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:

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:

tabla

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.

qPCR

Cosas del apartado 1, se necesita meter mas divs seguramente