Proof of Concept
After designing all of our switches, the engineering process of which is detailed in our engineering page, and the design details are outlined in our design page, we were keen to get into the lab to test our single trigger (gen2) switches, and double trigger (gen3) switch, as well as our isothermal amplification method, to show these work well with our switches. If the concept was proved to work, we would, as outlined in our implementation page, research other miRNAs in our own studies to create switches with even more triggers, to create a kit to detect preeclampsia using a cocktail of miRNA triggers.
Gen2 Toehold Switches
The aim for characterising the gen2 toehold switches was to show they could discriminate between concentrations of miRNA 4-fold higher, which was based on the data we gathered from research studies. This is outlined further in the biomarkers section of the design page and discriminate between similar miRNAs, or ‘homologs’, that were identified using our software tool, ‘ToeholdTools’. More information on the software tool can be found in our software page.
Gen3 Toehold Switches
The aim for characterising the gen3 ‘AND-gate’ toehold switch was to show it would discriminate between - both trigger miRNAs being 4-fold higher in concentration, one trigger being 4-fold higher in concentration, and neither trigger being 4-fold higher in concentration.
The goal of characterising miRPA was to show: it could discriminate between similar homologs, identified by ToeholdTools, and it would increase the concentration of our miRNAs, such that they could be detected by our toehold switch even at low concentrations.
In order to fulfill the goals of the toehold switches and miRPA, we had to conduct characterisation experiments.
Gen2 Toehold Switches
We ordered our switches and luciferase as linear DNA with 250 random base pairs before and after the prefix and suffix respectively on the advice of our TXTL kit supplier. We first suspended our gBlocks, consisting of a T7 promoter, upstream of the switch (containing an RBS), upstream of a firefly luciferase CDS, to reach an overall concentration of 6.7nM in TE buffer.
For each gen2 switch, we tested, at a constant DNA concentration, the target miRNA (miR-517-5p or miR-210-3p) at 9M and 2.25M to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia 1 2, as well as a homolog for each miRNA (miR-518f-5p for miR-517-5p and miR-6867-5p for miR-210-3p), which were determined to be the miRNAs have the highest probability of activating the switches aside from the target miRNAs themselves by our software tool (see software page). We then incubated the DNA, miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNAs) as well as a TXTL cell-free master mix for 1 hour, and then pipetted the contents of each tube into 8 wells of a 96 well plate, and took four measurements after D-luciferin in a buffer was injected, at different times. The luciferase assay kit told us to take a measurement ‘immediately’ after injection. But we found that taking the measurement a few seconds after injection caused no luminescence to be detected, so we took measurements at later times, to analyse how luminescence changed over time.
The results show a brief, high peak in luminescence for a high concentration of miR-210-3p. This is because, during the incubation time, more luciferase is produced due to an increased rate of translation, so there is a higher concentration of luciferase, so more luciferase-luciferin complexes form at the beginning, converting luciferin to oxyluciferin, producing luminescence. The results show that, compared to the negative control, the homolog ‘miR-518f-5p’ activates the switch more, causing an higher peak, sooner on, and there is only a small increase in luminescence between the control and the target RNA at reduced concentration.
The results for the miR-517-5p switch were very similar to that of the miR-210-3p switch. However, it showed that the difference in luciferase concentration after incubation was lower for the lower concentration of miR-517-5p than that of miR-210-3p. Furthermore, the homolog for this trigger miRNA, miR-518f-5p gave a higher concentration of luciferase than the homolog for miR-210-3p, suggesting that it induces translation better. This is what we would expect given it’s closer similarity.
Based on these sets of data, we decided that our kit should detect luminescence at some point between 100 and 200 seconds.
The graph on the left shows % increase in luminescence compared to the negative control (no miRNAs added) for the miR-210-3p gen2 switrch. This shows an 82% increase in expression for the target miRNA at 9M concentration, which drops to just 16% for a 2.25M concentration, showing how our toehold switch can clearly discriminate between these percentage differences in miRNA concentrations, and these concentrations are based on what we’d expect in patients with and without the condition at ten weeks 1. However, there is no data on actual concentrations of these miRNAs in the serum and the data we collected shows that if concentrations in the blood are lower than 2.25M, then an amplification technique would be necessary to discriminate between low concentrations of miRNA and leaky luciferase expression. The diagram on the right is similar to that of the miR-210-3p switch, and shows how the gen2 miR-517-5p toehold switch can discriminate clearly between miR-517-5p at 9M and 2.25M.
