All of the essential mechanisms in our system were verified in solution using
polyacrylamide gel electrophoresis. These mechanisms include:
walker-track binding,
triggering the walker,
walking from one track to another,
picking up cargo,
walking while carrying cargo,
triggering the cargo goal,
dropping off cargo, and
irreversibly walking from tracks to the walker goal.
Each gel has several control lanes (marked in green), where control
lanes are either single strands, or previously verified combinations of strands.
Positive controls are marked with (+) below the lane number, and negative
controls are marked with (-) below the lane number. We define a negative
control as one that should not appear in any reaction in that gel, even though
some may in practice appear due to stoichiometric errors etc. Lanes that
test the binding of certain strands are labeled in blue, whereas lanes that
test strand displacement reactions are labeled in red. Null experiments (ones
where we expect no reaction to occur, but provide comparisons for reactions
where we expect the end result to be the same or similar) are labeled in
black. All inputs in a lane were intended to be equimolar unless otherwise
noted. Probes were used when it seemed necessary to distinguish strands
or complexes of similar length (for example PTR2 was used with track 2
to distinguish it from track 1, although we found that TR1 always appears
slightly higher in the gel than TR2, and likewise when they are bound to the
walker, perhaps due to some minor secondary structure).
While the mechanisms all appear to behave properly in solution, there
were a few mysteries, which may or may not matter as the mechanisms are
translated onto origami. One such mystery is the apparent absence of TR2-PTR2
in lanes 6 and 7 of gel 4. There a couple possibilities
for this. One is that it is just very low intensity, and since it should be
close to the bands for W-TR1-C1, it blends in. Another is that there were
stoichiometric issues, so almost all of the TR2 is in W-TR2-PTR2-C1. In
any case, it does not seem possible for it to not exist if stoichiometry was
correct, given the results in those two lanes. Another mystery is that in
lanes 7 and 8 of gel 6
the bands that correspond to the walker
appear somewhat higher than expected. No logical explanation can be found
for this, and it seems ignorable, except for the fact that in a similar gel using
old cargo and cargo goal strands, the same effect appeared! Finally, in lane
4 of gel 7,
the walker goal appears much higher than a 47
nucleotide strand should appear (compare to the tracks). This was also seen
in other gels, which suggests there is probably some polymerization of the
strand (or otherwise some secondary structure), but since the rest of the gel
suggests the walker goal behaves as expected, it will be ignored.
Ability of the walker to bind to its tracks and the walker triggering mechanism.
Lane #
Input and Expected Reaction
Information from the gel
W | Control
Lane 2
TR1
Control
Lane 3
TR2
Control
Lane 4
WI
Control
Lane 5
WT
Control
Lane 6
W + TR1 → (W-TR1)
Walker binds to one of the track strands
Lane 7
W + TR2 → (W-TR2)
Walker binds to the other track strand
Lane 8
W + WI → (W-WI)
Walker Inhibitor Binds to the Walker
Lane 9
WI + WT → (WI-WT)
Walker Inhibitor Binds to the Walker Trigger
Lane 10
W + TR1 + WI → (W-TR1-WI)
Track binding to the Walker does not interfere with the binding between the Walker and Walker Inhibitor
Lane 11
(W-WI-TR1) + WT → (W-TR1) + (WI-WT)
Walker Trigger strips the Walker inhibitor off of the Walker
Gel 2
Random walking mechanism and initiation of walking by triggering the walker
Lane 1: W : Control
Lane 2: TR1: Control
Lane 3: TR2: Control
Lane 4: PTR2 : Control
Lane 5: TR2 + PTR2 → (TR2-PTR2): Track 2 binds with its probe. This will allow us to distinguish between Track 2 and Track 1 in future experiments.
Lane 6: W + (TR2-PTR2) → W-TR2-PTR2: Track 2's probe does not interfere with the binding of the Walker to track 2.
Lane 7: (W-TR1): Control
Lane 8: (W-TR1) + (TR2-PTR2) ↔ (W-TR2-PTR2) + TR1 : Walker can move between tracks, specifically from Track 1 to Track 2. The system should equalize with walkers on both tracks.
Lane 9: (W-TR2-PTR2) + TR1 ↔ (W-TR1) + (TR2-PTR2) : Walker can move between tracks, specifically from Track 2 to Track 1. The system should equalize with walkers on both tracks.
Lane 10: (WI-WT): Control
Lane 11: (W-WI): Control
Lane 12: (W-TR1-WI): Control
Lane 13: (W-TR1-WI) + (TR2-PTR2) : NULL experiment - Walker Inhibitor does in fact inhibit walking.
Lane 14: (W-TR1-WI) + (TR2-PTR2) + WT → (WI-WT) + TR1 + (W-TR2-PTR2) + (W-TR1-WI) +(TR2-PTR2): Walker Trigger initiates walking
In lanes 12, 13, and 14 there is 2x of WI, and in lane 14 there is 4x of WT.
Random walking mechanism: some parts of the gel was magnified. When track 1 and preannealed (walker – track2) complex are mixed together at room temperature for 2 hours, the solution reaches the equilibrium between track 1, track 2, (walker – track1), and (walker – track 2) [lane 7]. Similarly, when track 2 and preannealed (walker – track 1) complex are mixed together at room temperature for 2 hours, the solution reaches equilibrium with same ratio [lane 8]. This equilibrium provides an evidence of walker successfully moving from one track to another track in solution. In this gel electrophoresis data, we can see that walker prefers track 2 over track 1. This preference was previously anticipated by NUPACK simulation as shown below where 70% of the walker binds to track 2 while 30 % of the walker binds to track 1. The preference can be explained by energy difference between (walker – track 1) complex and (walker – track 2) complex due to dangling effect from fundamental structural difference.
