Chapter IV: A cargo-sorting DNA robot
4.2 Molecular implementation of the algorithm
This section describes implementing cargo sorting system using DNA-based molec- ular components. Using schematics, domain level descriptions and AFM character- ization, we hope to paint a picture of the robotic system designed to implement cargo-sorting.
The cargo-sorting robot is a single stranded DNA (53 nt) and addition of track locations covering a larger area of the nanostructure and addition of cargo and goal locations changes the system previously used for random-walking to a cargo-sorting system. The robot has a leg and two foot domains for walking and a hand and arm domain for picking up and dropping off cargo. Figure 4.2a is a schematic representation of the distribution of two different types of cargos and their respective goals. The scheme only shows the the top-layer of the double layer DNA origami (Appendix, fig.4.14). Staple ends on either layer of the double layer origami can be extended to form tracks and localize molecules. However, the staple design of the double-layer allows for one layer to have more possibilities of extensions than the other. We call this the top-layer. The top-layer has 95 3’ staple extensions, of which 6 locations are used to localize cargo molecules, 8 locations are used to localize goal molecules, 1 location to localize the robot allowing 70 locations to be used as tracks. The edge staples have hairpin motifs used to reduce stacking interactions
between nanostructures, and hence are not part of the cargo-sorting test ground.
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Figure 4.2: Mechanism of cargo-sorting. a, Schematic representation of cargo-sorting playground on DNA origami. Two different types of cargos and their respective goals are distributed on the opposite edges of the origami and the robot is fixed in the center at start.
b,AFM of the DNA origami showing cargo and the two different goals along the opposite edges of the structure. c, Domain level details of the two types of cargo and goals shown bound to the 3’ extensions of the origami staples. d,Basic mechanism of robot picking up cargo via the hand toehold from the cargo attacher ande, the basic mechanism of cargo drop offby the robot at the goal via the cargo toehold in an irreversible strand-displacement reaction.
The two types of cargo molecules are distributed interspersed with each other, along the top edge of the origami (fig.4.2a). The corresponding types of goal molecules are distributed on the opposite edge of the origami from the cargo and are separated
into individual piles as shown. While there are three cargo molecules of each type, there are four goal locations for each cargo type. The goal redundancy was included to ensure each cargo molecule has a goal, and that lack of a goal position (maybe due to lack of a staple incorporation into the origami) would not be the reason for incomplete sorting. The robot starting position was fixed symmetrically between the cargo and goal locations. The white dots on the schematic are 3’ staple exten- sions forming track1 and track2 as defined in the random walk system and cover the 2-dimensional origami surface as opposed to a linear track in the random walk system. The edge staples are omitted in the schematic to only show usable tracks.
Figure4.2b is the AFM image of the cargo-sorting playground. While depositing the origami sample on a mica substrate for imaging, the structures can land face- down (with extensions facing mica) or face-up (with extensions facing solution, away from mica). Structures that land face-down have staple extensions trapped between the mica and the origami. When imaging such structures, the DNA exten- sions produce higher contrast compared to the nanostructure. Structures that land face-up have staple extensions facing the solution resulting in some mobility of the extensions, thus may not register as high contrast as the trapped extensions. The AFM in fig.4.2b shows origami lying "face-down" on the mica substrate. This par- ticular DNA origami sample was annealed without tracks so the position of cargo and goals could be identified. The schematic in fig.4.2a shows cargo locations dis- tributed along the top-edge of the origami, with the first cargo location 12 nm from right edge of the origami, and the last cargo location 9 nm from the left-edge. The asymmetric distribution of cargo along the top-edge serves as a label to identify the right and left edges of the nanostructure while analyzing AFM images. Goal1 loca- tion is on the left bottom edge and goal2 on the right bottom edge of the origami.
Figure4.2c is a domain level description of the cargo-sorting system. Each track has a foot (f) as the toehold domain, and leg (l) as the branch migration domain.
The robot moves from track1 to track2 reversibly using the mechanism previously described (fig.3.2). The DNA sequence of the cargo-sorting robot is identical to the random-walking robot, without the quencher molecule on the 3’ end. The domains used for triggering the random-walking robot (hand (h),arm (a)) are also used for cargo-pickup. The h* (initiation toehold) and a* (branch migration) domain on both cargo-types are identical and hence indistinguishable by the robot. The two types of cargo have two spectrally different fluorophores on the 3’ end. Since the fluorophores are chemically different molecules, they may influence cargo-pickup.
At the domain level, the cargos are differentiated by thecargo1*(c1*) andcargo2*
(c2*) domains on the 5’ ends. These domains encoded in the cargo help identify the corresponding goal with its complementary domain. Both types of goal molecules have theh, adomains but differ by havingc1domain for goal1 (complementary to cargo1∗) andc2domain for goal2 (complementary tocargo2∗). The two domains act as identifiers for ensuring desired cargo-goal interaction.
The hand (h) toehold domain on the robot initiates strand-displacement reaction that picks up cargo (fig. 4.2d). The cargo binds to the robot via thehand andarm complimentary domains in an irreversible strand displacement reaction. The cargo- attachment site is left inert after transfer of cargo. In principle, the cargo should not interfere with the robot walk as the domains required for random walk are indepen- dent of the domains carrying the cargo molecule. Robot carrying a cargo molecule cannot pick up another until the cargo being carried is dropped offat the goal. The cargo dropping offmechanism is shown in fig.4.2e. During its random walk explo- ration when the cargo carrying robot bumps into a goal location, the cargo molecule on the robot identifies the goal via thecargo1 (c1)orcargo2 (c2)domain and binds irreversibly to its corresponding goal using complementary domain. After the cargo is dropped off, the robot continues its exploration and is free up to pick up another cargo. There is minimal interaction of robot and goal as they do not have comple- mentary domains.