Team:Madrid-OLM/AptCharacterization

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Biochemical characterization of the aptamers

One of the most important steps when you are working with aptamers, especially if you are looking for aptamers for a downstream application, is to demonstrate that aptamers have high affinity, specificity and selectivity for its substrate. It is logical to think that any aptamer with flexible conformational structure would also demonstrate interaction with many off-targets having similar motifs. However, aptamers with a defined ground state would bind only to their specific targets with high affinity.

Affinity is a term that makes reference to the strength of interaction that exists between a molecule (aptamer in this case) and its target. The key variable to measure if you want to assess the binding capacity of an aptamer is the association constant (Ka).

Aptamers that show high association constants have strong interactions with their targets. These high-affinity aptamers can bind low amounts of the target in samples.

In this case, we have developed aptamers from the start. For this, it was very important to know if it has really specific aptamers against a substrate and which was the affinity of the aptamers for its substrate.

To solve this problem, we decided to attempt to do an ELONA (Enzyme-Linked Oligonucleotide Assay). ELONA is a biochemical method based on enzyme-linked immunosorbent assay (ELISA). You have a plate with your target protein linked in the surface and instead of a first antibody (like in an ELISA assay), you use different concentrations of your aptamer.

It has been described different ELONA formats for aptamer-based protein detection. We have chosen one of them, which uses an anti-digoxigenin antibody to recognize an aptamer previously labelled with digoxigenin. This antibody is conjugated with a peroxidase enzyme, and once it adds ABTS with hydrogen peroxide, it will be responsible for the colourimetric reaction which will be detected by Varioskan Lux.

ELONA is a quantitative experiment and allows to calculate the Kd of the aptamers tested. This method improves the ones than previous iGEM teams have used to measure the affinity of aptamers like the Lyon team that uses polyacrylamide gels, a qualitative experiment that only tells if the aptamer binds to the target protein but doesn't give you further information about the interaction ( Kd), neither allow you to compare, once you have cloned and sequences your aptamers, to choose the one with the best affinity.

DIG-Labelling

We choose to mark our aptamers with digoxigenin, so the second antibody could detect the aptamers bind to the protein, as it would do in a normal ELISA assay. We choose this method, because it’s performed as a normal PCR, but with modified primers. We follow the steps describes in the “ELONA Protocol” and successfully label the round 6 of OLE-E1.

Figure 1: Agarose gel after 15 cycles of amplification: line 1 negative control; line 2 initial population; line 3 round 6 of OLE-E1, line 4 round 6 of OLE-E1.

ELONA

During the DIG-labelling, we only obtained enough ng of aptamers to test only one concentration, 1 ng/µL. Because of this, we did a triplicate, instead of a duplicate like in our protoco. We also didn’t use thrombin as a positive control, because we run out of it during the tunning of the protocol. We use another proven aptamer ceded from the Victor González research group./p> Elona Protocol

Results

1. We perform a successful ELONA assay, as it can be seen in a positive control, an aptamer that we know it has a high affinity, shows a high absorbance, proving we were able to measure aptamers binding to their target.

2. The absorbance from round 6 has increased in comparison to the initial population shows that we were able to recover the sequences that actually binds and discharge the ones that don’t.

3. The results also tell, together with the qPCR ones, that a few more rounds will be needed to achieve the desired affinity. Because if you compare the absorbance of our round 6 to the positive control, is still low.

We did an additional round of SELEX, restricting the time to half an hour to force the selection, but not repeat an ELONA with the new round because of the lack of time.

Figure 2: Absorbance of the initial population against negative control (BSA), round 6 of OLE-E1 against negative control, noise and positive control.