The extended actuator
Herein, we report on the extension of the scope of the DNA actuator by demonstrating that the actuator structure and mechanism can be applied to more complex devices. First, we have investigated the linear extension of the actuator. The extended actuator design and three-dimensional (3D) structure is shown in Figure 2 and it consists of two actuators each with their unique sequence design. The two actuators are fused by a third bridging roller strand. The bridging roller strand (Roller X) shares sequence homology with each of its neighbors and thus allows sliding motion of the actuator.
Figure 2. The extended linear DNA tile actuator in (a) a 3D illustration and (b) schematic illustration of the system locked in state 0, (c) state 5, and (d) state 11. Black marks regions of the strands with unique base sequences that only hybridize in one way that is static regions, or in the case of black piston termini, locking regions. Colors mark dynamic regions where the roller strands can base pair with each piston region of the same color. Vertical lines only illustrate connections, not nucleotides. Positions of fluorophores for FRET experiments are marked with stars in the schematic illustration and with cubes in the 3D rendering. Green is for Cy3b and magenta is for Cy5.
Download figure to PowerPoint
While the original actuator was designed with identical sequences in the two locking regions to enable use of the same lock strand (L) in both ends, the two ends of the extended actuator have different designs. Here we want to explore if locking the DNA actuator in one end of the structure will induce motion at the other end of the structure. Thus, in our experiments, we input a lock strand in the right hand end of the actuator (Figure 2) and monitor a change in conformation of the structure through a change in the FRET signal at the other end of the actuator.
The sequences were constructed utilizing a Python 2.7 script written for the purpose (see the Supporting Information). The script was designed to minimize unwanted complementarities while also suppressing the sequential occurrence of the same base more than twice in otherwise randomly chosen base sequences. All dynamic base regions (colored regions in Figure 2 b–d) were set to be 10 nt in length and Rollers 1 and 2 were fixed to a total length of 64 nt each. These lengths were consistent with the design of the original actuator.19 Roller X was given a length of 62 nt, and the lock strands a length of 20 nt each with the crossover set to be halfway into the sequence (see the Supporting Information for further information and sequences). The total length of the double stranded region of the extended actuator, including the lock strand is 10 helical turns, corresponding to 35 nm and the minimal distance from the dyes to the lock strand is 7 helical turns corresponding to 18 nm.
To verify that the extended actuator was correctly assembled it was analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE; Figure 3). The unlocked actuator monomer (Piston 2, 4, and Roller 2) is shown in lane 1 and the locked actuator monomer (S0) is shown in lane 2 and there is a clear mobility shift. The unlocked extended actuator can be observed in lane 6 and the locked version in lane 7. However, owing to the size of the extended actuator the insertion of the lock strand only results in a minor mobility shift. For the data shown in Figure 3, lanes 2, 3, and 7, the lock strands were nicked at the half-crossover and one of the two lock strands functionalized with biotin (lane 4). Upon binding to streptavidin the mobility of the lock strand is significantly retarded. When the lock-biotin-streptavidin strand was used with the actuator monomer (lane 3) and the extended actuator (lane 8) the binding of the lock strand was clearly verified by the decreased mobility of the complexes.
Figure 3. PAGE analysis (7.5 %) of the actuator monomer with Roller 2 and the extended actuator locked with biotin-modified nicked lock strands and streptavidin was added: Lanes from left to right contain: (1) unlocked actuator monomer (Roller 2), (2) Locked monomer, (3) Locked monomer with streptavidin, (4) Lock strand, (5) Lock strand with streptavidin, (6) unlocked extended actuator, (7) Locked extended actuator, (8) Locked extended actuator with streptavidin, and (9) a 100–1000 bp ladder on the far right. PAGE analysis (6 %) of the extended actuator locked with biotin-modified U-lock strands and incubated with streptavidin: lane 10) state S0, 11) unlocked, 12) state 5, 13) unlocked, and 14) state S10. The last lane 15) contains 25–500 bp DNA ladder.
Download figure to PowerPoint
The locking of the extended actuator in states S0, S5, and S10 is shown in Figure 3, lanes 10–14. The actuator was locked with biotin-modified lock strands and incubated with streptavidin before PAGE analysis. In this case the non-nicked U-lock strands were applied. In particular for the locked actuators (lanes 10, 12, and 14), independent of the locked state, we observed some weaker bands with lower mobility, which may arise from dimerization and/or aggregation of the extended DNA actuator.
