pubs.acs.org/NanoLett
DNA Machines: Bipedal Walker and Stepper Zhen-Gang Wang,† Johann Elbaz,† and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ABSTRACT The assembly of a “bipedal walker” and of a “bipedal stepper” using DNA constructs is described. These DNA machines are activated by H+/OH- and Hg2+/cysteine triggers. The bipedal walker is activated on a DNA template consisting of four nucleic acid footholds. The forward “walking” of the DNA on the template track is activated by Hg2+ ions and H+ ions, respectively, using the thymine-Hg2+-thymine complex or the i-motif structure as the DNA translocation driving forces. The backward “walking” is activated by OH- ions and cysteine, triggers that destroy the i-motif or thymine-Hg2+-thymine complexes. Similarly, the “bipedal stepper” is activated on a circular DNA template consisting of four tethered footholds. With the Hg2+/cysteine and H+/OH- triggers, clockwise or anticlockwise stepping is demonstrated. The operation of the DNA machines is followed optically by the appropriate labeling of the walker-foothold components with the respective fluorophores/quenchers units. KEYWORDS Nucleic acid, machines, molecular devices, pH, ions, i-motif
i-motif structure, by H+ and OH- stimuli, respectively, provide the signals for the activation of the machines. The direction of motion of the “walker”/“stepper” elements is dictated by the relative stabilities of the nucleic duplexes formed between the “walker”/“stepper” units and the respective footholds. Results and Discussion. Figure 1 depicts the DNA construct on which the bipedal walking of a DNA strand proceeds. The system consists of four interlinked footholds 1-4, acting as a track, where foothold 4 is rigidified onto the track by a complementary nucleic acid (5). Each of the footholds is blocked by complementary nucleic acids 1′-4′ that include sticky single-strand modified at their ends with the fluorophores F1 (Cy5), F2 (Cy3), F3 (Cy5.5), and F4 (ROX). The walker consists of two arms (6 and 7), held together by the blocker unit (8), that is modified at the 5′ and 3′ ends with the quencher units Q (Q1, Q2). The quencher units are
T
he development of DNA-based machines is attracting substantial recent research effort.1 DNA nanostructures that perform mechanical operations, such as “tweezers”,2 “walkers”,3 “gears”,4 “metronome operations”,5 and more,6 were reported. Recently, the mechanical translocation of nucleic acids on nucleic acid tracks associated with surfaces, such as electrodes7 or semiconductor quantum dots,7 or the programmed “walking” of DNA on DNA origami patterns8 were reported. Several limitations accompany, however, the development of DNA-based machines: (i) We lack diverse input signals for triggering the molecular machines, and most of the molecular devices rely on strand displacement by a nucleic acid activator. (ii) Often, triggering of the mechanical function involves a cleavage process, thus eliminating the reversible, cyclic, operation of the device. (iii) It is often difficult to ensure the intact structure of the device within its dynamic operation, and the diffusional exchange of components between different machines cannot be excluded. In the present study, we report on the construction of two DNA machines activated by H+/OH- or Hg2+ ions/ cysteine as external triggers. The devices perform a reversible bipedal walking function or a clockwise/anticlockwise stepper function on a DNA wheel. The engineered molecular devices ensure intact and nonseparable nanostructures during their mechanical activation. The principle for operating the machines involves the construction of four nucleic acid footholds on a DNA template and the signal-triggered motility of a “walker”/“stepper” nucleic acid tethered to two of the footholds in each state of the machine. The formation of the thimyne(T)-Hg2+-thymine(T) complexes and their separation by cysteine or the formation/dissociation of the
FIGURE 1. Fluorescence analysis of bipedal walker activated by Hg2+/ cysteine and H+/OH- inputs: panel I, walker immobilized on footholds I and II and position, imaged by high fluorescence of F3 and F4; panel II, walker positioned on footholds II and III imaged by high fluorescence of F1 and F4; panel III, walker positioned on footholds III and IV, imaged by high fluorescence of F1 and F2.
