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.

Selex

SELEX: Systematic Evolution of Ligands via Exponential Selection is the process of identifying specific aptamers.

To select a binding aptamer, you don't look for epitopes. This only simplifies the process as you don't design a determined structure but reduced little by little an already binding population.

The SELEX screening process starts with a random library of nucleotide oligomers of a fixed size with know sequences in each end (for further amplification by PCR). Then the library is incubated with the target molecule. A number of this random sequences will bound to the target and the unbound sequences will be discharged.

The bound sequences are separated now from the target molecule (elution step) and amplified to create a new library.

With every round, more and more oligonucleotides with low binding-affinity for our protein of interest will be discarded leaving only strong binding aptamers at the end:

This process used to imply high amounts of time, money and expertise to be performed correctly. This is why it has been so difficult to earlier iGEM teams to work with aptamers, or why they skip the aptamer developing process and works only with already discovered aptamers.

For the first time in iGEM we present a successful, affordable, and relatively fast SELEX process. You could see the details on how to perform it in our protocol page:

Selex Protocol

We hope that with this approach, in recent years, we will see the aptamers registry page getting larger and larger.

Our experiment:

To separate the bound sequences from the unbound ones, we decided to use a nitrocellulose membrane.

Nitrocellulose membranes can bind protein through hydrophobic interactions, so when applying the incubated library pool with the target protein to the membrane and forced the liquid to pass through it, the protein will stay within the membrane and as well the bound sequences.

The pore size was chosen after folding the aptamers and test both 0,22 um and 0.45 um size pore. After seeing the results we conclude that the 0.22 um pore was too small and the aptamers were being trapped because of their size and not by unspecifically interactions.

We tried in parallel two different proteins:

1. BSA

2. Ole-e1

BSA

We tested the detection limit of the ponceau dye to visualize the protein on the nitrocellulose membrane, which was 25 ug. This was decided to allow us to be able to detect, during the process, if the protein was being correctly binding with the membrane:

Figure 1. It is shown the OD600 measurement by the first calibration experiment. The corrected Abs600 is made subtracting H2O measurements from LUDOX CL-X ones. The OD600/Abs600 result (3,652) is the multiplication factor, which you have to use after a cell density measurement with the plate reader to have a correct analysis.

From that point on, the ratio between the aptamer/protein was 1:1. The recommended one is 1: 10, protein/aptamer, so there is competence for the binding sites and only the sequences with the best affinity are selected.

Using our ratio means that further rounds of selection will be needed in order to achieve the same range of affinity than other SELEX selection using less protein.

We start the Selex by dismissing the sequences that bind unspecifically to the system itself. We fold the aptamers and forced them through the membrane and keep the flowthrough.

With each round, we will repeat this process before incubating with the target protein and see that the amplified libraries did not bind to the membrane alone, only after incubation with the protein:

Table 1. Nmol of aptamers traped in the membrane and the % of sequences retain by unspecifically interactions for each round of Selex with BSA protein.

After the incubation with the target, we forced the mix through the membrane again but this time keeping the membrane. Because of the amount of protein, we use in each round and as in can be seen in figure 1, not all the protein was being trapped by the membrane and in all the measures done by the nanodrop the curves from the protein concentration and DNA concentration overlap, making impossible to measure the flowthrough.

During the consecutive rounds, the number of cycles to amplified by PCR were reduced from 30 to 10:

- Second round of selection.

Figure 2: A:Agarose gel after 10 cycles of amplification. B: Agarose gel after 30 cycles of amplification.

- Fourth round of selection:

Figure 3: Agarose gel after 10 cycles of amplification.

OLE-E1

We start the SELEX protocol as we did with BSA, with the exception that we followed the 1:10 ratio of aptamer/protein proportion, so for this experiment, we used 2 ug of protein for each round.

We had problems with the amplification of the rounds of selection so instead of checking the proper number of amplification cycles, we fixed 50 cycles. We eventually could manage to see enough DNA in the agarose gel, but the number of cycles boosts the creation of concatamers.Concatamers are long continuous DNA molecules that contain multiples copies of the same DNA sequences linked in series.

Not only were clearly seen in the agarose gels. The amount of DNA that was being trapped in the membrane before the incubation with the target protein increased too.

Figure 4. A: Agarose gel after the second round. B: Agarose gel after the sixth round.

Table 2. Nmol of aptamers traped in the membrane and the % of sequences retain by unspecifically interactions for each round of Selex with Ole-E1 protein.

At the end we finish for each protein:

- BSA: six rounds.

- Ole-1: seven rounds.

qPCR

The real-time PCR can show the evolution and enrichment of your selection process. When the amount of different sequences is very high, like in the initial population, the fluorescence star to grow and reaches a peak before decreasing.

This happens because as the SELEX id performed, the number of different sequences are drastically reduced, therefore the amplification can be done as usual and the characteristic sigmoid curve finally appears.

This happens because as the SELEX id performed, the number of different sequences are drastically reduced, therefore the amplification can be done as usual and the characteristic sigmoid curve finally appears. With each round of selection, we are able to reduce the number of sequences until the ideal curve it's achieved.

Figure 5: Graph with the cicles of the rounds of qPCR agains Relative Fluorescence Units (RFU) for the diferents round of Selex.