To ascertain that the different lock strands lead to the organization of the extended actuator in the expected states, FRET experiments were performed for the actuator locked in each of the possible twelve states. The fluorophores Cy5 and Cy3b were conjugated to piston strands 1 and 3 in positions placing them opposite each other when the dimer was locked in state S0 and furthest apart in state S11 (Figure 2). Because the lock strands hybridize with piston strands 2 and 4, the FRET data would thus only reflect a change in states if the locking induced a sliding motion which propagates across the Roller X link between the two actuators.
The observed FRET values are shown in Figure 4 a along with the theoretical FRET data in Figure 4 b, (see the Supporting Information for calculation details). There is generally a good qualitative agreement between the experimentally observed and theoretical FRET data. The amplitude changes of the FRET signal are relatively low compared to the expected values and this may be because only a certain share of the actuators undergoes the expected structural changes. This may, at least in part, be due to aggregation, as some undefined structures and aggregates are seen in lanes 10, 12, and 14 in Figure 3.
Figure 4. a) Relative FRET values for the 11 different states of the actuator using the dyes Cy5 and Cy3B that are each positioned on the Pistons 1 and 3 and located 16 nt from the left hand half crossover of the roller. b) Theoretical FRET values calculated based on our theoretical model, using R0=7.0 nm.
Download figure to PowerPoint
The FRET data show a non-monotonic dependence when locked from states S0 to S11 with a local minimum in state S6. The smallest FRET value is found for state S11 as expected. The local minimum in the FRET data relates directly to the rotational element in the movement of the fluorophores. A minimum at S6 is achieved in the model when fluorophores are rotated approximately 30 ° away from pointing directly toward each other (see the Supporting Information). In addition, a twist in the actuator plane resulting from torsional tension can contribute to a shift in the minimum.
The increase in signal from S0 to S4 in the observed FRET data may arise from the electrostatic repulsion between the DNA helices. If the model only takes into account the rotation of the fluorophores around the helices and the displacement along two parallel helices, the fluorophore–fluorophore distance is approximately constant in the first four states, S0–S3. However, the theoretical model where the helices are assumed to touch at the half-crossovers (an approximate helix center distance of 2 nm) and be 4 nm further apart at the midway point between half-crossovers results in an increase in the FRET values from state S0 to S2 as illustrated in Figure 4 b. In other words, the repulsion between the DNA double strands leads to a larger distance between the fluorophores in state S0 than in state S2. Our simplified model reproduces the observed FRET values which increase from states S0 to S3. The observed and modeled FRET values show similar trends which are consistent with the designed actuator structure at the molecular level.
The FRET results show that it is indeed possible to regulate the position of the actuator by using different lock strands that mechanically regulate the relative position of a FRET pair located 18 nm from the closest parts of the lock strands.
To further explore the scope of the actuator principle,19 we have investigated the possibility of expanding the structural features of the linear DNA tile actuator into triangular and quadrilateral actuators. The designs of the two new actuators are shown as 3D models in Figure 5. For simplicity and to form the actuators with a minimum of sequences, the two actuators were designed with maximal symmetry applying piston strands that are identical in all edges of the structure. The triangular actuator was designed with a roller strand of 96 nt containing three repeated regions of 32 nt complementary to the piston, and the quadrilateral actuator was formed with a roller strand of 128 nt containing four repeated regions of 32 nt complementary to the piston. In both cases the piston strands are 64 nt long and for each half-crossover there is 11 homologous bases around the junction providing the structures with their dynamic rolling ability.
Figure 5. 3D models of the triangular and quadrilateral actuators. In both models the roller sequence is shown in yellow, the identical piston strands are shown in blue, and the lock strands are illustrated in green. Symmetry around the half-crossovers allows the actuator to slide between states, defined in the absence of the lock strands and the lock strands enable the locking of the two actuators in 11 different positions.
Download figure to PowerPoint
To lock the actuators in specific positions and to demonstrate the ability to shift the actuator between states we have used lock strands (L) with a toehold for positions S0, S5, and S10. Each lock strand can hybridize with a part of both pistons extending from the half-crossovers. The toehold of the lock strand makes it possible to remove the lock strand again by applying a strand complementary to the lock strand (LC). This makes it possible to switch between states. The locked triangular and quadrilateral actuators are designed so that the helices are organized in one plane, which implies that the helices of the pistons are forced to bend at the junctions to allow for the half-crossover of the lock strand that locks the actuator. As shown below the triangular and quadrilateral actuators are indeed formed, however it should be noted it is not clear if the actual structures are planar as shown in Figure 5 or if they are twisted/bended owing to the intrinsic strain.
