment stands. Catalysts strands were premixed with attachment strands before adding into the walker and track solution. A 5 μL sample was taken out at each time for measurement.

strand tail with a three-thymine hinge. The single strand tail on W1 is designed to incorporate aptamer sequence which is available for capturing molecules. Two branches act like legs that could move alternately between three destinations. Five strands (T1, T2, T3, T4, and T5) hybridize with each other to form a rigid track, which are 70 base pairs in length. T1, T3, and T4 are also designed to have a 15 nt unpaired single strand domain that are labeled with FAM, ROX, and HEX at the 3′ end, respectively. They are evenly spaced and act as stators to hold the walker. Attachment strands A1, A2 and A3 contain two domains that are complementary to one branch of the walker and one of the stators, respectively. As can be seen in Scheme 1, the walker can be fixed on corresponding sites upon the addition of attachment strands. Similarly, upon the addition of detachment strands, the walker can detach from one site through branch migration. The single-stranded input is consumed in the course of the reaction and ends up in an inert double-stranded byproduct. Directional movement is possible because the sequence of anchorages has no inversion symmetry. This device displays motor behavior by coordinating the stepping cycle of two legs as it walks along its track. When a fuel strand attaches to one of the foothold strands and one leg of the walker, the other leg can only hybridize to the neighboring foothold to the adjacent site. It is unable to hybridize to the next available foothold further down the track. The working principle of the system is described as follows: the walker set on site S1 in the presence of A1, FAM on the 3′ end of T1, and BHQ on the 5′ end of W2 would be in close proximity, leading to fluorescence quenching of FAM. Then strand A2 is added in; both legs of the walker are fixed on the track. The fluorescence of HEX on the 3′ end of T3 is also quenched. When D1 displaces A1 through branch migration upon binding to a 6 nt toehold, the left leg of the walker would detach from site S1 and lift from the track. The right leg remains bound to its track. After the addition of strand A3, the left leg will rebind on the track. Similarly, the right leg would detach from site S2 in the presence of strand D2. The process could operate in both directions. The operating process is just like a postman delivering packages back and forth along a road. In our experiment, one of the branches is designed to incorporate a thrombin-binding aptamer, which is used as a model system. It could bind to thrombin and transport it to other sites. Native polyacrylamide gel electrophoresis and a fluorescence resonance energy transfer technique are used to confirm the assembly and operation of the DNA walker. Gel-electrophoretic experiments are performed first to demonstrate the construction of the track and the walker (Figure S1). Each complex migrates 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. Attachment strands and detachment strands are added alternatively to specify the capability and stability of the walker binding to each site. The gel exhibits bands with alternate slow and fast mobility (Figure S1, lane 3−lane 9). The bands with slowest mobility correspond to the states where W and T hybridize with attachment strands. Upon the addition of detachment strand D, the bands move slightly faster and a new band appears (Figure S1b). It demonstrates that attachment strand detaches from the complex and hybrid with strand D to form duplex A·D. Quenching effects of each leg with stator are individually characterized by electrophoresis (Figure S2). The gel displays clear sharp bands when exposed to UV trans-

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RESULTS AND DISCUSSION Our strategy is illustrated in Scheme 1. The designed structure contains four parts: a loading walker, track, attachment strands,

Figure 1. Analyzing the walking and loading performance of the walker. (a) A 10% gel was performed in TAE, MgCl2 buffer in cold room at 80 V and viewed by fluorescent imaging. Lane 1: Track (T); lane 2: Track (T) + Walker (W) +A1. Lane 3: lane 2 + A2. Lane 4: lane 3 + D1 + A3. Lane 5: lane 4 + D2 + D3. Lane 6: lane 5 + A2 + A3. Lane 7: lane 6 + D3 + A1. Lane 8: lane 7 + D2 + D1. (b) The walker loaded with thrombin verified by PAGE. Lane 1: W. Lane 2: T + (W + thrombin) + A1. Lane 3: lane 2 + A2. Lane 4: lane 3 + D1. Lane 5: lane 4 + A3. Lane 6: lane 5 + D2. Lane 7: DL 2000 (100, 200, 300, 400, 500, 750, 1000, 2000). (c) Fluorescent measurement to testify the loading of thrombin. Black line: walker fixing to the track in the absence of thrombin. Pink line: walker loaded with thrombin fixing to the track.

and detachment strands (all sequences used are listed in Supporting Information Table S1). The walker is composed of three strands: W1, W2, and W3 which are associated with each other to form a three branched junction through 12 complementary bases. Each branch has a 15 nt flexible single 14831

