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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.
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).
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.
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.
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.
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
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.
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.
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).
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.
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.