Chapter III: A random walk module for DNA robots
3.2 Track design for the random walk
The random walking robot combines dynamic DNA nanotechnology with structural DNA nanotechnology. The robot walks on the origami surface employing DNA strand-displacement technology to travel from one track localized on the origami to the next. DNA staples used to fold the origami structure have 3’ extensions (all extending from the same side on the origami) acting as tracks for the robot. To ensure bi-directional walk for the robot, two types of tracks were designed, track-1 and track-2. Track-1 and track-2 are both interspersed in the design fig.3.2a.
All staples for folding the origami get incorporated with high probability. How- ever, there can be some staples missing when the structure is annealed. In order to account for missing tracks, we use a linear track with width 3, so the robot does not get "stuck" in a location unable to progress towards the goal. Furthermore, we hypothesize that having 3 rows of tracks makes the central row more accessible to the robot as the border rows may provide sufficient electrostatic repulsion for the
central row of tracks to remain upright, which, in our opinion, could increase the efficiency of strand-displacement of the robot on tracks.
The robot makes a step by strand-displacing from track type one to the other. The two track types can be distinguished by the toeholds encoded in them (foot1*for track-1, foot2*for track-2). The robot contains both toeholds enabling it to make consecutive steps. For example, if the robot is on track-1 (fig.3.2b), its toehold do- main corresponding to track-1 (foot1*) is occupied and hence the next possible step for the robot would be onto track-2 usingfoot2*as the initiation toehold (fig.3.2c), branch migrating and finally disassociating via toehold on track-1, foot1*. Thus, the free toehold on the robot determines the next step of the robot. The track layout for implementing random walk is a 1-dimensional path with width 3, including a
"start" location and a "goal" location for the robot. The goal location is fixed for all experiments while the start location is varied to allow testing for different track lengths.
Earlier studies by Zhang et al. [71] showed that by varying the strength (length and sequence) of toehold length, the rate of strand displacement reaction (untethered in solution) can be controlled over a factor of 106, and begins to saturate with toehold length 6-7 nts for tested sequences. Toehold length in our study was chosen to be 6 nt to provide fast strand-displacement rate. The DNA sequences were chosen from a pool generated using a 3-base sequence (A, C and T) to reduce spurious interaction. Each track has afoot and aleg domain extending above a short poly- Thyamine (T) spacer above the surface of the DNA origami.
The spacer length was calculated based on the necessary length required for the track with robot to be able to make contact with the toehold on the adjacent track.
Using length of ssDNA as 0.43 nm, dsDNA as 0.34 nm and distance between two neighboring track locations as 6 nm, a spacer length corresponding to 5 nt was cal- culated for the track. Track-1 has the complimentary foot1* close to the origami surface and track-2 has thefoot2* domain further away from the origami surface.
5-Thyamines and 11-Thyamines were used as a spacer for track-1 and track-2 re- spectively. The six additional Ts were added to compensate for the foot position being higher with respect to the origami surface on track-2. The spacer lengths thus calculated allow the toeholds on the robot and the track to be aligned ensuring that the stands do not have to overextend in order to initiate and complete strand displacement (fig.3.2c).
In addition to the walking domains, the robot also has hand and arm domains,
which, for the purposes of the random walk module is only used to keep the robot stationary (inactive) at its start position (fig. 3.2d). The staple location on the origami chosen as the "start" location for the appropriate track length has a 5’ ex- tension of 20 nt. The tri-molecular robot start complex is localized onto the origami post-anneal via hybridization. The tri-molecular complex consists of (a) the robot;
(b) the robot-probe that hybridizes to the origami via the 20nt complementarity to the start location while also occupying foot1 toehold; and (c) the robot-inhibitor strand, which keeps the robot locked at the start location by occupying foot2toe- hold (fig.3.2d) The inhibitor strand also has a free toehold to aid in activation of the robot. Thus the robot in this state is stationary and inactive. Addition of a large ex- cess of the robot-trigger molecule removes the robot-inhibitor strand via a forward- biased toehold-mediated strand-displacement reaction. The triggering step frees the foot2activating the robot, enabling it to make a step. The robot moves from track-1 to track-2 reversibly until it reaches the goal. The goal molecule is a modified track and has both foot1andfoot2. Hence the robot reaching the goal is an irreversible strand displacement reaction. This reaction is monitored via fluorophore-quencher interaction between the robot and goal using a fluorescence spectrophotometer.
Figure 3.2: The random walk track design. a, Scheme of DNA origami surface with track layout for randomwalk. Pink and blue dots represent 3’ extension of staples acting as two types of tracks separated by 6 nm. The robot start location is represented by blue concentric circles with a cross running through. The robot goal location is represented by the white circle outlined in blue. b, Sequence and domain level representation of track 1 and track 2. The toehold domainsfoot1andfoot2are 6 nt each and the strand-displacement domainlegis 15 nt. Distance between two tracks on the origami surface is 6 nm and link- ers s1and s2are calculated to ensure that the robot can reach the necessary toe-hold to strand-displace between track 1 and track 2. c, Mechanism of toe-hold mediated strand- displacement reaction of robot from track 1 to track 2. The linkerss1ands2stretch and contract to aid displacement. d, Robot-start complex showing the inhibited form of the robot. Addition of robot trigger strand displaces the inhibitor strand reversibly. The ac- tivated robot has foot2toehold to start random walk on the track. e, The robot goal is a modified track with bothfoot1andfoot2so the robot is irreversibly bound to the goal.
In the process of implementing random walk, we experienced several challenges that influenced our design and experimental protocols. The observations made dur- ing this progress which eventually lead to a successful implementation of random walk were:
• Rigidity of DNA origami as a testing ground for the robot: Structural fluctu- ations of DNA origami in solution, influencing the extent of undesired reac- tions on its surface.
• DNA sequence of foot domains of the robot:influencing the rate of walking.
• Purity of DNA origami: Fraction of partially formed nanostructures interfer- ing with well-formed structures, influencing the completion level of desired reactions.
Each of the above observations are explored in more detail below.