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 and Definition:
Assumption: Switch MFE ∝ leakage
>> Switch MFE is the Gibbs free energy of a toehold switch folded in the most stable structure.
>> Leakage is the level of reporter expression when switch is not activated by trigger.
To activate toehold switch, an amount of energy is needed to open the toehold switch hairpin. Switch MFE reflects the difficulty for the toehold switch activation process. We assume that the more negative the Switch MFE, the harder for the activation to take place, and hence a lower leakage.
Assumption:∆ G RBS-Linker ∝ Dynamic range
>> ∆ G RBS-Linker is the Gibbs free energy of the sequence starting from the RBS to the linker(Figure).
>> Dynamic range is the ratio of reporter expression between non- activated switch and trigger- activated switch.
After the trigger opened the toehold switch hairpin, ribosome need to unwind the RBS- linker region to translate the RFP reporter gene. ∆ G RBS-linker reflects the difficulty for the unwinding process. It is assumed that the more negative the ∆ G RBS-linker , the harder for the translation to take place, and hence a lower dynamic range. It had already demonstrated that the ∆ G RBS-linker is correlated with the dynamic range in the original paper.
Assumption: MFE Difference ∝ Switch-trigger formation
>> MFE difference is defined as the difference between switch MFE and MFE of the dimner.
Since ∆ G = -RTlnK, MFE difference(∆ G) is proportional to equilibrium concentration (K). Therefore, we assume that the higher the MFE difference, the higher the dimer concentration and hence the expression level.
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 rare codon, stop codon and RFC illegal sites along the sequence. In addition, the built- in BLAST function also automatically screen for nonspecific region to avoid false positive detection. Ultimately, the program generated a list of possible Toehold Switch sequence according to their free energy using the embedded function of “Vienna RNA” (8). We ranked the ∆ G RBS- Linker as the most important parameter since it had already proven that it correlates with the dynamic range of switch. Below graph shows 394 possible H5 toehold switches generated by our software. We first chose the switches that with the highest ∆ G RBS- Linker (-3.8kcal/mol). Among those switches, we chose the 3 switches with low switch MFE and high MFE difference.
| Switch Candidate |
MFE RBS-Linker |
MFE Switch |
MFE Difference |
| 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.
| Thermodynamic parameters of chosen influenza switches |
| Switch Candidate |
MFE RBS-Linker |
MFE Switch |
MFE Difference |
| CGU’s SAT switch |
-9.2 |
-17.4 |
+46.8 |
| New SAT switch |
-4.1 |
-24.5 |
+23.1 |
