- The functionality of the CHA fuel system using kinetic simulation and fluorometric measurements
- The assembly of the walker system using acrylamide gel
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 walker
The first part of our walker system has been simulated using a kinetic model.
The following is a screencapture of the Kintek output.
The initiator is shown in black, fuel 1 in red, fuel 2 in teal, and the duplexed fuel 1 and fuel 2 in yellow.
Aside from this part of our system, the reactions present in the other parts of our system have unknown kinetic properties. In particular, the reaction rates around the exchange of two duplexes that occurs in our system have not been modeled to our knowledge. This presents an interesting possibility for studying our system to learn more about the kinetics of these interactions.
We used experimental method #9 to test our fuel kinetics.
Steady-state kinetics of F1:F2 hybridization. Performance of the fuel hairpins. The concentrations of F1, F2, and Reporter were all 200 nM. With our setting, roughly 101 Raw Fluorescence Units correspond to 1 nM unquenched RepF, which in turn corresponds to 1 nM F1:F2.
Initial kinetics of F1:F2 hybridization when the concentration of I was systematically varied. The concentration of F1, F2 and Reporter duplex are shown in the inset of each plot. The concentrations of I are color-coded as shown in the legend box to the right. Hollow symbols and solid lines represent raw data and linear regressions, respectively.
The catalytic activity (rate/[catalyst]) was about 4.19 × 10-3 min-1. The value is not as high as that of previous design. This is because we intentionally reduce the length of the toehold in F2, so as to reduce the speed of hairpin F2 being opened, which will reduce the chances of walker dissociation from the track.
To form 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.
Conclusion: There is considerable interaction between FA,h and I,h, but not between FC,h and I,h. There is also a slight interaction between FA,h and FC,h even when I,h is not present. However, this interaction increases dramatically in the presence of I,h. For this reason, FA,h and FC,h will serve as suitable fuels for the walker system, although there will be some leakage.
Conclusion: Lanes 1 through 3 illustrate that Substrate 1 (Aa,h+Tb+bB,h) is successfully being formed. Lane 4 illustrates that the walker itself (W1+W2) is being formed. Lanes 5 through 9 illustrate that Substrate 2 (cA+Ta+dB+eA+f) is being formed. Therefore, it is at least possible to assemble the large components of the walker system.
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.
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.
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 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.