Team:Thessaloniki/Proof Of Concept

☰







Proof of Concept






Overview


In order to prove that our idea can be implemented, we had to show that 3 of our toehold switches are operating properly. And more specifically, we had to do so for one toehold from each miRNA that we chose from the literature research and bioinformatics analysis. The dry lab had created from scratch, 16 toehold switches (4 for hsa-miR-143-3p, 4 for hsa-miR-1246, and 8 for hsa-miR-30e-5p), and the wet lab tested them in the lab. More specifically, for our experiments, we used them in the form of g-Blocks -that is double stranded DNA molecules. To test our toeholds, we used miRNA mimics and not urine samples from patients, because biological samples can be dangerous and difficult to handle. Moreover, in urine samples there are present a lot of different microRNAs, that could affect our results, and before we proceed with something complicated we first had to ensure that our toeholds work correctly. It is worth mentioning that all of the experiments in the sector of the "Proof of Concept" have been carried out in our new plate reader. The reason for that was because our last plate reader had technical issues. Moreover, to increase the sensitivity of our method, despite the samples that will be used, we chose an isothermal amplification method called EXPAR. The final products of the EXPAR should be compatible with the toehold switches. To ensure that they work properly we tested the best of our toeholds with the EXPAR products and the results exceeded our expectations with a very good outcome.



Operation


Our sequences had specific parts that end with a gene that encodes the enhanced Green Fluorescent Protein (eGFP). This protein is the key part of the quantification. The quantification of fluorescence is carried out through the test of the different quantities of microRNA with keeping the value of the parameter of the toehold switch constant. The increased quantities of the miRNA should increase the fluorescence proportionally.

In order to prove the above, we have to:

  • ~Prove that only in the presence of the miRNA, our toehold switches produce fluorescence. This means that they have a complementary sequence that is binding with miRNA and thus the eGFP is expressed.
  • ~Quantify the miRNAs through fluorescence. The fluorescence produced should increase proportionally with the concentration of miRNA. So the higher the miRNA quantity used, the higher the fluorescence produced.
  • ~Prove that EXPAR amplifies the microRNAs and the end-product of the reaction opens the toehold switches. This step is necessary because in urine the concentration of the miRNAs is very low both in cancer patients and in healthy individuals.



Prove that with the presence of the miRNA, we have fluorescence


The experiments that have been carried out, showed that many toehold switches have been binding with the miRNA and give us fluorescence. But especially the following sequences give us the best results:

The experiments that have been carried out, showed that many toehold switches have been binding with the miRNA and gave us fluorescence. But especially the following sequences gave us the best results:

  • ~Toehold switch 1 from hsa-miR-143-3p
  • ~Toehold switch 1 from hsa-miR-1246
  • ~Toehold switch 8 from hsa-miR-30e-5p


The reagents used in the experiments are in Table 1.

Reagents Volume
Solution A 10 ul
Solution B 7.5 ul
RNAase inhibitor 0.5 ul
Template DNA 30 ng
miRNA 10 ul from 10 nM
Nuclease free water to 50 ul


The reaction was carried out up to 25 or 50 ul and was incubated for up to 2 hours at 37 ℃.

Solution A and B are the parts of the cell-free system.

The protein expression was measured using a 96-well plate in a plate reader. To measure eGFP, excitation at 485 nm was used, while detection was at 535 nm; the protein’s maximum fluorescence emission point. The results of the assay can be seen in the graph below:


Figure 1. This graph presents the fluorescence produced by Toehold switches in the presence of the complementary miR mimic (Thermofisher - mirVana® miRNA mimic #4464066) in comparison with the fluorescence produced by the Toehold switch in the absence of miRNA (Negative Control).


As expected, with the presence of miRNA in the ON phase we have higher fluorescence in the three different systems of miRNA. The results imply that our three-miRNA system is working, meaning that the miRNAs bind with the Toehold switches and express eGFP.






Quantification of miRNA through fluorescence


After we ensured the functionality of the system with the three different miRNAs, we proceeded with the quantification of the fluorescence through different concentrations of miRNAs.

Concentration miRNA 143-3p miRNA 30e-5p miRNA 1246
1 nM 100 98 99
10 nM 110 105 103
100 nM 180 150 140
1000 nM 210 160 250
Table 2: Fluorescence results (units) from the quantification experiments of the miRNAs
Following the same protocols as before, we tested a wide range of trigger concentrations and we changed the final volume of the reaction, to find out in which concentration we have the best results. Moreover, we realized that the incubation for 2 hours was not enough and we did not have the results that we expected. Thus, we performed the reaction in an incubator, at 37 ℃ for 4 hours this time. The results of the quantification of the miRNAs are depicted in the following figures:

Figure 2. This graph shows the fluorescence produced from toehold switch T-143-3p-1 (BBa_K3727016) of hsa-miR-143-3p according to the quantities of miR-143-3p from 10nM to 1000nM.


