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: ΔG_RBS-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 MFE difference, 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 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.