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Figure 2. Time-dependent fluorescent intensity changes upon the fixing and lifting a leg from the track. Fluorescence time courses were recorded instantly after each addition of attachment strands and detachment strands. The sample was excited at 497, 585, and 538 nm and monitored at the wavelength of 520, 602, and 555 nm, respectively. The slits were both set to 5 nm. Fluorescence intensities are normalized by the initial fluorescent intensity value of track. (a) The fluorescence intensity change of T1 upon the fixing and lifting, the processes were initiated by adding strand A1 and D1, respectively. (b) The fluorescence intensity change of T3 upon the fixing and lifting processes, the processes were initiated by adding strand A2 and D2, respectively. (c) The fluorescence intensity change of T4 upon the fixing and lifting processes; the processes were initiated by adding strand A3 and D3, respectively. All the measurements were performed continuously. During the measurements, at least one leg was fixed to the track all the time.

study local environments changes and nanoscale motion. The energy transfer efficiency is very sensitive to the distance between one pair of fluorophores.22 In order to investigate the kinetics during the walking process, we measured the time course of the process through monitoring the intensity change of the corresponding fluorophores. The fluorescence intensity of track is only slightly influenced by the addition of walker but significantly influenced by the attachment strands. That suggests that the walker and track are not bind with each other in the absence of attachment strands. All the following experiments are performed in the presence of thrombin. The binding of thrombin is further verified through the fluorescence kinetics (Figure 1c). In the process of anchoring to site S1, the fluorescence intensity change of FAM is recorded in the absence and presence of thrombin, respectively. The slower quenching kinetics is due to the spatial hindrance of thrombin which binds to the walker. The quenching and recovering of fluorescence at each step are demonstrated clearly in Figure 2, and spectra changes of each dye are also shown in Figure S3. After the addition of attachment strand A1, the walker is fixed at site S1. FAM on the 5′ end of T1 is in close proximity with BHQ on the 3′ end of W2, resulting in fluorescence quenching of T1. When strand A2 and D1 are added, the right leg bound to site S2 and the left leg of the walker detached from site S1. During this process, the fluorescence intensity of T1 recovers and that of T3 is quenched. Similarly, upon the addition of strand A3 and D2, the left leg of the walker anchors to site S3 and the right leg detaches from site S2. The fluorescence

illuminators. The operation of the walker is performed by mixing equal molar W and T with the addition of attachment strands. The mixture is allowed to incubate adequately to reach a plateau, where A2 is added in and further incubated at room temperature. Strands D1, A3, D2, and D3 are added in the same manner. A 5 μL sample is pipetted out at each step for gel analysis. As can be seen from the gel, a single and clear band (Figure 1a, lane 1) corresponds to the track. A band with slower mobility corresponds to walker/track complex with one leg fixed (lane 2). After the addition of strand A2, an even slower band appears. It corresponds to the state where both legs are fixed (lane 3). The movements are operated in both directions. From lane 2 to lane 5, the walker moves from site S1 to S3. Lane 5 to lane 8 corresponds to the walker moves from site S3 to S1. The experiment is also carried out in the presence of thrombin to verify its ability to transport molecules. The walker was incubated with thrombin in buffer for 3 h to make sure the loading.21 As can be seen in Figure 1b, when one leg of the loading walker is fixed on the track, the complex has much slower mobility than the walker itself (lane 2, lane 4, and lane 6). After both legs are fixed, the complex migrates even slower (lane 3 and lane 5), which means that the complex is too large to penetrate the gel. The gel analysis confirms that the walker can load proteins as well as attach and detach from track as expected. To gain further support of the proposed design and construction, fluorescent measurements are used to monitor the real time locomotion of the walker. FRET is an ideal tool to 14832

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Figure 3. Schematic illustration of loading strand F. Strand F is thio modified at 5′-end that could attach 5 nm gold nanoparticles. The track is assembled from strands T1, T2, T3, T4, and T6. The walker is assembled from W2, W3, and W4. W4 and T6 could hybridize with strand F.

particles could act as reporters to demonstrate the state of the walker (as is shown in Figure 3). We make a theoretical calculation by assuming each strand is well extended. As is shown in Figure S4, when one foot is fixed on site S1, the distance between two particles is estimated to be 30.8 nm. The distances are estimated to be 27.2 and 26.5 nm when the walker is fixed on S2 and S3, respectively. TEM is carried out to confirm each state of the process. As is shown in Figure S5, the TEM images clearly show the formation of the desired structures in high yields, and the primary products exhibit as dimer when the leg anchors to different sites. The interparticle spacings of the AuNPs assemblies are analyzed from the TEM images. To avoid the confusion in finding the correct particle pairs, only assemblies with easily distinguishable dimer structures are chosen for the measurements. As is shown in Figure S6, over 200 pairs are measured to make a histogram. The observed interparticle distances have a majority distribution in the range of 28−55 nm. We attributed the spacing variation to the flexibility of the linkage part between the gold nanoparticles and the oligonucleotide strands, which could

