Chapter IV: A cargo-sorting DNA robot
4.3 Demonstration of the robot picking up cargos
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.
state. One inactive robot per origami is also localized on the origami. All goal locations were inert in this setup, meaning the origami was annealed with staples without extensions for the goal location.
Figure 4.3: Domain level representation for cargo pickup. a, Basic mechanism of activating the robot using a robot trigger. The active robot then picks up cargo1 via toehold hand b, and similarly cargo2 via toehold hand, separating the cargo from the quencher molecule on the attacher strand.
Figure 4.3a is a domain level diagram showing an inactive robot and cargo1-F in a quenched state bound to its attachment strand with a quencher. Once the robot trigger is added,rttoehold binds to the robot inhibitor strand and strand-displaces it from the robot. The triggering reaction is a reversible strand-displacement reaction as thehandtoehold is still available on the "waste" complex to re-initiate the robot inhibition reaction. Addition of a large excess of trigger strands (20x trigger strands to 1x robot), ensures the reaction is forward biased. The active robot useshandtoe-
hold domain to initiate strand-displacement reaction separating the cargo1-F from its attachment strand. The cargo pickup is an irreversible strand-displacement reac- tion via a 6 nt toehold. Once the robot and cargo are bound, the attachment strand is left inert. Figure4.3b shows the domain-level design of cargo2-F pickup by the robot. The mechanism is the same as cargo1-F with thehandacting as the initiation toehold for the pickup reaction.
a
b
O1 (Origami with cargo1)
O2 (Origami with cargo2)
Single cargo1 picked up
Single cargo2 picked up
Excess robots added
Excess robots added
initial state example final state
initial state example final state
Figure 4.4:Demonstration of cargo pickup. a,Layout of position of cargo1, cargo2 and the robot on a schematic of the DNA origami.b,Fluorescence kinetics data of robot picking up cargo1 and cargo2 performed as separate experiments. Each origami has a single robot that picks up a single cargo leaving behind 2 quenched cargoes per origami.
Figure4.4a shows the layout of quenched cargo1-F on the origami. Goal positions are inert. The robot is fixed at the center, initially inactivated. The robot is sta- tionary and inactive, as the domains necessary for walking and cargo-pickup are inhibited. Once activated, the robot can explore the entire testing ground of the origami surface and can pick-up cargo at any location. When the robot reaches a cargo molecule, the cargo gets picked-up and is no longer in the quenched state (as indicated by the star-like fluorescence signal on the robot). Each origami carries a single copy of the robot, and in the absence of goal locations, picked-up cargo cannot be dropped-offand hence remains on the robot. In this setup, the robot is incapable of performing further iterations of pick-up and hence two of the three available cargo on each origami remain quenched as seen in the schematic.
Figure4.4a also shows the fluorescence kinetics data for cargo1-F being picked up by the robot after being activated by the trigger molecule. Y-axis represents cargo1- F concentration. As seen in the schematic, each origami carries three copies of cargo1-F and thus the total cargo concentration is 9 nM. The kinetics data shows three stages of equilibrium. The first stage is with all copies of cargo1-F quenched while bound to its attachment strand. After establishing the quenched state fluores- cence of cargo1-F for ~1hr, 20x (60 nM) of robot trigger was added to activate the robot.
Once the robot is activated, it begins its random walk exploration to pickup cargo1- F which is seen as the fluorescence signal increase until an equilibrium is reached at ~3 nM. The increase in fluorescence is due to the fluorophore on the cargo1-F molecule separating from the quencher on its attachment strand. With the absence of goal molecules, the robot and cargo1-F remain bound and hence 2x of cargo1-F molecules remain at their initial location, in a quenched state (hence only increasing fluorescence signal to 3 nM, instead of 9 nM). A large excess (200x) of free-floating active robots was added at approx 20 hr time point to unbind remaining cargo from their attachment strands, resulting in the increase in signal to 9 nM. The final com- pletion level is helpful in estimating the fraction of cargo that was picked-up by the random walking robot and confirming the presence of roughly three copies of cargo molecules on each origami.
The above test was repeated to observe pick-up reaction for cargo2-F (fig. 4.4b).
The schematic shows the location of cargo2-F initially bound to its attachment site with a quencher molecule and an inactive robot. When activated, the robot performs its exploratory walk of the test ground and picks up a single copy of cargo2-F.
The absence of goal molecules leaves the picked-up cargo2-F bound to the robot, leaving two copies of cargo2-F bound in their initial quenched state, evident from the fluorescence kinetic data.
The kinetics of pick-up of cargo1-F and cargo2-F show qualitative similarity in reaction rate. The overall pickup rate is similar in both cases for two reasons, the first where the mechanism of cargo pick-up being initiated via hand toehold and completed viaarm domains for both cargo types. The second being the distance between the cargo in both cases, where the robot maybe within reachable distance from either cargo location. With these design considerations, we expected to see no bias in the robot performance for either case. Taking the total concentration of the cargos from the data into consideration, we observe 30.54% of cargo1-F and 29.1%
of cargo2-F being picked-up by the robot in the two separate tests conducted, thus demonstrating unbiased pick-up mechanism by the robot.