Team:GreatBay SCIE/Engineering

Engineering

During the implementation of our project, it is crucial to measure the binding affinity of aptamer against HER2 receptor. At the beginning of the experiment, we ran into some difficulties. However, utilizing the design, test, and learn cycle at experimental design, testing, analyzing and learning steps, We continue to enhance our design, optimize the experimental process and improve detection methods，in order to improve detection sensitivity and accuracy. We tried many variations of ELONA methods. All of our efforts can be found here.


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 aptamer 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 also performed MB-ELONA with no-protein conjugated. This proves the validity of MB-ELONA, as aptamer binds non-specifically to MB, which means that proteins were actually coated onto MB.

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 traditional 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 aptamer 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.

All of the parts, we used in our engineering, have been added to the iGEM parts registry. You can get more detailed messages from the following links:
- HR2 Aptamer; Part:BBa_K3897000
- DNA nanoswitch; Part:BBa_K3897001
- pH-sensitive HR2 Aptamer; Part:BBa_K3897005


Testing Method for Aptamer-conjugated Liposome

When we first tried to test the conjugation of our liposomes with FAM, NH2 pre-modified aptamer, using the method of electrophoresis and microplate reader to test the fluorescence of doxorubicin and FAM, we found that doxorubicin has a quite bright fluorescence under a range of wavelength and that covers the wavelength that FAM would be triggered to give out fluorescence.

Figure 7 Agarose gel-electrophoresis of ED-lip formulation Samples are loaded onto the agarose gel. UV light visualized the gel. A, B, and C are 3 repeats of aptamer conjugated liposome running on Agarose gel-electrophoresis.

We tried to find another fluorescence tag that can be conjugated on the aptamer and has a different excitation wavelength. Sadly, after a series of researches, we found that the wavelength range of the excitation of doxorubicin is too wide that almost covers all the excitation wavelength of current fluorescence tags. Moreover, through electrophoresis, we found that even the excitation wavelengths of doxorubicin and tags were different, the fluorescence of doxorubicin is too strong and will cover the faint fluorescence of tags. In other words, the background interference is too high. This bad news directed us to find another method for the testing of conjugation of liposomes and aptamer.

In order to eliminate the effect of high fluorescence of doxorubicin, we decided to just avoided using it during testing. This means that we only have to test the fluorescence of FAM and that can be much more clear to distinguish whether the aptamer has been conjugated on our liposomes. We quickly decided to prepare another set of liposomes using the same preparation method - Microfluidic - but without doxorubicin in it. So the doxorubicin-PBS solution we used before is replaced by pure PBS solution.

However, our ultimate goal is to prepare a complex of PEGylated liposome encapsulating doxorubicin conjugated with aptamer. We have to develop another method that can somehow put doxorubicin into our prepared liposome. Meanwhile, we were searching on some protocols of increasing encapsulation rate, and we came up with an idea of ammonium sulphate gradient method, which is usually used to increase the drug encapsulation rate by dragging free drugs into the liposomes and forming precipitate inside them. This method works on the different concentration of ammonium sulphate between solution inside the liposomes and the outside. To us, we only need to make sure that we add ammonium sulphate when preparing liposomes and remove the free ammonium sulphate outside our liposomes as much as we can. We believe this method can work thus we can diffuse doxorubicin after conjugation of liposome and aptamer, and conjugation test.

Then our protocol underwent considerable revision. We substituted the doxorubicin-PBS solution we used before when preparing liposomes into the same amount of 200mM ammonium sulphate dissolving in double distilled water. Then for the empty liposomes we produced, they went through the same conjugation and test process and fortunately we got the results that can answer our expectation.

For the process of doxorubicin diffusion, we purified the liposome solution thoroughly which can result in almost zero concentration of ammonium sulphate outside our liposomes. After that, we added doxorubicin-PBS solution to the outside of our liposomes, heated the solution using metal dry bath, and centrifuged it. We concluded the success of our protocol because of the red precipitate we saw after centrifugation, showing that the doxorubicin had been dragged into our liposomes.

Figure 8 The Doxorubicin sulphate shown as red precipitate after centrifugation

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