First, we investigated the formation of the triangular actuator. A number of titration experiments were performed to investigate the exact stoichiometry. Second, the magnesium concentration was varied from 1 mM to 30 mM (see Figure S4 in the Supporting Information). At low magnesium concentrations significant amounts of the complex with only two pistons (Piston/Roller=2:1) were observed. At magnesium concentrations of 12.5 mM and above, the complex with three pistons (Piston/Roller=3:1) was abundant and 12.5 mM was chosen to avoid unnecessarily high salt concentrations in PAGE analysis, as well as aggregation problems.
To test the dynamic locking and unlocking of the actuator, the initial piston and roller 3:1 complex (PR) was first formed separately, followed by consecutive addition of L0. The mixture was then heated to 37 °C for 20 minutes while shaken, resulting in the formation of the locked complex, S0, with a lower electrophoretic mobility. Subsequently, the complementary LC0 was added, unlocking the system and regenerating the PR complex. The unlocked actuator is locked in S5 by addition of L5, and subsequently it is unlocked by addition of LC5. Finally, L10 generated the locked complex S10 and LC10 regenerated PR. Initially, the experiments were performed with lock sequences containing a 10 nt toehold extension, however, this resulted in problems with aggregation of the structures and low quality of the PAGE. As an alternative, we used lock strands with only 5 nt toeholds in the 3’ end, which resulted in successful formation of the locked and unlocked complexes. The resulting assembly of the triangular actuator is shown in Figure 6. Lanes 2 and 3 contain the piston and the roller sequences, lane 4, the PR complex. The band just below the formed three-way complex is a complex with only two pistons. Additional unhybridized pistons were observed in the bottom of the lane. Lanes 5–10 demonstrate the locking and unlocking of the actuator progressing with the same sample from state 0 to state 10. In lanes 5–10 some additional bands were observed, and as indicated in the right of the figure, these are believed to be excess piston and piston hybridized with excess lock strands.
Figure 6. Non-denaturing PAGE (7.5 %) of the triangular actuator. Lock strands with 5 nt toehold regions were used, resulting in the formation of all the locked and unlocked complexes. Lanes from left to right contain: (1) 25–500 bp ladder, (2) piston, (3) roller, (4) unlocked PR, (5) Locked PR complex S0, (6) unlocked PR, (7) Locked S5, (8) unlocked PR, (9) Locked S10, and finally (10) unlocked PR.
Download figure to PowerPoint
The locked and unlocked complexes were formed successfully as shown in Figure 6, however, for some of the locked structures, various aggregation products were observed. This may result from lock strands inter-linking the actuator complexes. Indeed, the inherent bending of the piston helices at the roller half-crossovers is caused by the lock strand half-crossover, and this strain may favor the sharing of the lock strands with another actuator, causing dimerization and polymerization of the actuators. To test this hypothesis, we nicked the lock strand at the half-crossover and thus used two half lock strands to lock the actuator. PAGE analysis of the actuator formed with the nicked lock strands showed significantly less aggregation (Figure S5). However, the actuator is not securely locked when the lock strand is nicked due to the homology around the half-crossover. In this case, the crossover can be considered as a traditional Holliday junction in which one sequence is nicked. The position of the actuator that places the nick at the junction is probably the most stable; however, we have no evidence to confirm this.
The quadrilateral actuator was formed by extending the roller sequence to 128 nt, introducing an additional 32 nt unit with complementarity to the piston. The symmetric four-way junction was expected to be formed from four pistons and one roller stand. As for the triangular actuator, the quadrilateral actuator has sequence homology around the half-crossovers allowing the actuator to slide between states defined by the lock strands. The dynamics of the quadrilateral actuator was demonstrated using lock strands with 5 nt toehold regions (Figure 7 a). Formation of the unlocked actuator complex and insertion and removal of lock strands was demonstrated by PAGE. The non-denaturing PAGE showed the formation of all the locked and unlocked complexes, however, it was accompanied by a significant degree of aggregation and partly folded actuators. Thus, it was also attempted to form the actuator with the nicked lock strands for the quadrilateral actuator and as shown in Figure 7 b the PAGE analysis revealed much cleaner products through the insertion and removal of lock strands.
Figure 7. Non-denaturing PAGE (7.5 %) of the quadrilateral actuator (a) formed with full length locks with 5 nt toehold regions (b) formed with the nicked lock strands each with 5 nt long toehold regions. Lanes from left to right contain: (1) 25–500 bp ladder, (2) piston, (3) roller, (4) unlocked PR, (5) Locked PR complex S0, (6) unlocked PR, (7) Locked S5, (8) unlocked PR, (9) Locked S10, and finally (10) unlocked PR.
Download figure to PowerPoint