The gen2 toehold switches cause a noticeable increase in translation of luciferin with increased miRNA concentration, and, with software, they would be able to binarily discriminate between levels of miRNA at concentrations indicative of the condition, given they are above 2.25M in ‘normal’ patients. The results also confirmed that an amplification technique, such as miRPA, should be used in order to ensure the concentration is greater than 2.25M in patients without the condition.
Gen3 Toehold Switches
We ordered our switch and luciferase as linear DNA with 250 random base pairs before and after the prefix and suffix respectively on the advice of our TXTL kit suplier, as well as a gBlock containing our anti-miRNA under the control of a T7 promoter. We first suspended our Toehold switch, consisting of a T7 promoter, upstream of the switch (containing an RBS), upstream of a firefly luciferase CDS, and anti-miRNA gblock to reach an overall concentration of 6.7nM in TE buffer.
We tested, at a constant DNA concentration, the target miRNAs (miR-517-5p and miR-210-3p) at 9M and 2.25M combinations to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia 1 2. We then incubated the DNA for the switch and luciferase, the DNA for the antiRNA (aRNA) (See Design page), miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNAs) as well as a TXTL cell-free master mix for 1 hour, and then pipetted the contents of each tube into 8 wells of a 96 well plate, and took a measurement after 155s as this was in the region of high expression for high miRNA concentration samples observed in the single trigger switches.
Having tested the single trigger switches, and thereby demonstrating how our concept of a toehold switch works as hypothesised, the main purpose of testing the AND-Gate Switch was to see whether it could discriminate between a high concentration of one miRNA, and a low concentration of another, and a high concentration of both. This was in case a patient had increased levels of one miRNA and not the other, which would not be indicative of Preeclampsia.
The graphs show how, compared to the negative control, which shows leaky expression of the toehold switch, the luminescence output of the sample with 9M concentration of both miRNA triggers is 92% higher. Interestingly, the percentage increase in luminescence of the miR-210-3p at 9M and miR-517-5p at 2.25M is twice more than twice as high as the miRNAs at flipped concentrations - 28% compared to 12%. This could be because the miR-210-3p binding site is further downstream than the miR-517-5p binding site, so miR-210-3p is able to bind and partially unfold the switch in the absence of miR-517-5p, increasing the rate of translation. However, as the miR-517-5p binding site is further upstream, it is completely bound up in hydrogen bonds, so it is not accessible to the miRNA unless it binds as the switch is being transcribed.
The gen3 toehold switch also causes a noticeable increase in translation when both miRNAs are at a high concentration (9M), compared to when one of both of them is at a low concentration (2.25M), which gives strong evidence that our AND-gate design works. This framework could therefore be scaled up to create more than 2 input sensing switches.
Based on results from our toehold switch characterisation, we decided to find an isothermal amplification strategy. Read more about it on our Design page.
We were supplied miRPA characterisation protocols by Dr O’Sullivan (see Human Practices page) after we told her about our project. To show that miRPA can discriminate between our miRNAs and homologous miRNAs, we tested our trigger miRNAs with their respective closest homologs, as determined by our software tool.
In order to design probes for miRPA, we designed a python script using Nupack’s design functions in its API to find probes which would bind to the miRNAs, but have overhangs which didn’t bind within themselves, to ensure primers could easily anneal to them.
We ordered our probes and primers on IDT and then resuspended them in a TE buffer and added them to our trigger and homologous miRNAs, with DNA ligase to bind the probes together. We amplified the miRPA product with PCR.
We performed a gel electrophoresis on the PCR products. We did not have access to gel ladders which could discriminate between <100bp DNA strands, but our gel showed that the target miRNAs miR-210-3p and miR-517-5p were amplified more than the homologs. Furthermore, the fact that the homologs moved further through the gel suggested that the probes had not bound together, which would suggest they did not bind to the homologs.
Using miRPA in our test would allow us to ensure there is an adequate miRNA concentration, and our characterisation of the technique showed it is specific to our miRNAs.
We were able to design two generations of toehold switches which we proved to be able to discriminate between the ratio of concentrations typical of patients with and without preeclampsia, as well as similar, or ‘homologous’ miRNAs.
We were able to design a method of amplification for our switch and proved it not only worked, but could also discriminate between homologous miRNAs.
We successfully designed a luminometer and extraction method, such that the concept of our test as a whole is thought through from start to finish.