RNA thermodynamic modeling:
Designing Toehold Switch
Background
According to Green et al., the optimal length of RNA to be detected by a toehold switch is around 30 bp. In other words, a target RNA with 1000 bp in length can have 970 possible switches. However, the performances of each possible switch will be different, since switches that target different region will have different thermodynamic characteristic and structure, which can affect the performance of the switch. Therefore, we modeled the thermodynamic and structure of our toehold switch during designing stage and simulate the expression of activated switch in silico. Our modelling helped us a lot in gaining insight.
Toehold switch structure:
We adopt the toehold switch design from the original paper. Our toehold switch contains 15nts “toehold domain”, 21nts stem-loop that contains a start codon and a RBS B0034 loop at that toop. A 21nts linker and mRFP reporter sequence is present downstream the toehold switch. The linker is used to separate the coding sequence in the toehold switch and the reporter to prevent interference of protein folding.
Assumptions
Assumption: Switch MFE (Minimum Free Energy) correlates with the expression leakage
>> Switch MFE is the minimum Gibbs free energy that a toehold switch could have among all the possible structures.
>> Expression leakage is a phenomenon where the reporter (i.e. Red Fluorescent Protein in our project) is expressed in the absence of trigger RNA. The level of leakage can be measured as:
To activate toehold switch, an amount of energy is needed to open the toehold switch hairpin. The Switch MFE reflects the difficulty for the toehold switch unwinding process. We assume that the more negative the Switch MFE, the harder for the unwinding to take place, and hence a lower leakage.
Assumption: ΔGRBS-linker correlates with the duplex expression
>> ΔG
RBS-linker is the Gibbs free energy of the RNA sequence starting from the RBS to the linker in the switch-trigger duplex (Figure).
>> The duplex expression is the reporter expression of the switch-trigger dimer RNA.
After the switch RNA hairpin is unwound after binding to the trigger RNA, a switch-trigger dimer RNA would be formed. The RBS-linker region of the MFE structure of this dimer RNA should have minimal base pairs. This makes it easier to unwind the RNA for this region, allowing ribosomes to bind to the RBS and move along the RNA for translation of the RFP reporter gene to occur. ΔG
RBS-linker reflects the difficulty for the unwinding process of the RBS-linker region. It is assumed that the more negative the ΔG
RBS-linker, the harder it is for the unwinding to take place, leading to lower translation rates. Thus, the duplex expression would be reduced.
Assumption: ΔMFE correlates with the duplex expression
>> ΔMFE is defined as the difference between (switch RNA MFE + trigger RNA MFE) and MFE of the switch-trigger duplex RNA.
Since ΔMFE = –RTlnK, where:
R=gas constant
T=temperature
K=equilibrium constant
Switch RNA+Trigger RNA↔Duplex RNA
Therefore, we assume that the higher the ΔMFE, the higher the switch-trigger duplex RNA concentration compared to that of the switch RNA when in equilibrium.
Consequently, increased equilibrium concentrations of the switch-trigger duplex RNA would provide an increased number of active mRNAs for the translation of the reporter RFP.
Screening by our software
To minimize the manpower on screening of the switches, we constructed an online toehold switch design program. Apart from basic thermodynamic parameters, it also screens for other factors(link to the software page of the wiki). Ultimately, the program generated a list of possible toehold switch sequences according to many different free energy parameters using the ViennaRNA library[2]. The graph below shows 394 possible H5 toehold switches generated by our software. The assumptions motioned earlier stated 3 very important parameters for the selection of switch candidates with the greatest possible performances: Switch MFE, ΔGRBS-linker, and ΔMFE. We applied these parameters to our switch selection process: We first chose the switches that with the highest ΔGRBS-linker (-3.8 kcal/mol). Among those switches, we chose the 3 switches with the lowest switch MFE and the highest ΔMFE.