* To whom correspondence should be addressed,
[email protected]. † These authors contributed equally to the study. Received for review: 11/24/2010 Published on Web: 12/17/2010
© 2011 American Chemical Society
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black-hole quenchers (Q1 ) Iowa black RQ and Q2 ) black hole 2) capable of quenching all fluorophores (F1-F4). The walker can bind or be released, under appropriate conditions, from all of the footholds, and its positions on the track are controlled by the relative stabilities of the duplexes generated between the walker and the respective footholds. In the initial state, the walker is stabilized on footholds I and II due to preferred hybridization. This is reflected by the quenching of the fluorophores F1 (λex ) 648 nm, λem ) 660-700 nm) and F2 (λex ) 545 nm, λem ) 555-580 nm) and high fluorescence intensities of fluorophores F3 (λex ) 680 nm, λem ) 700-750 nm) and F4 (λex ) 588 nm, λem ) 600-650 nm), as evident in the results shown in panel I. Treatment of the system with Hg2+ ions results in the walkover of the strand associated with foothold I to foothold III due to increased stabilization of the duplex between 6 and the sticky end of 3′ in addition to the base-pairing that is synergetically stabilized by the T-Hg2+-T bridging unit between the components.9 Accordingly, the fluorescence of F1 is triggered-on, the fluorophores F2 and F3 are quenched, and F4 is still switched on (panel II, right). The “walk-over“ process is a strand-displacement mechanism, where the resulting T-Hg2+-T bridges between foothold 3′ and 6 leads to a duplex of enhanced stability as compared to the duplex between 1′ and 6 that includes the respective A-T bases.9a (See Figure S1 in the Supporting Information for sequences and schematic design details.) Treatment of the system shown in panel II with cysteine results in the removal of the Hg2+ ions10 from the duplex structure on foothold III (due to the formation of the Hg2+-cysteine complex10), and, hence, the ”arm“ of the walker, represented by strand 6, walks back to foothold I, restoring the system shown in panel I. In turn, treatment of the system shown in panel II with H+ ions (pH ) 5.2) results in the rearrangement of strand 2′, into the i-motif,11 C-quadruplex, structure. This releases the walker ”arm” (7) from foothold II and enables its walk over to foothold IV that yields the duplex between the sticky end (4′ and 7), panel III. Accordingly, the fluorescence of F3 and the fluorescence of F4 are quenched, while the fluorescence intensities of F1 and F2 are high (panel III, right). Neutralization of the system shown in panel III dissociates the i-motif structure of strand 2′, thus favoring the step back of the “arm” (7) from foothold IV to foothold II, where the duplex formation, between 2′ and 7, is energetically stabilized (For optimization of the nucleic acid sequences, see Tables S1 and S2 in the Supporting Information). As we know from the fluorescence intensities of the fluorophores F1 to F4 in the absence of the “walker” elements (6-8) and the fluorescence intensity of the free fluorophores in the different walking steps, we can estimate the efficiency of translocation of the walker along the different footholds. We find that ca. 65 ( 2% of the 6 walk from foothold I to foothold III. The ”walk-over“ process of 6 to foothold I by means of cysteine proceeds, however, with an efficiency corresponding to 100%. Similarly, the unit 7 walks over to © 2011 American Chemical Society
FIGURE 2. Time-dependent fluorescence changes upon the reversible activation of the bipedal walker. The start rest position is when the “walker element” is bound to the footholds I and II. On the upper part of the figure, the respective inputs (Hg2+/cysteine or H+/OH-) that activate the “walker” are marked. The fluorescence of the four fluorophores (F1-F4) is simultaneously recorded in the presence of each input. The figure follows the forward (f), backward (r) and forward (f) walking processes.