Figure 3. This graph shows the fluorescence produced from toehold switch T-30e-5p-8 (BBa_K3727027) of hsa-miR-30e-5p according to the quantities of miR-30e-5p-8 from 10nM to 1000nM.


Figure 4. This graph shows the fluorescence produced from toehold switch T-1246-1 (BBa_K3727028) of hsa-miR-1246 according to the quantities of miR-30e-5p from 10nM to 1000nM.



All of the three diagrams show that we have achieved quantification and the results are promising.







EXPAR Experiments


For the amplification of the miRNAs in the urine samples, we decided to use the EXPAR amplification method. This is a simple amplification technique ideal for small sequences that produces DNA products. However, since our toehold switches are better suited to detect RNA sequences, we also designed a new protocol based on the EXPAR technique. You can find more information about these protocols on our “Experiments'' page.

For the amplification of the miRNAs in the urine samples, we decided to use the EXPAR amplification method. This is a simple amplification technique ideal for small sequences that produces DNA products. However, since our toehold switches are better suited to detect RNA sequences, we also designed a new protocol based on the EXPAR technique. You can find more information about these protocols on our “Experiments'' page.

Experiments





For our proof of concept, we decided to test both of these protocols.

After the design of our DNA Template sequences, we confirmed that our EXPAR protocols at 37 ℃ are functioning (EXPAR-DNA & EXPAR-RNA protocol).

The following diagrams show that our EXPAR protocols at 37 ℃ for 30 minutes, amplify the microRNAs even by x10^2 times, with very good results!

Amplification plots with negative controls for EXPAR – DNA Protocol at 37 ℃:





Amplification plots with negative controls for EXPAR - RNA Protocol at 37 ℃:




After we ensured that both of our EXPAR protocols work, we tested the reaction products with the best of our toeholds.

For more information, on the procedure that has been followed, you can check our EXPAR Lab Book.

EXPAR LabBook




In Table 3, you can see the results of the experiments with toehold switches and EXPAR products, which confirmed the results shown in the amplification plot.

miR-30e Samples Fluorescence miR-143 Samples Fluorescence miR1246 - Samples Fluorescence
miR-30e (2 nM) 95 miR-143 (2 nM) 110 miR-1246 (2 nM) 98
DNA of miR-30e (400 nM) 100 DNA of miR-143 (400 nM) 99 DNA of miR-1246 (400 nM) 92
ed miR-30e (x nM) 120 ed miR-143 (x nM) 120 ed miR-1246 (x nM) 130
ed miR-30e negative control (0 nM) 110 ed miR-143 negative control (0 nM) 110 ed miR-1246 negative control (0 nM) 100
er miR-30e (x nM) 150 er miR-143 (x nM) 140 er miR-1246 (x nM) 190
er miR-30e negative control (0 nM) 110 er miR-143 negative control (0 nM) 150 er miR-1246 negative control (0 nM) 160
Empty well 95 Empty well 94 Empty well 93
Empty well 95 Empty well 94 Empty well 95
Empty well 95 Empty well 94 Empty well 93
Empty well 96 Empty well 94 Empty well 93
Empty well 95 Empty well 94 Empty well 94
Empty well 95 Empty well 94 Empty well 94
Table 3: Fluorescence results from the experiments with Toehold switches and EXPAR products at 37 ℃.
*Abbreviations: ed = EXPAR - DNA amplification protocol products | er = EXPAR - RNA amplification protocol products | x nM = unknown concentration



In all cases, the fluorescence produced by the amplified products was higher in the samples with microRNA than in the negative controls, as expected, and higher than the positive controls used (miR-30e 2 nM and DNA of miR-30e 400 nM) which is in accordance with the concentration used.

Unfortunately, we can’t quantify the exact amount of the amplified products only based on the above fluorescence results because the EXPAR products contain SYBR Green I which exhibits fluorescence at 520 nm, according to Sigma Aldrich [1].

The concentration of the SYBR Green I is very low in the above samples (final concentration = 0.01x) and in the final form of our diagnostic tool it will not be needed. This is mainly because EXPAR will be standardized and the quantification will be done only by the toeholds. Nevertheless, more experiments are required with the proper amount of SYBR Green I in the positive control samples of each microRNA because it can affect the final fluorescence.






References