.
The expected fluorescence(determined by the ratio of suboptimal structures with open/closed toehold domains) exhibited by E. coli co-transformed with the switch and trigger RNA after 12 hours was compared with the actual observed fluorescence during the experiment.
From the table above, we observed that only 3 out of 15 of our predictions were incorrect, showing promising accuracy of this prediction method. None of the 3 incorrect predictions were false negatives, indicating that the suboptimal structure prediction method should be mostly applied to filtering out switch sequences with low predicted performances.
Comparing thermodynamic parameters with experimental result
Correlation of switch MFE (Minimum Free Energy) and expression leakage
The plot shows some kind of weak positive correlation between expression leakage and the MFE of a switch, with an outlier N9-2. This suggests that the MFE of a switch might not have a large contribution to the expression leakage.
Correlation of switch MFE (Minimum Free Energy) and the expression by Switch-Trigger duplex
Although the switch MFE correlates with the expression leakage, this plot showed that the switches with lower MFE also have lower RFP signal in the presence of trigger. This suggests that a low MFE of the switch could also be a hindrance to detection that it increases the energy input to the system (activation energy), so it takes more energy for switch-trigger dimer formation to occur. This could lower the expression level of the switch-trigger dimer.
Correlation of MFE difference and the expression by Switch-Trigger duplex
We initially thought that the larger the MFE difference between the sum of the MFE of switch and trigger and that of the switch-trigger duplex, the more favourable the duplex structure will be. However, there seems to be a weak positive correlation between the expression level of the switch+trigger dimer and the MFE difference of a switch. This could be because that a low MFE difference could indicate a more similar conformation of the switch/trigger RNA to the switch+trigger dimer RNA. This could lead to less conformational changes in the dimer formation process, making the process faster. Thus, a small MFE difference could lead to an increased expression level of the switch+trigger dimer.
Correlation of toehold domain base pair and the expression by Switch-Trigger duplex
The plot shows some kind of negative correlation between RFP signal in the presence of trigger and the number of base pairs in the toehold domain. Some exceptions were observed, they are N1-3, N9-3, N9-1. H5-3 and N1-2. This supports the findings from the suboptimal structure modelling.
References:
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