Figure 2: ΔGRBS-linker of switch candidates generated from an example RNA sequence input by our software
| Switch Candidate |
ΔGRBS-linker |
MFE Switch |
ΔMFE |
| H5-1 |
-3.8 |
-19.1 |
+32.1 |
| H5-2 |
-3.8 |
-21.1 |
+34.8 |
| H5-3 |
-3.8 |
-24 |
+37.9 |
| H7-1 |
-3.8 |
-24.5 |
+41.4 |
| H7-2 |
-3.8 |
-17.8 |
+34.2 |
| H7-3 |
-3.8 |
-16.3 |
+26.3 |
| N1-1 |
-3.8 |
-17.1 |
+34.4 |
| N1-2 |
-3.8 |
-17.1 |
+24 |
| N1-3 |
-3.8 |
-19.4 |
+25.9 |
| N9-1 |
-3.8 |
-22.2 |
+34.5 |
| N9-2 |
-3.8 |
-17.9 |
+30.8 |
| N9-3 |
-3.8 |
-21.6 |
+27 |
| PB2-1 |
-3.8 |
-12.5 |
+34.6 |
| PB2-2 |
-3.8 |
-24.7 |
+38 |
| PB2-3 |
-3.8 |
-16 |
+28.2 |
To improve CGU’s oral cancer toehold switch, we employed the screening function of our program. A toehold switch was chosen that is predicted to outperform the CGU’s switch according to the MFE RBS-Linker.
| Switch Candidate |
ΔGRBS-linker |
MFE Switch |
ΔMFE |
| CGU’s SAT switch |
-9.2 |
-17.4 |
+46.8 |
| New SAT switch |
-4.1 |
-24.5 |
+23.1 |
Suboptimal Structure Modelling
The toehold domain (first 15 nucleotides) of the switch RNA is crucial for the binding of the trigger RNA to the switch RNA: this domain must have minimal paired bases in the switch RNA to ensure the successful binding of this domain with the complementary sequence in the trigger RNA, which allows the unwinding of the switch RNA and permits translation of the reporter protein, RFP, to occur.
Our program can only calculate the minimal free energy structure (MFE) for each target RNA region to reduce calculation workload. In reality, different conformations of RNAs with the same sequence coexist in solution, whose and the concentrations of those populations are determined by their structures and free energy. Therefore, we manually checked the predicted structures and equilibrium concentrations of the ten suboptimal structures of each influenza switches with the lowest MFEs on the web tool developed by ViennaRNA package(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/barriers.cgi)[2]. Then we predicted the performance of each influenza switches and compared with experimental results.
The table below shows the different suboptimal structures of each switch RNA sequence:
Each suboptimal structure is labelled by numbers 1 to 10, where structure 1 has the lowest free energy(the MFE structure), and structure 10 has the highest free energy. In the "Suboptimal structures" column, only suboptimal structures with non-negligible equilibrium concentrations are considered and shown. (The equilibrium concentrations were calculated by the ViennaRNA webserver[2]: example for H5-3 switch{1}) We checked the status of the toehold domain for each suboptimal structure from the ViennaRNA webserver output[2] to see if it was open or closed.(example for H5-3 switch{2}) The structures with an open toehold domain are shown in green, and ones with a closed toehold domain are shown in red.
The expected fluorescence(determined by the ratio of suboptimal structures with open/closed toehold domains) exhibited by E. coli cotransformed with the switch and trigger RNA after 12 hours was compared with the actual observed fluorescence during the experiment.
| Switch Candidate |
Suboptimal structures |
Expected fluorescence |
Experimental fluorescence(relative) |
Conclusion |
| H5-1 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| H5-2 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| H5-3 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| H7-1 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| H7-2 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| H7-3 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| N1-1 |
1, 2, 6, 7 |
low |
11.676366 (low) |
true negative |
| N1-2 |
-3.8 |
-17.1 |
+24 |
| N1-3 |
-3.8 |
-19.4 |
+25.9 |
| N9-1 |
-3.8 |
-22.2 |
+34.5 |
| N9-2 |
-3.8 |
-17.9 |
+30.8 |
| N9-3 |
-3.8 |
-21.6 |
+27 |
| PB2-1 |
-3.8 |
-12.5 |
+34.6 |
| PB2-2 |
-3.8 |
-24.7 |
+38 |
| PB2-3 |
-3.8 |
-16 |
+28.2 |