intensity of T4 is quenched and that of T3 recovers. Traversing the track from one end to the other and back, the fluorescent signal from each foothold could respond specifically to the addition of attachment and detachment strands. More importantly, this DNA-based walking device could be cycled between well-defined states through sequential addition of fuel strands and performed in both directions. Each foot can act as a temporarily fixed pivot for the other under different sets of conditions. The loading ability of the walker is also testified by loading a complementary strand. A strand complementary to one branch of the walker is introduced as cargo. To facilitate the observation, we use a 5′ thiol-modified strand F as cargo. The thiolated DNA strand F is first incubated with 5 nm AuNPs.23 The track is assembled from strands T1, T2, T3, T4, and T6. The walker is assembled from strands W2, W3, and W4. Strand T6 and strand W4 both have 15 unpaired bases which are complementary with strand F. The basic principle is to hybridize Strand F-functionalized AuNPs with track and walker to form the desired structures, and the gold nano14833

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Figure 4. Schematic illustration of the walker fixing to track in the presence of catalyst.

cause some variation in all the distances. Also, the variation in the angle between the two domains could generate a range of distances between all pairs of junction arms. The TEM results serve as complementary evidence along with gel electrophoresis and FRET studies and clearly demonstrate the feasibility of using this system as functional devices. Hybridization on reactions between structure-free DNA strands with complementary sequences typically occurs fast, whereas hybridization between strands with strong secondary structure becomes exceedingly slow. Strong binding of the cargo with capturing strands also has the effect of slowing down transfer of the cargo between anchorages.24−26 So far, the walker is relatively slow. It takes nearly 30 min to fix each foot or lift it up from the track. Consequently, it is of great importance to push forward the reactions with catalyst strands. Its stepping rate is determined by the fuel hybridization rate. The rate-limiting step in the pathway is the binding of the walker to the track. Hybridization of attachment strands with walker strands and stators is limited by the topological possibility of pulling two strands close in a dissipative environment which requires expenditure of free energy. The reaction can be speeded up by competitive fixing of at least one end of the attachment strand to the stator through an assisting strand. A useful assisting strand should incorporate a toehold domain that can initiate the fixing of the leg by hybridizing either to the single-stranded extension at the free end of the attachment strand or to the first few bases in the stator. The

hybrid complex can react more rapidly to form attachment strand/stator duplex. Herein, we introduce short single strands which composed of two domains as catalysts. One domain is complementary to the 5′ end of the attachment strand and the other domain can hybrid with the 3′ end of foothold strand. Each domain has five bases that complementary to attachment strand and foothold strand. As shown in Figure 4, the catalyst can pair with the foothold strand to trigger a rapid branch migration process that leads to an intermediate complex and the attachment of the strands A. Then one leg of the walker binds to the intermediate, and the stator strand displaces the catalyst by branch migration. The catalyst is then available for another round of catalysis. In each reaction cycle a catalyst strand undergoes a transition from a random coil configuration into a stretched metastable complex. The energy consumed per step is the free energy change for hybridization between the fuel and toehold strands. The free energy is derived from the potential of forming base pairs or releasing strands and is provided by the reactants. The reaction is therefore limited by the amount of reactants that supplied initially and the time it takes for the system to reach equilibrium. The complexes are kinetically trapped in metastable configurations. They collectively store the energy that drives the catalyzed reaction forward thermodynamically. Complete hybridization of the attachment strands with the foothold strands displaces the catalyst. The freed strand can then hybridize to other strands and accelerate the formation of double-stranded product. As can be seen in Figure 5, the speed 14834

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Figure 5. Time-dependent fluorescence changes in the presence of catalyst strands. Fluorescence intensities are normalized by the initial fluorescent intensity value of track. (a) Catalyst accelerates the process of walker fixing to site S1. Three independent traces with different catalyst concentration are shown: red line (0.1 μM), green line (0.2 μM), and blue line (0.5 μM). (b) Catalyst accelerates the process of walker fixing to site S2. Two independent traces with different catalyst concentration are shown: red line (0.1 μM), green line (0.5 μM). (c) Catalyst accelerates the process of walker fixing to site S3. Two independent traces with different catalyst concentration are shown: red line (0.1 μM), green line (0.5 μM).

