We use several methods to determine information about how our design is working. First we needed to simulate the design using Kintek Explorer. This allowed us to determine relative times for assembling and testing. Next we used polyacrylamide gels as an analytic device as well as a purification method.
Kinetic simulation of walker system
The rate at which our walker proceeds across the track is dependent on the rates of the toehold exchanges. These toehold exchanges in turn are mediated primarily on the size of the toeholds being exchanged. It is therefore possible to model how the walker should ideally proceed by using calculated rate constants and a kinetic simulation program. For the calculated rate constants, we refer to the paper “Control of DNA Strand Displacement Kinetics Using Toehold Exchange” by David Yu Zhang and Erik Winfree, where the following figure allows us to calculate estimated rate constants.
Reactions of the walker system were drawn up and the difference in toehold sizes was compared to calculate the various rate constants. These reactions and rate constants were then put into KinTek Explorer for kinetic simulation.
To for the gel, it is cross-linked using 10% APS and TEMED between two glass plates, then clipped to an electrophoresis rig which uses voltage to drive the negatively charged DNA down the cross-linked polyacrylamide gel. As the strands of DNA are moving through the gel, the shorter ones will pass faster than longer ones. This means there is a separation of the DNA based entirely on molecular weight. If the DNA is pure enough, bands will be present in the fluorescent image of the gel. These bands can be correlated to a known standard, a DNA “ladder” that contains specific sequence lengths, that is run alongside the samples. Using the number and location of the bands allows us to reach conclusions about how our design is working. We chose to test two buffer conditions for our system that are specifically designed to help large DNA structures form and maintain stability.
Walker System Test, 10-27-2012 - Buffer: TMgK
Fuel Test 1, 8-13-2012 - Buffer: TMgK
Conclusion: There is significant hairpin leakage between FA,h and FB,h, and also between FA,h and FD,h, but this decreases noticeably in the absence of the initiator. From the presence of excess lines in both lanes that contain FD,h, it is clear that there are impurities in FD,h, which will require the test to be run again after the sample is purified.
Fuel Test 2, 8-14-2012
Conclusion: There is significant hairpin leakage between FA,h and FB,h, and also between FA,h and FD,h, regardless of whether the initiator is present. While this trend does decrease slightly when temperature of incubation is decreased and concentration of FA,h is increased, the change is not dramatic enough to make these hairpins viable as fuels in this buffer.
Fuel Test 2, 8-14-2012
Conclusion: There is significant hairpin leakage between FA,h and FB,h, and also between FA,h and FD,h, regardless of whether the initiator is present. There is no noticeable change in this trend when temperature of incubation is decreased or concentration of FA,h is increased. Therefore, these hairpins are not viable as fuels in this buffer.