We have developed an alternative Western Blot protocol based on AptaBodies replacing antibodies in the detection of proteins. AptaBodies consist of a DNA aptamer having a high affinity and specificity against its target molecule that is bound to a hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP DNAzyme). Already known aptamers or aptamers generated by our newly developed software MAWS were applied to build AptaBodies. As a proof of principle we applied AptaBodies to detect His-tagged proteins. Using the AptaBody-based approach, the same protocol as for conventional antibody-based Western blotting can be applied. Our innovative technology shows a variety of advantages. AptaBodies are a cheap, fast and a simple alternative to antibodies. Furthermore, time-consuming animal experiments to generate antibodies can be soon a thing of the past. Therefore, AptaBodies provide a promising alternative compared to antibody-based Western blotting.
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
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
Methods and Results
As a proof of principle we first applied already pre-established two anti-His-tag aptamers that are known to interact with His-tag of a protein of interest (POI) and differ in their DNA sequence. In the following we call them Anti-His I AptaBody and Anti-His II AptaBody. (
Analysis of binding properties of AptaBodies that vary in their poly-A linker length
To ensure a specific binding of the AptaBody to the protein of interest (POI) the linker length between the Aptamer and the HRP DNAzyme is highly important. We tested two anti-His Aptamers, which differ in the poly-A linker length (3 to 10 adenosine). To study the influence of the linker length with respect to the detection limit and signal to noise ratio, we applied a purified His-tagged Endolysin from Enterobacteria phage lambda as target protein. Instead of primary and secondary antibody, we applied our established AptaBody-Western blot protocols to detect Endolysin. We can show in Figure 2 that the poly-A linker length influences the flexibility between the aptamer and the HRP-mimicking DNAzyme.
All two tested anti-His AptaBodies specifically bind to the His-tagged Endolysin with high affinity. Already 5 pmol of the protein could be successfully detected by our AptaBodies (Fig. 2). However, the Anti-His I AptaBody with a 10A-linker showed a relatively high background signal. Thus, for further experiments, we decided to use the 5A-linker version, which provided the best signal-to-noise ratio (Fig. 2).
In addtion, we analyzed the influence of freezing and thawing cycles on the properties of the AptaBody (Fig. 3). If the AptaBody was frozen and thawed in presence of hemin no change in the signal of our Anti-His I AptaBody was observed (Fig. 3).
Identification of the detection limit of AptaBodies
To achieve an optimal signal-to-noise-ratio, we tested different concentrations of the AptaBody in the Western blot protocol. In Dot Blots as well as in Western Blots the AptaBodies show the same detection limit as the anti-His Antibody. We tested 0.03 µM, 0.08 µM, and 0.16 µM anti-His I AptaBody (Fig. 4). 0.03 µM AptaBody shows a weak signal in Western Blots and almost the same background noise comparable to 0.08 µM AptaBody. With 0.16 µM AptaBody the signal to noise ratio was best. Therefore, a concentration of 0.08 µM AptaBody was best suited under these Western blot conditions.
Influence of Blocking Reagents on the signal to noise ratio
To analyze the influence of the classical Western blot blocking step, we tested the effect of blocking with bovine serum albumin (BSA). As sown in figure 4 no unspecific bands could be observed even without blocking. The signal to noise ratio was comparable with and without blocking with BSA. These data points to another potential advantage of the AptaBody based-protocol for Western blots: the faster method.
After we have shown the functionality, the high affinity as well as the good detection limit of our AptaBodies, we tested their specificity in protein lysates. For this purpose we performed a Western blot using a purified his-tagged protein and an E. coli cell lysate with overexpressed His tagged T7 RNA polymerase. The blots were incubated with either Anti-His I AptaBody or Anti-His II AptaBody. Already after one hour incubation with the AptaBody a defined signal from the HRP DNAzyme was observed (Fig. 5 and Fig. 6). After overnight incubation, we observed and enhanced signal of our protein of interest (T7-RNA polymerase). However, the background signal was increasing as well (Fig. 7 and Fig. 8). In comparison to commercial antibody, our AptaBodies show reduced unspecific binding to other proteins within the cell lysate (Fig. 9). Moreover we tried to improve our signal to noise ratio. Thus, we were blocking our membranes with milk and salmon sperm DNA or incubated the blot in a mixture of AptaBody and milk in TBST (Fig. 10) to reduce the background noise. We can show that a combination of AptaBodies with Southern blot buffers such as SSC and Denhart’s solution gives a high specific signal (Fig. 10).
To show if DNA or RNA, which are present in cell lysates, influences the specific binding of our AptaBodies to our protein of interest, we treated the samples either with DNAse, RNase or both. We showed that samples for AptaBody Western Blots do not need and additional DNase or RNase treatment before blotting. The AptaBodies do not interfere with DNA or RNA from the cell lysate.
To show the general feasibility of our AptaBody approach, we generated new aptamers by MAWS that should detected a variety of different interesting biological targets. In this context we analyzed the specific binding of the newly designed AptaBody p53-His. In Figure 11 we blotted p53-His, xylanase, G-actin, lysozyme-His and stained the blots with the MAWS-predicted Aptabody p53-His (A) and with MAWS-optimized Anti-His I AptaBody (B), respectively. Ponceau staining was applied as loading control.
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:
- 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)
- 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.
- MAWS can generate specific AptaBodies against each and every protein of interest.
- In contrast to antibodies the production of AptaBodies do not require animal experiments.