Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Gel Verification

Tuesday, April 16, 2024 Home Members Project Protocols Progress Discussion References

Gel Verification 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. Gel 1: Ability of the walker to bind to its tracks and the walker triggering mechanism. Lane 1: 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 In lanes 10 and 11 there is 2x of WI and in lane 11 there is 4x of WT. 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 in solution 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. NUPACK simulation; equilibrium between (walker – track 1) and (walker – track2) after random walking in solution 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