During this competition, we have obtained inspiration from different sources. One of the most important iGEM teams that worked with aptamers was iGEM Bonn 2017.
Although their work trying to develop aptamers which are active in saliva is remarkable, when we tried to replicate it, we have come across the following problems:
The way they separate aptamers from the target proteins is more expensive than what we could afford. It also needs special laboratory equipment which is not standard in a normal wet lab.
The aptamer/SELEX round characterization method,, which is through Next Generation Sequencing, is expensive and requires more than one month of waiting to be adapted to a particular case. This is the reason why It is not feasible for checking frequently the success of SELEX rounds. Additionally, It also does not give any kind of information on aptamer specificity or affinity.
The lack of results on the achievement of the SELEX process. This is an intricate process and having success on the aptamer discovery during an iGEM competition is not as easy at it seems. In their wiki page, we could not find any kind of result that indicates the accomplishment of their SELEX process.
Inspired by their work, we have developed a new SELEX process that is fully documented in our protocol section and explained in the aptamer discovery page. To make a brief description, we have solved the above-mentioned issues with the following actions:
For making an easy and affordable SELEX process, we have designed a 3D printed nitrocellulose spin columns. The only mandatory requirement is to place the aptamer solution, which has been previously incubated with the target protein, on the top of a fully assembled column. Then centrifuge it at 8000rpm and purify and amplify the bound DNA through the nitrocellulose membrane. You could see a more detailed explanation of the system in our aptamer discovery page.
To analyze the success of the SELEX rounds, we have established and described a qPCR-based system made with the purified DNA from the nitrocellulose membrane. It enables us to estimate the number of different aptamer sequences in a SELEX round, through the shape of the qPCR results.
To analyze the success of the aptamer binding capacity, we have documented a new qualitative method called ELONA. It is the aptamer’s version of an ELISA test. With this method, we are able to determine the specificity and the affinity of the developed aptamer, defining some of its parameters like the affinity constant.
We have the results of the previous two methods (qPCR and ELONA) for our selection process, demonstrating the success of our SELEX protocol.
Aptamer Discovery page