Base Pair Ex
Figure 1: bpdists
Base Pair Probabilities:
The Foldase program generates these base pair distribution files which are set up in a two column orientation with the i,j base pairs on the left and the squared probabilities on the right. When it comes to interpreting them we read it as the probability that base 42 and 75 will pair together are 0.95 such that the higher the value the more likely the two will bind together. We can compare these results with what we dubbed as the “ground truth” which is the IRE folded by itself, looking at the pairs that will more likely bind. Using these we compare those values to the shifted values and see how the likelihoods compare the closer to the ground truth the better of a candidate that sequence is. Since it will most likely fold into the desired structure.
Modeling of TOP, IRES, Unreg:
After imaging all 60 wells of the three plasmids given to us by Dr. Albeck, the translational rate of each plasmid was measured by measuring the fluorescence value in each well. To account for cell death, any cell that reached a fluorescence intensity above 500 was tracked even if the value drops below 500. The average fluorescence and a heat map was measured over time. A line of best fit was added to the average fluorescence graphs to determine the slope and translational rate. The heat map shows fluorescence values of individual cells over time. Time stamps were added at the point of TMP introduction. The data was then normalized by subtracting each cell’s own minimum intensity over time. This makes the graphs show a change in intensity overtime starting from a baseline.
Unreg-H2B-DD-Card:
Figure 2: bpdists
Figure 3: bpdists
TOP-H2B-DD-YFP:
Figure 4: bpdists
Figure 5: bpdists
IRES-H2B-DD-YFP:
Figure 6: bpdists
Figure 7: bpdists
From the images, it is clear to see that there was an immediate translation shift after the addition of TMP at the 5.8hr mark, except for the wells that only received DMSO. The heat maps also show this trend as few cells show fluorescence before the addition of TMP. This could be due to random mutations in the DD domain that inactivated its function. Looking at the translational rates, slope of each mean graph, it was hypothesized that their slopes should have been similar without the addition of DTT and Torin2. IRES and Unreg have similar values with a 0.1 difference between them. TOP on the other hand showed a much higher translational rate to the other plasmids. When an mTOR inhibitor binds to TOP, it should downregulate translation. To fully understand these graphs, next steps would be to image transfected cells with the addition of Torin2 and DTT. Currently we have proven that we can accurately model translational rates.