absence of catalyst is about 1.32 × 103 M−1 s−1 and increases to 7.57 × 103 M−1 s−1 after the addition of catalyst (C2). The process of fixing to site S2 accelerates nearly 6 times. The rate constant is also increased about 2 times when binding to site S3 in the presence of catalyst. We noticed that the accelerated rates of the leg anchoring to S2 and S3 are relatively slower compared to the leg binds to S1. We attribute this to following reasons. First, the track and walker when simultaneously present would cooperatively bind to attachment strand A.27 When the catalyst presents, the catalyst strand would cooperatively bind to attachment strand and track. At the first step, the catalyst could greatly accelerate the anchoring process through facilitating the hybridization of attachment strand and track which are away from each other. In the steps of fixing one leg to S2 and S3, the other leg has already anchored to the track. The track and the walker are in closer proximity at the beginning of the following steps. The metastable complex between catalysts, attachment strand, and track may have a constraint effect over the whole structure.28 So the extent of acceleration is kind of weaker compared to the first step. Second, catalysts form former steps are always present in the solution; they may have some nonspecific interactions and make the rate acceleration effects less obvious compared to the first step. Overall, the catalysts C1, C2, and C3 used in the experiments could promote the performance of the designed walker. This suggests that the transfer of the walker can be triggered by binding of the catalysts to the toehold of attachment strands and subsequent binding with stators.

of a leg fixing on the track is faster than before. By fitting the time course curves shown in Figure 5, we deduce a secondorder rate kinetic constant for locomotion. We model this system using the following reduced reaction set: k1

[A] + [C] → [A·C] k2

[A·C] + [S] → [I] k3

[I] + [W] → [A·S·W] + [C]

(1) (2) (3)

The time course of the catalyzed reaction over a wide range of catalyst concentration is accurately reproduced by this reduced system of rate equations. Here C is the catalyst, and I is a reaction intermediate which is a three-stranded complex. A stand for attachment strands; S and W stand for the stator and walker, respectively. Second-order rate constants k2 were determined by fitting the fluorescence intensity decay curve to the function17 FI(t) = {FI(0) − FI(∞)}/{1 + k2ct} + FI(∞). The interaction between catalysts fit well to second-order kinetics giving a rate constant of 1.63 × 104 M−1 s−1, as compared with a rate constant of 2.41 × 103 M−1 s−1 in the absence of catalyst (C1) when anchoring to site S1. The reaction rates are related to the concentration of catalysts. The kinetics of the catalyzed reaction over a 50-fold range of catalyst concentration is measured. According to this model, the addition of catalyst can accelerate the reaction by over 10-fold. When the leg is switching to site S2, the rate constant in the 14835

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Interaction between the catalyst and these fuels opens a fast pathway for the rearrangement of the fuels into products. In essence, the catalyst could reduce the kinetic barrier19,20,29 and accelerate the leg binding to the track, which could speed up the walker remarkably.

CONCLUSIONS In summary, we have shown a strategy to speed up DNA walking devices through the use of DNA catalyst system. The DNA walker is designed to move bidirectional on a threefoothold molecular track much faster with alternate addition of corresponding attachment and detachment strands. The walking machine reported before that using strand displacement are limited by the time-consuming operational cycles since the stepping rate of strand displacement is determined by the fuel hybridization rate, which is relatively slow. By combining rationally designed catalysis reactions with DNA walkers, current work is useful in accelerating the movement of the DNA walker. The motor could be halted at a desired location by a simple control, and the locomotion is about 1 order of magnitude faster than previous hybridization-based walker. Additionally, one branch of the walker can be designed to incorporate kinds of cargo recognition sequence, which make our engineered DNA system more versatile. It can be designed to capture and transfer bimolecular or some other inorganic molecules along a designed track with easy control. Importantly, it is conceivable to engineer tracks to allow DNA device to move in a complicated geometry with faster speed. We hope to produce more efficient artificial, linear molecular motors that could move directionally along tracks to transport cargoes and perform tasks resemble biological motor proteins in the future. Fast and efficient walking devices are crucial for their applications, and this area remains largely unexplored. This article represents one step in this direction. By choosing the proper sequence, we believe that the accelerating of the speed could be pushed further. Therefore, this work is an important step forward in obtaining artificial nanodevices with fast and precise motion control and will be highly beneficial for future applications and complex operations in diverse areas ranging from drug delivery to nanoscale assembling or patterning. The field of molecular robotics is still emerging. It is possible that these tiny creations may someday have important medical applications. This work one day may lead to effective control of chronic diseases. ASSOCIATED CONTENT

S Supporting Information *

Table S1 and Figures S1−S6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax (+86)0431-85262625, e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by the National Basic Research Program of China (Grant 2011CB936004) and the National Natural Science Foundation of China (Nos. 20831003, 90813001, 20833006, 90913007, and 21072182). 14836

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