Engineering

Testing Methods for HER2-Targeting Aptamers

To test the binding affinity of our aptamer, we tried our many variations of ELONA methods. All of the protocols can be found here.

Iteration 1: MB-ELONA

First, we used Magnetic Beads-ELONA.

This assay employs Magnetic Beads, which is modified with -NH₂. This method uses MB to localize proteins for the ligand to bind on. HER2 ECD solution is added to activated-MB, and incubated for 1hr. Then it is placed on a magnetic rack, and the supernatant is removed. It is washed several times to remove unbound protein and biotinylated aptamers of different concentrations are added to incubate with protein-coated MB. The MB is then washed again to remove unbound aptamer and HRP-conjugated streptavidin is added for incubation, followed by washing and the addition of TMB solution. At last, stopping solution is added after incubation. MB is removed from the resulting yellow solution(if positive) and can be tested for absorbance at OD450 under the microplate reader.

We employed this method testing the binding affinity of HR2 aptamer. However, our results were not strong enough to prove its specificity (Figure 1).

Figure 1 A qualitative test for the binding affinity of HR2 aptamer using MB-ELONA.

As can be observed in the result, BSA-coated MB showed OD450 values of roughly 0.2 in both aptamer concentrations, proving that our aptamer did not bind to BSA protein; whereas HER2-coated MB showed an increase in OD450 value when aptamer concentration increased by 0.1 when aptamer concentration increased from 0.0 to 1.0 μm. This provided evidence that our aptamer does have some degree of affinity for HER2 protein. However, we were not satisfied with this result as we think the experiment group is not very different from the control group. We also performed MB-ELONA with no protein-coated, this gave us insight into possible reasons for the lack of difference between the control and experiment groups.

We ascribe the lack of difference between control and experiment groups to the high amount of non-specific binding on MB, in other words, the background value is too high. During the experiment, even a group of no protein and aptamer showed a yellow color, meaning HRP-conjugated streptavidin bound to MB none specifically.

Figure 2 Tested well-plate of MB-ELONA.

Iteration 2: Traditional ELONA

To solve the issue of high background values. We returned to the traditional methods of ELONA. The method remains very much the same, but instead of coating protein onto MB, it is coated onto the well-plate.

Figure 3 Result of tradional ELONA.

However, the result is not expected. All wells showed extremely low OD450 values, similar to that of blank wells. We speculated that perhaps none of the protein is coated onto the well-plate thus, no aptamer was bound to the plate. The binding ability of MB was too high, and too low for well-plate.

Iteration 3: ELISA-Kit modifed ELONA

To obtain the surface with the suitable binding ability we need for ELONA, we bought ELISA kits and modified them to suit our needs. We designed our experiment based on a sandwich ELISA kit which is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human HER2 in serum, plasma, and cell culture supernatants. Our assay employs a well-plate with an antibody specific for human HER2 receptors coated on a 48/96-well plate. Recombinant Human HER2 standard is pipetted into the wells and is bound by the immobilized antibody. The wells are washed and this time we used FAM-labeled aptamers to reduce the rounds of incubation needed to increase the efficiency of the experiment.

Before any experiment, we evaluated the sensitivity of the Kit by testing with its standard sample provided (Figure 4).

Figure 4 Standard test of the ELISA Kit.

We chose to coat 4 ng/mL of protein onto the provided well-plate as shown in result it showed a significant level of value, meaning it contain enough HER2 to bind with aptamer.

Our design were successful and we were able to obtain a qualitative result to prove the specificity of our aptamer, followed by a full quantitative evalutation of HR2 aptamer. (See more in Result)

Figure 5 Qualitative result of ELONA (left). Quantative evaluation of HR2 aptamer (right).

As we moved on to the next stage of our lab, we designed pH-sensitive ssDNA sections to add to the aptamer. But the design of the ssDNA is too long (145 bp) to be synthesized by companies through chemical synthesis. So we researched a method called asymmetric PCR to try and replicates ssDNA strands ourselves. (More can also be found in Results)

Figure 6 The agarose gel for asymmetric PCR. As shown in the picture, when the F : R primer ratio is above 1 : 1, a blurry new band appears above the original band, which is identified as ssDNA.

Reference

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5. Thompson, I., Zheng, L., Eisenstein, M., & Soh, H. T. (2020). Rational design of aptamer switches with programmable pH response. Nature communications, 11(1), 2946. https://doi.org/10.1038/s41467-020-16808-2