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Introduction

Western blotting (or Immunoblotting) 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:

  • antibodies are often quite expensive
  • production of specific antibodies is time consuming
  • even if antibodies are available they might not work very well for the protein of interest
  • their applicability is limited to a subset of proteins with sufficiently high molecular weight and certain biochemical properties
  • for many proteins no antibodies are available
Figure 1. Classical design of an aptabody.

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.

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).

Main advantages arguing for the application of aptamers for Western blotting are:

  • AptaBodies are very cheap (see cost)
  • They are easy and fast to get

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 wang_highly_2015 (Wang, Wang, & Huang, 2015). In the presence of potassium ions, hemin can bind to the G-quadruplex, which then catalyses the luminol-H2O2 chemiluminescence (CL) reaction. These two functional parts of the AptaBody are fused together via a poly-A linker (Fig. 1).