Results
Tau protein is the central part of our project and, therefore, we first had to produce our wanted protein before we could use it for further experiments. We used a plasmid in which the tau sequence (human tau 0N4R with an N-terminal His-tag) was fused to enhanced green fluorescent protein (eGFP) sequence, resulting in His-tau-eGFP protein. This was helpful as tau could easily be visualised by the naked eye. As can be seen in figure 1, there was a definite green colour to our samples which confirmed the production of tau.
Figure 1: Cultures with Tau expressed samples (left) and pellets (right).
After eluting tau from the nickel column through IMAC, we visualised it in an SDS-PAGE gel to check the identity and purity of the protein. We tested a sample of E. coli culture after tau induction but before cell sonication, the flowthrough and the wash from IMAC as controls, side by side with successive elutions of tau. The results can be seen in figure 2.
The wells contained the following:
Figure 2: SDS- PAGE gel on different tau elutions. Expected molecular weight of the His-tau-eGFP protein (tau) was 70.6 kD.
The results showed that there was no impurity in elution 4 and 5. However, the tau yield was small. Impurities are seen in elutions 1,2 and 3, while the yield is much bigger. The wash doesn’t seem to have many impurities. This means that the washing step wasn’t able to remove most of the impurities. However, it is also visible that the wash didn’t remove a lot of tau. The flowthrough had a lot of impurities and even had some tau. The sample from after induction shows a thin tau band, even though sonication hadn’t been performed yet at that point. These results were also supported by a fluorescence scan performed on another unstained SDS-PAGE gel (figure 3).
Since the tau elutions contained impurities, it was decided to dialyse them.
Figure 3: Fluorescence scan on SDS-PAGE gel.
Recognition sequence is a p53 sequence taken from Marciniak et al. 2008 (see Parts), that we have added to our aptamer construct via a linker, and that is recognized by the padlock probe. We ran RCA-LAMP on the recognition sequence alone first to make sure that LAMP worked. RCA-LAMP consists of three main parts: phosphorylation of padlock probe, preparation of ligation mix, preparation of amplification mix.
Figure 4: Positive result from LAMP using the recognition sequence (p53).
There was one problem that was persisting throughout the samples. If the sample was removed from the PCR machine and amplification occurred and a pH change took place, the sample would turn back to its original pink colour. It was decided to run a PCR cleanup on the samples. After this cleanup step was incorporated the positive samples stayed the yellow colour after it was removed from the heat. In figure 5, it can be seen that a sample is turning back to its original pink colour.
Figure 5: Positive yellow sample tuning back to its original pink colour.
After seeing that RCA-LAMP is successful, the next step was to see whether the aptamer would detect tau. To capture tau, we used magnetic nickel beads (figure 6). Tau binds to the beads thanks to the his-tag. Aptamers were added to tau bound to beads and then LAMP was performed. This procedure was done for both aptamer 1 and 2.
Figure 6: Eluate of tau and aptamer
Both showed colour changes to different degrees, where aptamer 1 showed the most colour change. To verify the results, an agarose gel was run. Figure 7 shows the agarose gel, where well 1 contains aptamer 1, well 2 aptamer 2 and well 4 contains a DNA ladder. The rest of the wells are empty. From the image it is clear that the colour change was a result of DNA amplification and not simply pH change. Moreover, LAMP performed with aptamer 1 has the biggest amount of amplification. Therefore, it was decided to continue the rest of the experiments with aptamer 1.
Figure 7: Agarose gel showing amplification using different aptamers.
Finally, the procedure was repeated again with a negative control. No tau was added to the magnetic beads and the rest of the steps remained unchanged. The reason for this was to make sure that aptamers didn’t bind to the magnetic beads. The results can be seen below in figure 8.
Figure 8: Positive and negative control LAMP.
Synthetic tears were prepared to mimic human tears with some of the most basic electrolytes and proteins. Synthetic tears of different tau concentrations were tested on magnetic beads using LAMP. Unfortunately, the results weren’t satisfactory. All synthetic tears (even the ones without any tau), had a colour change which was due to DNA amplification as confirmed by agarose gel results (figure 9 and 10).
Figure 9: Agarose gel of LAMP from synthetic tears of different tau concentrations (from left to right: no tau, 10 ug/mL, 10 ng/mL, 5ng/mL, ladder).