foothold IV and back with an efficiency that corresponds to 82 ( 2%. Figure 2 shows the time-dependent fluorescence changes of all four fluorophores upon the cyclic forward and backward activation of the bipedal walker by the respective triggering signals. It should be noted that the fluorescences of the chromophores F1-F4 are unaffected by the pH changes from 7.2 to 5.2, and are not affected upon the addition of Hg2+, 1 × 10-5 M, or cysteine, 2 × 10-5 M. Also, we are able to activate the walker forward and backward for at least three times with no noticeable loss in the efficiencies of translocation of the ”walker” element. Careful examination of the fluorescence intensities upon the repeated activation of the walking process along the scaffolds with the footholds, Figure 2, indicates that the biggest fluorescence changes are observed for ROX (less than 6%, taking into account the dilution in each walking steps). While this phenomenon may be partially attributed to the partial bleaching of ROX, the results imply that a substantial number of “walking” cycles may be activated on the scaffolds. Realizing the mechanistic operation of the bipedal walker, one may envisage the design of a circular bipedal walker by linking the ends of the track and triggering the clockwise or anticlockwise motion of the walker. The configuration and generation of such a bipedal “stepper” is depicted in Figure 3. The system consists of a circular DNA (9) on which four footholds V-VIII are constructed. Footholds V and VI are parts of a nucleic strand (10) that includes the domain that hybridizes with the sequence R of the circle (9). Similarly, footholds VII and VIII are part of the nucleic acid strand (11) and they associate to the circle (9) through hybridization of the domain with the sequence γ of the circle. The nucleic acids 12 and 13 are hybridized with the other singlestranded domains of 9, in order to rigidify the circular 305
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FIGURE 3. Fluorescence analysis of the bipedal stepper activated by Hg2+/cysteine and H+/OH- inputs: state “A”, stepper associated with footholds V and VI, state reflected by high fluorescence of F3 and F4; state “B”, generated upon treatment of state “A” with Hg2+, resulting in high fluorescence of F1 and F4; state “C”, generated from state “B” by the addition of H+, resulting in the stepper “legs” on footholds VII and VIII, and reflected by high fluorescence of F1 and F2; state “D”, generated from state “C” by the addition of cysteine, resulting in the stepper “legs” on footholds V and VIII, and reflected by high fluorescence of F2 and F3. The figure also shows that state “D” can be generated from state “A” by the addition of H+, and that all states can be reversed by the application of the respective inputs.
structures (for the need to rigidify the structure, vide infra). The system includes several of the units that were used to design the “bipedal walker”, where the fluorophore-labeled strands 1′, 2′, 3′, and 4′ hybridize with footholds V, VI, VII, and VIII, respectively. The “stepper” unit consists of the subunits 14 and 15, which are rigidified by the duplex structures with the bis-quencher nucleic acid 8 and the strand 16. The “arms” of the subunits (14 and 15) include “sticky ends” that can bind, at the appropriate conditions (appropriate inputs), to the different footholds. The position of the “stepper” on the circle is then read-out by the quenching of the respective fluorophore. It should be noted that the rigidification of the circular structure of 9 by 12 and 13 is essential in order to prevent undesired quenching of the fluorophores in “collapsed” structures of the device. The cyclic operation of the bipedal stepper is depicted in Figure 3. In state “A” the bipedal stepper is linked to footholds V and VI, a structure that results in the quenching of F1 and F2, while retaining the high fluorescence intensities of F3 and F4. Treatment of the system with Hg2+ results in the clockwise “stepping” of 14 to foothold VII that yields state “B”, © 2011 American Chemical Society
where a duplex structure of enhanced stability between 14 and 3′ is formed due to the formation of Hg2+-thymine bridges. As expected, in state “B” the fluorophores F2 and F3 are quenched, while the fluorescence intensities of F1 and F4 is high. Treatment of state “B” with acid, pH ) 5.2, results in the folding of 2′ into the i-motif (C-quadruplex), leading to the release of the sticky end of arm 15 that binds favorably to foothold VIII to yield state “C”. In this configuration, the walking element moves clockwise, leading to the quenching of F3 and F4, while triggering-on the fluorescence of F2 and F1. The final state “D” is formed either by treatment of state “A” with acid and driving the anticlockwise stepping or by the treatment of state “C” with cysteine and stimulating the clockwise stepping. In state “D”, the fluorophores F1 and F4 are quenched, while the fluorophores F3 and F2 are switchedon. The four states “A” to “D” can be interconverted clockwise or anticlockwise using the appropriate triggers, Hg2+/ cysteineorH+/OH-.Thedynamicsofthecyclicinterconversion across the different states is shown in Figure 4. The sequences of the nuleic acids used to assemble the “walker” 306
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FIGURE 4. Time-dependent fluorescence changes upon the activation of the bipedal stepper: (a) in the clockwise direction; (b) in the anticlockwise direction. Both the stepper directions are initiated at the same state where the “pedals” are confined to footholds V and VI. The respective inputs that trigger the stepper are marked on the top of the figures. The fluorescence of all fluorophores F1-F4 is simultaneously recorded for each of the stepper states.
