We used several methods to investigate the progress of our walker. We simulated the design using Kintek Explorer and this allowed us to determine the relative times for assembling and testing. Next we used electrophoresis gels to purify the strands and to analyze the design. What follows is a summary of those methods.
Kinetic simulation of catalyzed hairpin assembly
The following is a screencapture of the Kintek output. Time in seconds is on the x-axis and concentration in μM is on the y-axis.
The initiator is shown in black, fuel 1 in red, fuel 2 in teal, and the duplexed fuel 1 and fuel 2 in yellow.
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 - Buffer: TMgK
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 - Buffer: TNaK
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.
We have shown:
- The assembly of the walker system using acrylamide gel
- The functionality of the CHA fuel system using kinetic simulation and fluorometric measurements
We will continue working to build this walker system. To do this we need to verify walker functionality using fluorometric measurements, calibrate the results for leakage, and determine optimum buffer conditions. The nicking enzyme will be tested using an acrylamide gel to determine validity. We also will work to redesign the fuel strands so the walker is capable to reverse its direction.