Figure 10: Agarose gel of LAMP from synthetic tears without tau and (From left to right: ladder, synthetic tears with no tau).
The first suspicion we had at seeing these results was that some contamination of the synthetic tears was possible. However, since the result is the same for the human tears, we also suspected that the aptamer might be too sensitive and that we needed to reduce its concentration.
To see the difference in composition of human tears versus the synthetic tears with varying concentrations of tau, an SDS-PAGE was run. The samples loaded in the SDS gel wells contained:
Well 1 - ladder
Well 2 - synthetic tears 1 ng/mL tau
Well 3 - synthetic tears 2.5 ng/mL tau
Well 4 - synthetic tears 5 ng/mL tau
Well 5 - synthetic tears 10 ng/mL tau
Well 6 - synthetic tears 10 ug/mL tau
Well 7 - synthetic tears 0 ng/mL tau
Well 8 - synthetic tears 0 ng/mL tau
Figure 11: SDS-PAGE of synthetic tears of varying tau concentrations.
From the image above, the results show that there is definitely some contamination in the synthetic tears as they all appear to be the same even though two of them don’t contain any tau.
His-tag antibodies were used to capture tau in tears. High-binding 96-well plates were coated with antibodies, solution containing tau was added, aptamer with linker was added and then LAMP was performed. To get reliable results triplicates of antibody + tau were prepared, as well as triplicates of just tau coating wells. Finally, blanks were also used. ELISA was run twice to verify results. In the first run, LAMP was performed on 3 wells coated with antibody and 3 wells directly coated with tau (as a positive control) in the fourth row, as well as three blanks in the last row containing antibody, PBS buffer and LAMP mixes (figure 12.a). From the results in figure 12.a, it seemed that the aptamer in the blank wells was not completely washed off. Therefore, ELISA was repeated with a few changes: aptamer was diluted in BSA instead of water, washing was done with PBS-T instead of PBS. In the second run, LAMP was tested on the row with three blanks containing buffer and LAMP mix, and the column with two tau-coated wells (figure 12.b).
Figure 12: First ELISA run (a) and second run (b) with negative results.
From the results seen above, both runs gave unsatisfactory results. While it’s a good thing that there’s a colour change in the wells containing tau, the blanks, which also changed colour even though they were not supposed to, indicate that the positive results are not reliable. For this reason, an agarose gel was run using the samples from the second run only. As can be seen from figure 13, there has been no amplification, meaning that the colour change was simply a false positive.
Figure 13: Agarose gel results of ELISA (from left to right: ladder, blank, tau-coated).
Since LAMP results from synthetic tears, human tears and ELISA were unsatisfactory, a series of experiments were performed to verify that the detection method was working properly. These experiments consisted of repeating the protocol in “LAMP detection of tau in magnetic beads” while removing a component of the LAMP one at a time to see the effects each had. The results were similar for most of them. The following samples were in the wells left to right.
Figure 14: Agarose gel results of Troubleshoot LAMP experiments
The removal of each component resulted in amplification, as seen in figure 14, except for the cases where at least one of the primers was not present in the mix. Consequently, the problem with the detection kit generating false positives resided in the primers being faulty. This might be either a manufacturer’s defect or a problem with storage and handling.
MST is a method to check the affinity between a fluorescent-labelled protein and a non-fluorescent ligand. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. Interactions are detected by changes in molecular diffusion or fluorescence intensity. In this case, the target whose change in fluorescence is observed is the tau protein, which was labelled using Red-tris-NTA dye (interacting with the His-tagged tau protein), and the non-fluorescent ligand is the aptamer.
Firstly, the affinity between tau and the dye was checked. The result was a dissociation constant (Kd) of 240 nM showing that we have successfully detected His-tagged tau protein.
After seeing that tau interacts strongly with the dye, the protocol was followed for the labelling of tau with the dye. Then MST was repeated for the labelled protein and aptamer. Unfortunately, the results were inconclusive and did not confirm any affinity between tau and the aptamer. The reasons may be that the fluorescence change of the RED-tris-NTA-tau protein interacting with the aptamer is not sufficient due to the dynamic properties of the intrinsically disordered tau protein.