Proof of Concept
Detection of brain damages and other neurodegenerative diseases is a time, cost, and work-intensive process. Patients often go to check-ups once they experience symptoms and must undergo either invasive methods where spinal fluids or blood samples are analysed, or they have complicated and expensive tests such as MRI and brain scans. The problem is that these methods are either uncomfortable for the patients or expensive for our health care system. Due to long queues, patients also have to wait a long time to get their results from such tests.
This is where AptaTear comes into play. Our project aims to be a quick, non-invasive, and easy-to-use detection method for mild traumatic brain injuries (mTBI) that would provide the user with results in less than two hours and would be of minimal invasiveness for the patients. The risk of undetected mTBI resulting in neuronal damage is in the long term associated with neurodegenerative disease, e.g. chronic traumatic encephalopathy (CTE).
AptaTear was developed to detect the biomarker tau in patient derived tears, through the use of tau-specific aptamers and an amplification method known as “Rolling Circle Extension-Actuated Loop-Mediated Isothermal Amplification” (RCA-LAMP). Using RCA-LAMP would allow us to amplify a target sequence causing a pH change which in return leads to a visible colour change. Tau protein is normally an intraneuronal protein not present in circulation in a healthy brain and is therefore a marker of neuronal injury.
Our project was divided into several steps. The first step was to express and purify the protein tau by using the E.coli strains BL21/DE3 and DH5-alpha as our genetically modified organisms (GMO).
The second step was to find the right aptamer for our biomarker and establish a functional LAMP reaction.
The last step was to assemble every part of the kit and use it on synthetic “patient-derived” tears containing either no tau or tau at different concentrations.
The E. coli strains BL21/DE3 were used for the tau expression. The plasmid contained recombinant human tau 383 with a His-tag on its N-terminal (0N4R) which was fused with enhanced green fluorescent protein (eGFP). The His-tag allowed it to be captured by magnetic beads.
We purified tau from the modified E. coli by lysing the bacteria and then proceeding to elute them through an IMAC (Immobilized metal affinity chromatography) column. The immobilized Tau protein fused to eGFP allowed us to separate our biomarker from other proteins in an easy way through the purification process.
As shown in figure 1, an SDS page was used to confirm the collection of tau proteins. Tau has a molecular weight of ca. 70.6kDa.
Figure 1: SDS page showing the collected Tau with a molecular weight of ca. 70.6kDa, indicated by an arrow.
Our project is based on previous studies confirming the affinity of aptamers for tau and the success of RCA-LAMP in detection methods [1]. We used the aptamers IT2e and IT2d (shown in figure 2). Based on the article, it was already confirmed that our aptamers have an affinity for tau. T231 and T231P stand for Human tau and phospho tau. As IT2e only had one epitope on tau to which it could bind, it was eventually decided that the experiments would proceed exclusively with this candidate.
We also performed molecular modelling to understand the mechanism of aptamer-tau complex formation.
Figure 2: Table of several different binding molecules and their potential tau isoforms as targets.
As mentioned earlier, using RCA-LAMP allowed us to amplify a target sequence causing a pH-induced colour change of our samples. We got this idea from the article “Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences" [2], in which a p53 DNA-sequence functioned as a padlock-probe-recognition-site that was amplified by having a padlock probe binding to it. The primers FIP and BIP would amplify the padlock probe and with it also the target sequence which once amplified and accumulated in the samples would lead to a pH change. The sample would turn from a reddish colour to a yellow colour. This yellow colour would indicate a positive sample. We used the amplification approach and linked our aptamers together with the p53 sequence so that the presence of our aptamers would lead to an amplification and a pH change. Since the presence of the aptamer together with the p53 amplification-sequence would lead to a colour change we had to introduce a washing step to wash away the aptamers in absence of tau.
We confirmed the success of RCA-LAMP and our aptamers affinity for tau by using magnetic beads capable of capturing tau. This was possible due to the His-tag on our biomarkers N-terminal. We had one tube containing magnetic beads and tau and one tube containing only magnetic beads. Aptamers were introduced to both tubes and after incubating the tubes for one hour we washed our samples with an equilibration buffer. If tau was present and bound to the magnetic beads, the aptamers would be bound to the tau and therefore wouldn't be washed away. If no tau was present and the aptamers were not bound to anything, then they would be washed away.
Using an elution buffer, the components bound to the beads would be eluted and could be used for our RCA-LAMP reaction. We added the padlock probe and primers to each elute. If aptamers were present in one of the samples, then the recognition site of the padlock probe linked to the aptamer would be amplified. If all aptamers are washed away, then no colour change would occur. The result was that the sample containing tau changed colours while the sample without tau proteins remained of reddish colour (Shown in Figure 3). This proved that the aptamer binds to tau as well as the reaction can only occur in the presence of aptamers with the recognition site linked to them.
Figure 3: Positive sample (yellow sample on the left) and negative sample (reddish sample on the right).
Our goal with AptaTear is to offer a quick, non-invasive detection method of brain damage. During our time in the laboratory, we managed to create a functional detection method specifically for tau and hope that this would lead the way for alternative detection methods for brain damage and neurodegenerative diseases. The eyes are an extension of our brain and therefore can tell us a lot about the condition of our most essential organ. We in LiU iGEM believe that tears as test samples as well as aptamers will play a key role in the fight against neurodegeneration and will be crucial for modelling such diseases in patients. This makes us proud to work on a project which contributes to this change.