Difference between revisions of "Team:Madrid-OLM/AptCharacterization"

 
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                         <a href="#diglab" class="inner-link" data-title="DIG-Labelling"></a>
 
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                         <a href="#qPCR" class="inner-link" data-title="qPCR"></a>
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                         <a href="#elona" class="inner-link" data-title="ELONA"></a>
 
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                             <h1 id="Teamtittle">Biochemical characterization of the aptamers</h1>
 
                             <h1 id="Teamtittle">Biochemical characterization of the aptamers</h1>
−
                             <p class="lead">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.</p>
+
                             <p class="lead">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 specific targets with high affinity.</p>
−
                             <p class="lead">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).</p>
+
                             <p class="lead">Affinity is a term that makes reference to the strength of the 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 dissociation constant (Kd). </p>
−
                             <p class="lead">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.</p>
+
                             <p class="lead">Aptamers that show low dissociation constants have strong interactions with their targets. In this case, we have developed aptamers from scratch. Because of this, it was very important to know if we ended up with a specific aptamer against his target and which was their affinity. </p>
−
                            <p class="lead">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. </p>
+
                             <p class="lead">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 you test different concentrations of your aptamer instead of doing so with a first antibody (like in an ELISA assay).</p>
−
                             <p class="lead">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.</p>
+
                             <p class="lead">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.</p>
−
                             <p class="lead">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.</p>
+
                             <p class="lead">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 <a href="https://2016.igem.org/Team:INSA-Lyon">Lyon team</a> that uses polyacrylamide gels. This is a qualitative experiment that only tells if the aptamer binds to the target protein but does not give you further information about the interaction ( Kd),  neither allow you to compare between different sequences, to choose the one with the best affinity.</p>
−
                             <p class="lead">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 <a href="https://2016.igem.org/Team:INSA-Lyon">Lyon team</a> 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.</p>
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             </section>
 
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             <section id="diglab" class="text-center">
 
                 <div class="container">
 
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                         <div class="col-md-10 col-lg-8 boxed boxed--border bg--secondary boxed--lg box-shadow">
 
                             <h2>DIG-Labelling</h2>
 
                             <h2>DIG-Labelling</h2>
−
                             <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 choose to mark our aptamers with digoxigenin, so the second antibody could detect the aptamers binding to the protein, as it would do in a normal ELISA assay. We choose this method because it is performed as a normal PCR, but with modified primers. We followed the steps described in the “ELONA Protocol” and successfully labelled the sixth round of OLE-E1:</p>
−
                             <img alt="Image1" src="https://static.igem.org/mediawiki/2018/5/52/T--Madrid-OLM--Aptamer--Characterization--DIGlabeling.png" style="width:80%;"/>
+
                             <img class="figureimage" alt="Figure1" src="https://static.igem.org/mediawiki/2018/5/52/T--Madrid-OLM--Aptamer--Characterization--DIGlabeling.png" style="width:80%;"/>
−
                             <p class="lead" style="margin-left:10%; margin-right:10%;">Figure 1: Explicacion</p>
+
                             <p class="lead" style="margin-left:10%; margin-right:10%;">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.</p>
 
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             <section id="elona" class="text-center">
 
                 <div class="container">
 
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                             <h2>ELONA</h2>
 
                             <h2>ELONA</h2>
−
                             <p class="lead">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 protocol ( LINK AL PROTOCOLO). We also didn’t use thrombin as 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>
+
                             <p class="lead">During the DIG-labelling, we only obtained enough nanograms of aptamers to test only one concentration, 1 ng/µL. Because of this, we did a triplicate, instead of a duplicate like in our protocol. We also didn’t use thrombin as positive control, because we run out of it while tuning up the protocol. We used another proven aptamer ceded from the Victor González research group.</p>
 +
                            <a class="btn btn--primary-2 btn--sm type--uppercase" href="https://2018.igem.org/Team:Madrid-OLM/AptamerProtocols#characterization">
 +
                                <span class="btn__text">
 +
                                    Elona Protocol
 +
                                </span>
 +
                            </a>
 
                             <h5>Results</h5>
 
                             <h5>Results</h5>
 
                             <ol class="ourlist">
 
                             <ol class="ourlist">
−
                                 <li><p class="lead">We perform a successful ELONA assay, as it can be seen in the 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. </p></li>
+
                                 <li><p class="lead">We performed a successful ELONA assay, as it can be seen in the positive control. As the positive control is an aptamer that we know that has a high affinity, it shows a high absorbance, proving that we were able to measure aptamers binding to their target. </p></li>
−
                                 <li><p class="lead">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. </p></li>
+
                                 <li><p class="lead">The absorbance from round 6 increased in comparison to the initial population. This fact showed that we were able to recover the sequences that actually binds as well as discard the ones that don’t.</p></li>
−
                                 <li><p class="lead">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.</p></li>
+
                                 <li><p class="lead">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, it is still low.</p></li>
 
                             </ol>
 
                             </ol>
−
                             <p class="lead">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. </p>
+
                             <p class="lead">We did an additional round of SELEX, restricting the time to half an hour to force the selection, but we did not repeat an ELONA with the new round because of the lack of time.</p>
−
                             <p class="lead">We follow the steps describes in the “ELONA Protocol” and successfully label the round 6 of OLE-E1:</p>
+
                             <img class="figureimage" alt="Figure2" src="https://static.igem.org/mediawiki/2018/8/81/T--Madrid-OLM--Aptamer--Characterization--Elona.png" style="width:60%;"/>
−
                            <img alt="Image1" src="https://static.igem.org/mediawiki/2018/8/81/T--Madrid-OLM--Aptamer--Characterization--Elona.png" style="width:60%;"/>
+
                             <p class="lead" style="margin-left:20%; margin-right:20%;">Figure 2: Absorbance of the initial population against negative control (BSA), round 6 of OLE-E1 against negative control, noise and positive control.</p>
−
                             <p class="lead" style="margin-left:20%; margin-right:20%;">Figure 2: Explicacion</p>
+
−
                           
+
−
                           
+
 
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Latest revision as of 03:15, 18 October 2018

Madrid-OLM

Aptamer Characterization

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 specific targets with high affinity.

Affinity is a term that makes reference to the strength of the 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 dissociation constant (Kd).

Aptamers that show low dissociation constants have strong interactions with their targets. In this case, we have developed aptamers from scratch. Because of this, it was very important to know if we ended up with a specific aptamer against his target and which was their affinity.

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 you test different concentrations of your aptamer instead of doing so with a first antibody (like in an ELISA assay).

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.

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. This is a qualitative experiment that only tells if the aptamer binds to the target protein but does not give you further information about the interaction ( Kd), neither allow you to compare between different sequences, 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 binding to the protein, as it would do in a normal ELISA assay. We choose this method because it is performed as a normal PCR, but with modified primers. We followed the steps described in the “ELONA Protocol” and successfully labelled the sixth round of OLE-E1:

Figure1

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 nanograms of aptamers to test only one concentration, 1 ng/µL. Because of this, we did a triplicate, instead of a duplicate like in our protocol. We also didn’t use thrombin as positive control, because we run out of it while tuning up the protocol. We used another proven aptamer ceded from the Victor González research group.

Elona Protocol
Results

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

  2. The absorbance from round 6 increased in comparison to the initial population. This fact showed that we were able to recover the sequences that actually binds as well as discard 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, it is still low.

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

Figure2

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