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                         <h3 id="Introduction" class="basicheader"> Introduction </h3>
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                         <h3 class="basicheader"> Discussion and Outlook </h3>
 
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                                 <p class="basictext">In this project we show a new, efficient, and specific Western blot assay based on AptaBodies for the detection proteins. The fusion of a HRP DNAzyme with an aptamer, that binds to a POI, so called AptaBody is a promising alternative that could complement and sometimes even replace classical antibodies so far applied in Western Blot experiments. </p>
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                                    Western blotting is a common analytic technique to detect and quantify proteins. Traditionally, primary and secondary antibodies are widely used for this purpose. Although nowadays tens of thousands of antibodies are available for Western blotting, dependency from antibodies also raises several limitations:
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As a proof of principle we targeted His-tag proteins using antiHis AptaBodies. Even in cell lysates the AptaBody is able to detected its target protein with a high specificity.
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Using AptaBodies instead of Antibodies may potentially have the following benefits:</p>
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<li> antibodies are often quite expensive </li>
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<li> production of specific antibodies is time consuming </li>
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<li>The protocol is cost and time saving. The costs for an AptaBody are just those for an oligo DNA strand. Furthermore, no blocking step is needed to achieve a specific signal. (Fig. 12)</li>
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<li>    even if antibodies are available they might not work very well for the protein of interest </li>
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<li> The generation of new AptaBodies is much faster than development of new antibodies. The design and production of the AptaBodies takes 14 days maximum. Please note that AptaBodies can be readily designed for proteins for which no antibodies are available yet.</li>
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<li>    their applicability is limited to a subset of proteins with sufficiently high molecular weight and certain biochemical properties </li>
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<li> MAWS can generate specific AptaBodies against each and every protein of interest.</li>
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<li> for many proteins no antibodies are available </li>
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<li> In contrast to antibodies the production of AptaBodies do not require animal experiments.  </li>  
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Having these limitations in mind, our idea was to introduce aptamers into Western blotting. We decided to develop an “AptaBody” as a new tool for protein detection. AptaBodies are short DNA oligos, which combine the capabilities from primary and secondary antibodies within one molecule. At its 5’-end the AptaBody consists of an aptamer, targeting a specific molecule. Through this aptamer, the AptaBody can bind to an immobilized protein blotted on a membrane (Fig. 1).
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Figure 1. Classical design of an aptabody.
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AptaBodies consist of an aptamer, which binds to an immobilized protein of interest, a poly-A linker coupled to a HRP-mimicking DNAzyme forming a G-quadruplex.  
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                                  Main advantages arguing for the application of aptamers for Western blotting are:
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<li> AptaBodies are very cheap (<a href="Fig.12) </li>
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<li> they are easy and fast to get </li>
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Using our MAWS software we are able to design aptamers containing nucleotide sequences that optimally target every specific molecule of interest. On its 3’-end, the AptaBody contains the sequence of the HRP-mimicking DNAzyme. This oligonucleotide forms a G-quadruplex <x-ref>wang_highly_2015 </x-ref> (Wang, Wang, & Huang, 2015). In the presence of potassium ions, hemin can bind to the G-quadruplex, which then catalyses the luminol-H<sub>2</sub>O<sub>2</sub> chemiluminescence (CL) reaction. These two functional parts of the AptaBody are fused together via a poly-A linker (Fig. 1).
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Latest revision as of 19:55, 19 November 2015

Discussion and Outlook

In this project we show a new, efficient, and specific Western blot assay based on AptaBodies for the detection proteins. The fusion of a HRP DNAzyme with an aptamer, that binds to a POI, so called AptaBody is a promising alternative that could complement and sometimes even replace classical antibodies so far applied in Western Blot experiments.

As a proof of principle we targeted His-tag proteins using antiHis AptaBodies. Even in cell lysates the AptaBody is able to detected its target protein with a high specificity. Using AptaBodies instead of Antibodies may potentially have the following benefits:

  1. The protocol is cost and time saving. The costs for an AptaBody are just those for an oligo DNA strand. Furthermore, no blocking step is needed to achieve a specific signal. (Fig. 12)
  2. The generation of new AptaBodies is much faster than development of new antibodies. The design and production of the AptaBodies takes 14 days maximum. Please note that AptaBodies can be readily designed for proteins for which no antibodies are available yet.
  3. MAWS can generate specific AptaBodies against each and every protein of interest.
  4. In contrast to antibodies the production of AptaBodies do not require animal experiments.