Difference between revisions of "Team:Madrid-OLM/AptCharacterization"
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<p class="lead">We follow the steps describes in the “ELONA Protocol” and successfully label the round 6 of OLE-E1:</p> | <p class="lead">We follow the steps describes in the “ELONA Protocol” and successfully label the round 6 of OLE-E1:</p> | ||
<img alt="Image1" src="https://static.igem.org/mediawiki/2018/5/52/T--Madrid-OLM--Aptamer--Characterization--DIGlabeling.png"/> | <img alt="Image1" src="https://static.igem.org/mediawiki/2018/5/52/T--Madrid-OLM--Aptamer--Characterization--DIGlabeling.png"/> | ||
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| − | <h2> | + | <h2>ELONA</h2> |
<p class="lead">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.</p> | <p class="lead">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.</p> | ||
<p class="lead">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.</p> | <p class="lead">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.</p> | ||
Revision as of 09:15, 16 October 2018
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 follow the steps describes in the “ELONA Protocol” and successfully label the round 6 of OLE-E1:
ELONA
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.