Difference between revisions of "Template:Heidelberg/project/standardization/HRP"

 
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                         <h3 class="basicheader"> HRP-mimicking DNAzyme </h3>
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                         <h3 class="basicheader"> HRP-mimicking DNAzyme </h3>
 
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                                     The HRP-mimicking DNAzyme<x-ref>travascio1998</x-ref> is our favorite part and as such we used it as modular part in different setups. It forms a G-quadruplex structure in which hemin can bind (Fig.2). This enables it to catalyze the fission of hydrogen peroxide to water and a reactive oxygen species (ROS). Thus it can be used to catalyze chemiluminescence and a series of colorimetric reactions, known from the horseraddish peroxidase from <i>Amoracia rusticana</i>.
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                                     HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it. Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H<sub>2</sub>O<sub>2</sub> to H<sub>2</sub>O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.
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                                </p>
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<p>&nbsp;</p>
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                        <h3 class="basicheader"> HRP DNAzyme in the AptaBody </h3>
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             <img class="img-responsive" src="https://static.igem.org/mediawiki/2015/1/18/HeidelbergFSFig3_AptaBody.png">
 
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                 HRP-mimicking DNAzyme
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                 Figure 3. AptaBody
 
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                 HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it. Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H<sub>2</sub>O<sub>2</sub> to H<sub>2</sub>O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.
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                 An aptamer connected via a linker to a HRP-mimicking DNAzyme that detects protein an a Western blot.
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                                    Initially we have connected the HRP-mimicking DNAzyme via a linker region to a His-tag aptamer<x-ref>Bartnicki2014</x-ref> (Fig.3). This way we showed that these two parts together with a linker that connects both can be applied to detect different His-tagged proteins from cell lysate on a Western blot. The aptamer part of this construct can easily be exchange by any other aptamer as we could show for p53. We generated an aptamer for p53 and were able to detect p53 with its AptaBody. We have calculated several other aptamers for proteins that need to be tested with our software MAWS. Aptamers generated by our software MAWS can be fused to the versatile HRP DNAzyme to produce a library of AptaBodies.
 
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The transformation of a terminal label into an internal label, one can be achieved by splinted ligation using a DNA template that is complementary to the two RNA templates that are to be connected to each other <x-ref>Kershaw2012</x-ref>.
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                Figure 2. HRP-micking DNAzyme
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                HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it (Fig. 2). Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H<sub>2</sub>O<sub>2</sub> to H<sub>2</sub>O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.
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Latest revision as of 23:07, 18 September 2015

HRP-mimicking DNAzyme

HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it. Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H2O2 to H2O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.

 

HRP DNAzyme in the AptaBody

Figure 3. AptaBody

An aptamer connected via a linker to a HRP-mimicking DNAzyme that detects protein an a Western blot.

Initially we have connected the HRP-mimicking DNAzyme via a linker region to a His-tag aptamerBartnicki2014 (Fig.3). This way we showed that these two parts together with a linker that connects both can be applied to detect different His-tagged proteins from cell lysate on a Western blot. The aptamer part of this construct can easily be exchange by any other aptamer as we could show for p53. We generated an aptamer for p53 and were able to detect p53 with its AptaBody. We have calculated several other aptamers for proteins that need to be tested with our software MAWS. Aptamers generated by our software MAWS can be fused to the versatile HRP DNAzyme to produce a library of AptaBodies.

The transformation of a terminal label into an internal label, one can be achieved by splinted ligation using a DNA template that is complementary to the two RNA templates that are to be connected to each other Kershaw2012.

Figure 2. HRP-micking DNAzyme

HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it (Fig. 2). Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H2O2 to H2O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.