Gel 3
Cargo goal triggering mechanism
Lane 1: CG1 : Control
Lane 2: CGI : Control
Lane 3: CGT : Control
Lane 4: CG1 + CGI → (CG1-CGI) : Cargo goal inhibitor binds to the cargo goal.
Lane 5: CGI + CGT →(CGI-CGT) : Cargo goal trigger binds with the cargo goal inhibitor
Lane 6: (CG1-CGI) + CGT → (CGI-CGT)+ CG1 : Cargo goal trigger is capable of stripping the Cargo goal inhibitor off of the Cargo goal
Gel 4
Picking-up mechanism
Lane 1: C1, TR1 : Control
Lane 2: TR2 : Control
Lane 3: W, CA : Control
Lane 4: C1 + CA →(C1-CA) : Cargo binds to the Cargo Attaching strand
Lane 5: W + C1 → (W-C1) : Cargo binds to the Walker
Lane 6: W + (C1-CA) →(W-C1) + CA : Walker picks up the Cargo by stripping off of the Cargo Attaching strand
Lane 7: (W-TR1) : Control
Lane 8: (W-TR2) : Control
Lane 9: W + TR1 + C1 →(W-TR1-C1) : Track 1 does not interfere with the binding between the Walker and Cargo.
Lane 10: W + TR2 + C1 →(W-TR2-C1) : Track 2 does not interfere with the binding between the Walker and Cargo.
Lane 11: (W-TR1) + (C1-CA) →(W-TR1-C1) + CA : Track 1 does not interfere with the Walker picking up the Cargo off of the Cargo Attaching strand.
Lane 12: (W-TR2) + (C1-CA) →(W-TR2-C1) + CA :: Track 2 does not interfere with the Walker picking up the Cargo off of the Cargo Attaching strand.
Lane 13: (W-TR1-C1) + CA : NULL experiment. Picking up mechanism is irreversible
Lane 14: (W-TR2-C1) + CA : NULL experiment. Picking up mechanism is irreversible
Gel 5
Walker walking while carrying a cargo
Lane 1: C1 : Control
Lane 2: TR1 : Control
Lane 3: (TR2-PTR2) : Control
Lane 4: (W-TR1-C1) : Control
Lane 5: W + TR2 + PTR2 + C1 → (W-TR2-PTR2-C1): Control
Lane 6: (W-TR1-C1) + (TR2-PTR2) ↔ (W-TR2-PTR2-C1) + TR1 : Walker walking while carrying a cargo. Reached equilibrium
Lane 7: (W-TR2-PTR2-C1) + TR1 ↔ (W-TR1-C1) +(TR2-PTR2) : Walker walking while carrying a cargo. Reached equilibrium
Lane 8: (W-C1) : Control
Lane 9: (W-TR1) : Control
Lane 10:(W-TR2) : Control
Lane 11: (W-TR2-C1): Control
Lane 12: (W-TR2-PTR2): Control
Gel 6
Dropping off mechanism
Lane 1: W : Control
Lane 2: CG1 : Control
Lane 3: PCG1 : Control
Lane 4: CG1 + PCG1 → (CG1-PCG1): cargo goal binds to the probe for the cargo goal
Lane 5: C1 + CG1 + PCG1 → (W-C1-PCG1) : cargo binds to the cargo goal with the probe
Lane 6: (W-C1) : Control
Lane 7: (W-C1) + (CG1-PCG1) ↔ (C1-CG1-PCG1) + W : Dropping off mechanism
Lane 8: W + (C1-CG1-PCG1) - NULL experiment : Dropping off mechanism is irreversible
Lane 9: (W-TR1) : Control
Lane 10: (W-TR1-C1) : Control
Lane 11: (W-TR1-C1) + (CG1-PCG1) ↔ (C1-CG1-PCG1) +(W-TR1): Dropping off mechanism with track 1
Lane 12: (W-TR2) : Control
Lane 13: (W-TR2-C1) : Control
Lane 14: (W-TR2-C1) + (CG1-PCG1) ↔ (C1-CG1-PCG1) + (W-TR2): Dropping off mechanism with track 2
Gel 7
Termination of random walking by reaching the walker goal
Lane 1: W : Control
Lane 2: TR1 : Control
Lane 3: TR2 : Control
Lane 4: WG : Control
Lane 5: PWG : Control
Lane 6: (W-TR1) : Control
Lane 7: (W-TR2) : Control
Lane 8: W+WG → (W-WG) : Walker binds to a Walker goal.
Lane 9: WG + PWG → (WG-PWG) : Walker goal binds to the probe. Works as a control here.
Lane 10: W+WG + PWG → (W-WG-PWG) : Control
Lane 11: (W-TR1) + (WG-PWG) → (W-WG-PWG) + TR1 : Walker walks from track 1 to a Walker goal.
Lane 12: (W-TR2) + (WG-PWG) → (W-WG-PWG) + TR2 : Walker walks from track 2 to a Walker goal.
Lane 13: (W-WG-PWG) + TR1 → NULL experiment : Walker goal is irreversible
Lane 14: (W-WG-PWG) + TR2 → NULL experiment : Walker goal is irreversible