The main goal of our iGEM project was to design an accurate and accessible diagnostic test to simplify the diagnosis of multiple sclerosis (MS). We divided our main goal into sub-parts to work efficiently. Our team investigated microRNA (miRNA) amplification, designed an RNA threshold system, and designed and tested in vitro miRNA-targeting toehold switches. Throughout our project, we used the Design-Build-Test-Learn (DBTL)-cycle to effectively work towards our goals. The two DBTL-cycles that we would like to highlight are the cycle of making accurate miRNA-targeting toehold switches and the cycle of developing an RNA threshold through toehold-mediated strand displacement.

miRNA-targeting toehold switches

We performed three iterations of the engineering cycle, focusing on the computational design of miRNA-targeting toehold switches and their performance in vitro. During the first and second cycle, we further improved the software programme SwitchMi Designer, built by the 2021 Uparis_BME team, to enable the miRNA and toehold switch to fully hybridise. During the third cycle, we measured the performance of the designed toehold switches over time and fitted the results against our model to gain insight on leakiness of the toehold switch output.

Introduction

miRADAR focused on miRNAs as potential biomarkers to diagnose MS patients. In our test design, we used toehold switches to detect RNA levels that are associated with MS. For a proof-of-concept of our test, we computationally designed miRNA-targeting toehold switches and tested the predicted designs in the lab. We specifically focused on detecting hsa-miR-484 since this miRNA is shown to be significantly upregulated in patients with relapsing-remitting MS (RRMS).¹
The SwitchMi Designer software tool, built by the Uparis_BME team of 2021, generates toehold switch sequences targeting miRNAs. We predicted three toehold switch sequences for hsa-miR-484 using this tool and verified these in NUPACK, a web-based software which predicts how two sequences hybridise based on base-paring and minimum free energy (MFE) calculations.² NUPACK generates a figure which shows the predicted secondary structure of the hybridised miRNA and toehold switch. We found that not all predicted toehold switch sequences fully anneal to the miRNA (Figure 1). A fully annealed miRNA results in a larger difference in MFE between the native secondary structures of the toehold switch and miRNA, and the hybridised structure. A large MFE difference makes hybridisation energetically favourable, which is expected to improve the performance of the toehold switch in vitro. Therefore, we aimed to first improve the SwitchMi Designer software tool to ensure complete hybridisation of the miRNA and the toehold switch, before testing the designs in vitro.

NUPACK hybridisation analysis for hsa-miR-484 and a toehold switch predicted by SwitchMi Designer. hsa-miR-484 (shaded) partially anneals to the toehold switch sequence. The colour of the nucleotides indicates the probability of the nucleotide’s position in the complex from low (blue) to high (red) probability.

Round 1

Design
The SwitchMi Designer tool allows the user to set restrictions to the length of the miRNA sequence that anneals to the paired and unpaired regions of the toehold switch (Figure 2). However, we found that defining these parameters leads to predicted toehold switch sequences that do not always fully anneal to the miRNA. Therefore, we decided to replace these parameters with a fixed requirement in the code of the software that takes into account the total length of the miRNA to anneal to the toehold switch.

Schematic representation of a miRNA (green) and toehold switch (light and dark purple), showing the top- and bottom stem, the bulge, the loop, and the linker. Our adjusted software scans the miRNA sequence to find the stem base (orange box), after which the miRNA sequence is split downstream of the stem base (dotted line). The downstream miRNA part anneals to the unpaired region of the toehold switch and the upstream miRNA part anneals to the paired region of the toehold switch.

Build
To design the toehold switch, the SwitchMi Designer tool scans the miRNA sequence to find two A or T nucleotides and one G or C nucleotide together, to form the base of the bottom stem (stem base) (Figure 2). We kept this first step. Then, we adjusted the software to split the miRNA sequence downstream of the stem base into two parts (Figure 2). The 3’ end of the miRNA sequence anneals to the unpaired region of the toehold switch while the 5’ end of the miRNA sequence anneals to the paired bottom stem of the toehold switch, thereby opening up the bottom stem (Figure 2). We coded the software to then implement the reverse complement sequence of both parts of the miRNA in the toehold switch design.

Test
We used our adjusted software to predict three toehold switch sequences for hsa-miR-484. The hybridisation of the toehold switches and miRNA was tested in NUPACK. We found that the trigger mainly stays free in solution and that there is a low chance of the toehold switch annealing to the miRNA (Figure 3).

NUPACK hybridisation analysis of the trigger miRNA hsa-miR-484 (Trigger) and three predicted toehold switch sequences (Toehold 1-3), provided by the improved software. The concentration (\muM) of each (hybridised) component or complex present in the system is shown. A higher concentration of a component or complex indicates a more favourable state.

Learn
We found that a very small amount of the miRNAs annealed to the predicted toehold switches. Complete hybridisation would lead to a lower MFE compared to the native secondary structures of the toehold switches and miRNA, and thus a favourable hybridisation reaction. Therefore, it was assumed that complete hybridisation was not possible with these toehold switches. Based on our results, we revisited the software. Round one revealed that the software joined the two reverse complement sequences of the miRNA parts in an inside-out manner, which led to an incorrect miRNA-recognition sequence in the toehold switch. We focused on resolving this error to work towards software that predicts toehold switch sequences that fully anneal to the miRNA.

Round 2

Design
The software should design toehold switches for the trigger miRNA sequence by correctly implementing the reverse complementary miRNA sequence in the predicted toehold switch sequence. We aimed to implement this requirement while maintaining the basic requirements of the software.