TABLE 1. List of Nucleic Acid Sequences and the Appropriate Labels in the “Bipedal Walker” and “Stepper” Constructs
* The color of each sequence corresponds to the respective strand shown in Figure 1 and Figure 3. © 2011 American Chemical Society
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and redissolved in 50 µL of ultrapure water; (iv) finally, the concentration of the solution was increased using a Microcon centrifugal filter (cut-off MW 10000). The concentration of DNA was evaluated with UV-vis microscopy. Operation of the Stepper. The stepper consists of 8-16 and 1′-4′, in which 9 existed in a circular configuration, Figure 3. The stepper was operated in a MES buffer solution (50 mM, that includes 500 mM NaNO3. This salt concentration was found to perform the activation of the walker in the best condition), which included 0.5 µM of each nuleic acid components. The solution was incubated at 90 °C for 5 min, and cooled instantly to 25 °C for 40 min to hybridize the respective components. Acetic acid (20 vol %) and ammonia (10%) aqueous solutions were added to adjust the pH of the solution for triggering the movement of the “arm” (7) (the pH oscillated between pH 5.2 and pH 7.2). To activate the “arm” (6) of the walker, Hg(CH3COO)2 and cysteine were added into the solution to generate 8.0 µM of Hg2+ for bridging the thymine units and 16.0 µM of cysteine for complexing Hg2+ ions. The operation of the stepper was conducted at 25 °C.
and the “stepper” are available in Table 1, which also provides the optimized sequences for the processing of the “walker”. In conclusion, the present study has introduced Hg2+/ cysteine and H+/OH- as triggers for the activation of bipedal walking or stepping within supramolecular DNA nanostructures. The mechanical devices enable the cyclic and reversible, forward, and backward walking/stepping of the molecular units. By the introduction of additional triggering signals, for example, other ions and synthetically modified DNA (lingandosides),9b other mechanical DNA devices of enhanced complexity may be envisaged. Furthermore, the incorporation of such DNA devices into cells may provide a means to sense pH changes6f or harmful ions. Experimental Section. Materials. 2-(N-Morpholino)ethanesulfonic (MES) acid potassium salt, MES acid hydrate, sodium nitrate, and mercury(II) acetate were purchased from Sigma-Aldrich, Inc. L-Cysteine hydrochloride was purchased from Fluka, Inc. Modified DNA oligonucleotides 1′-4′ and 8 were purchased from Integrated DNA Technologies, Inc. All other oligonucleotide sequences were purchased from Sigma-Genosys. Quick Ligation Kit (M0202L) was purchased from New England Biolabs (NEB). Ultrapure water from a NANOpure Diamond (Barnstead) source was used in all of the experiments. Instrumentation. Light emission measurements were performed using a Cary Eclipse fluorimeter (Varian Inc.). The excitations of Cy3, ROX, Cy5.5, and Cy5 were performed at 545, 588, 680, and 648 nm, respectively. Operation of the Walker. The walker was operated in a MES buffer solution (50 mM, that includes 500 mM NaNO3), including 1-8 and 1′-4′, 0.5 µM each. The solution was incubated at 90 °C for 5 min and cooled instantly to 25 °C for 40 min to hybridize the respective components. Acetic acid (20 vol %) and ammonia (10%) aqueous solutions were added to adjust the pH of the solution for triggering the movement of the “arm” (7) (the pH oscillated between pH 5.2 and pH 7.2). To activate the “arm” (6) of the walker, Hg(CH3COO)2 or cysteine was added into the solution to generate 4.0 µM of Hg2+ for bridging thymine and 8.0 µM of cysteine for complexing Hg2+ ions. The activation of the walkers was performed at 25 °C. Preparation of the Circle Template (9). To prepare the circle template, 2.0 µM of 9 was treated with 5.0 µM of ligation template 12a in Quick ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM dithiothreitol, pH 7.5) for 40 min. The synthesis was completed with the Quick Ligation Kit, using the manufacturer-supplied protocol. The enzyme was denatured by heating at 65 °C for 10 min. The circlular DNA (9) was then extracted from the solution using the ethanol extraction DNA protocol: (i) 150 µL of cold ethanol (kept in ice) was added to 50 µL of the circle/ligation template solution, which was then stored in the freezer (-20 °C) for 12 h; (ii) the solution was centrifuged at 10000 rpm at 4 °C for 30 min; (iii) the white precipitate was separated © 2011 American Chemical Society
Acknowledgment. Part of this research was supported by the MOLOC EC project and by the Volkwagen Stiftung, Germany. Supporting Information Available. A figure depicting the sequences and the schematic design of the bipedal walker system and tables of different sequences of a DNA arm to optimize the operation of the bipedal walker. This material is available free of charge via the Internet at http://pubs. acs.org. REFERENCES AND NOTES (1)
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