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 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.)
This gel tests the ability of the walker to bind to its tracks and the walker triggering mechanism. Lane 1: W; 2: TR1; 3: TR2; 4: WI; 5: WT; 6: W + TR1 → (W-TR1); 7: W + TR2 → (W-TR2); 8: W + WI → (W-WI); 9: WI + WT → (WI-WT); 10: W + TR1 + WI → (W-TR1-WI); 11: (W-WI-TR1) + WT → (W-TR1) + (WI-WT).