Team:Heidelberg/Project/AB

Abstract

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.

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).

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. (aptamers_pat_1 aptamers_pat_2. To apply them in our Western blot assays, we fused those anti-His-tag aptamers to a HRP DNAzyme, to have a read out system based on chemiluminescence.

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.

Figure 2. Study of different Anti-His AptaBodies that vary in their poly-Adenosine (A) linker lengths.

Anti-His AptaBodies with 3A-, 5A-, 10A-linkers or no linker recognize His-Endolysin well, similar to our positive control (anti-His antibody) (right panel). Already 5 pmol of His-Endolysin, can be detected.

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).

Figure 3. Identification of the detection limit of our AptaBody approach.

AptaBody concertation was titrated from 0.05 µM to 0.2 µM. Freezing and thawing cycles do not affect the performance of the AptaBody.

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.

Figure 4. Determination of the best suited AptaBody concentration as well as the influence of blocking reagents on the AptaBody Western blot.

The effect of BSA on the signal to noise ratio was tested. As protein of interest His-Endolysin was applied. In addition, different Aptabody-concentrations ranging from 0.033 µM to 0.166 µM were applied. Best detection was achieved in presence of 0.166 µM AptaBody. A blocking step with BSA is not necessary.

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.

Figure 5.Detection of His-tagged proteins in cell lysates using Anti-His I AptaBody.

AptaBody Western Blot to detect his-tagged T7-polymerase (T7-His) in E. coli cell lysates. The blots were incubated with Anti-His I AptaBody for one hour. A defined signal from the HRP DNAzyme was observed. The whole lysate as well as the supernatant of the lysate were treated with DNase or RNase before blotting. An additional DNase or RNase treatment before blotting is not needed.

Figure 6. Detection of His-tagged protein in cell lysates using Anti-His II AptaBody.

AptaBody Western Blot to detect his-tagged T7-polymerase (T7-His) in E. coli cell lysates. The blots were incubated with Anti-His II AptaBody for one hour. A defined signal from the HRP DNAzyme was observed. The whole lysate as well as the supernatant of the lysate were treated with DNase or RNase before blotting. An additional DNase or RNase treatment before blotting is not needed

Figure 7. Detection of His-tagged protein in cell lysates using Anti-His I AptaBody.

AptaBody Western Blot to detect his-tagged T7-polymerase (T7-His) in E. coli cell lysates. The blots were incubated with Anti-His I AptaBody over night. In comparison to shorter incubation times (1 hour, Fig. 5), signal intensities increased. DNA or RNA do not interfere with the AptaBody and do not influence the readout.

Figure 8. Detection of His-tagged protein in cell lysates using Anti-His II AptaBody.

AptaBody Western Blot to detect his-tagged T7-polymerase (T7-His) in E. coli cell lysates. The blots were incubated with Anti-His II AptaBody over night. In comparison to shorter incubation times (1 hour, Fig. 6), signal intensities increased. DNA or RNA do not interfere with the AptaBody and do not influence the readout.

Figure 9. Detection of His-tagged protein in cell lysates using antibodies.

As a positive control we detected his-tagged T7-RNA polymerase (T7-His) in E. coli cell lysates. The blots were incubated with a commercial antibody, which detects the his-tag as well. The blot shows an increase of the background signal in comparison to our AptaBody approach (Fig. 5-8).

Figure 10. Optimization of buffer conditions for AptaBody Western Blot.

To improve our signal to noise ratio we were blocking our membranes with milk and salmon sperm DNA. In addition, blots were incubated in a mixture of AptaBody and milk in TBST (Fig. 10) to reduce the background noise.

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.

Figure 11: DotBlot using the MAWS-predicted Aptabody p53-His (A) and MAWS-optimized Anti-His I

AptaBody (B). 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.