Build
We adjusted the model to first generate the reverse complementary sequence of the whole miRNA sequence. This sequence was then used as input for finding the stem base and implementing the sequence in the paired and unpaired region of the predicted toehold switch sequence.

Test
After adapting the software, we again predicted three toehold switch sequences for hsa-miR-484 using the software tool. NUPACK analysis showed that all predicted sequences fully annealed to the miRNA sequence (Figure 4). In addition, all miRNA annealed to a toehold switch during hybridisation analysis in NUPACK (Figure 5). This indicated a clear improvement compared to the toehold switches in round 1, which did not have a favourable hybridisation reaction, leading to free trigger in solution (Figure 3).

NUPACK hybridisation analysis for hsa-miR-484 and three toehold switches (a,b, and c) predicted by our improved software tool. hsa-miR-484 (shaded) fully anneals to the toehold switch sequences. The colour of the nucleotides indicates the probability of the nucleotide’s position in the complex from low (blue) to high (red).

NUPACK hybridisation analysis of the trigger miRNA hsa-miR-484 (Trigger) with three predicted toehold switch sequences (Toehold 1-3), provided by the improved software. The concentration (nM) of each complex present in the system is shown.

Learn
Round two revealed that our software can successfully predict toehold switch sequences that fully anneal to the miRNA hsa-miR-484 and that the hybridisation reaction is energetically favourable. Based on these findings we decided to test our toehold switches in vitro, to confirm their ability to be accurately activated by annealing to the trigger miRNA.

Round 3

Design
We tested two of our three predicted toehold switches in the lab, which we refer to as toehold switch A (THA) (BBa_K5106001) and toehold switch B (THB) (BBa_K5106002). Our third in silico tested toehold switch had a less desired native secondary structure. Here, we aimed to design an accurate toehold switch with a fast visible output signal, and a high signal-to-noise ratio. The toehold switches were tested in vitro. This required a T7 promoter (BBa_K1614000) upstream of the toehold switch sequence, a reporter gene downstream of the toehold switch, and a downstream T7 terminator(BBa_K5106019). LacZ(BBa_K1444017) was chosen as reporter gene since it provides a clear visible output signal which promotes the accessibility of the test.

Build
For each toehold switch we built a plasmid containing the composite parts (BBa_K5106004) and (BBa_K5106005) for THA and THB respectively, under control of the T7 promoter and T7 terminator. We used the PURE cell-free system to test the toehold switches in vitro. A negative control which did not contain the trigger miRNA was added to investigate the possible leakiness of the system.

Test
When the trigger miRNA anneals to the toehold switch, the ribosome binding site (RBS) and start codon become available which allows translation of the LacZ coding sequence. The produced enzyme \beta-galactosidase converts the yellow substrate chlorophenol-red \beta-d-galactopyranoside (CPRG) to a purple product. After verifying that the toehold switches produced a purple output after two hours, we evaluated the performance of the switches over time in a new experiment to study the increase in output and the leakiness of the toehold switches. The conversion by \beta-galactosidase can be monitored over time by measuring absorbance at 570 nm. We expected to see an increase in output over time for the reactions with trigger miRNA and no output for the reactions without trigger miRNA. THB with the trigger miRNA showed a significantly higher increase in absorbance over time than THB without the trigger miRNA (Figure 6). However, there was still an increase in absorbance over time for THB without the trigger, which indicates that translation of LacZ is also possible without hybridisation of the trigger miRNA to the toehold switch. THA with the trigger miRNA also showed a significantly higher increases in absorbance than without the trigger; however, the final abosrbance is lower than observed for THB (Figure 6). This indicates that the toehold switch is activated less effectively by the miRNA trigger.

Performance over time of our designed toehold switches THA and THB *in vitro*. Absorbance at 570 nm over time (min) for cell-free reactions with THA and THB constructs, with (+) and without (-) miRNA trigger added. **: p < 0.01.

Learn
Round three revealed that THA and THB can be activated by the trigger miRNA to produce \beta-galactosidase, although THB showed more effective activation. THB was however leaky since the toehold switch without trigger miRNA also showed an increase in absorbance. This means that translation of LacZ is possible without miRNA trigger present. We wrote a model to quantify the leakiness of THB and fit the model to the results obtained for THB, with and without trigger miRNA. We found that the 35.8 % of the signal output of THB is caused by leakiness. To further improve our toehold switches, we aim to minimise the leakiness in the future. This could be done by optimising the toehold switch structure to get a more energetically favourable native secondary structure of the toehold switch. The possibility of the toehold switch linearising by chance, enabling translation, could then be minimised. This may be achieved by elongating the stem of the toehold switch for instance.

(1)

Regev, K.; Healy, B. C.; Paul, A.; Diaz-Cruz, C.; Mazzola, M. A.; Raheja, R.; Glanz, B. I.; Kivisäkk, P.; Chitnis, T.; Jagodic, M.; Piehl, F.; Olsson, T.; Khademi, M.; Hauser, S.; Oksenberg, J.; Khoury, S. J.; Weiner, H. L.; Gandhi, R. Identification of MS-Specific Serum miRNAs in an International Multicenter Study. Neurology Neuroimmunology & Neuroinflammation 2018, 5 (5), e491. https://doi.org/10.1212/NXI.0000000000000491.

(2)

Fornace, M. E.; Huang, J.; Newman, C. T.; Porubsky, N. J.; Pierce, M. B.; Pierce, N. A. NUPACK: Analysis and Design of Nucleic Acid Structures, Devices, and Systems, 2022. https://doi.org/10.26434/chemrxiv-2022-xv98l.

Proof of concept