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DESIGN

Biomarkers for Breast Cancer

A variety of biomarkers are associated with breast cancer, such as CA 15.3, MUCIN 1, and CA 27.29. These biomarkers are present in blood and saliva. Although no protein has yet been recommended as a satisfactory biomarker for the early detection of breast cancer, the three proteins mentioned above are being proposed as potential candidates. Therefore, we have sought to construct a proof-of-concept using these three biomarkers, in the hopes that future advances in the early detection of breast cancer may use the same technology and techniques as we do here. Furthermore, the proteins shown here have established reference ranges in literature, which makes them promising targets for future breast cancer diagnosis.

Aptamers

Aptamers are single-stranded oligonucleotides that can bind tightly and specifically to almost any molecule. Also known as synthetic antibodies, these short strands of DNA hold great promise as alternatives for traditional antibodies, and have the potential to be used in a variety of clinical settings.

At the beginning of this year, we settled on using a homogeneous immunoassay to fulfill our goal of a simple kit that can be easily administered in a clinical setting, offering a suitable alternative to costly and uncomfortable mammograms. Initially, we were intent on adapting lateral flow assays in pursuit of this goal; however, technical challenges and the relative sensitivity required to measure small fluctuations in biomarker concentration indicated that this was not the best course of action. Therefore, we instead turned to highly sensitive homogeneous immunoassays, such as the AlphaLISA test from Perkin-Elmer for inspiration.

We settled on using a homogeneous immunoassay due to the fact that such an assay does not require the time-intensive washing steps required by a traditional heterogeneous immunoassay, such as ELISA or DELFIA. Considering that this test kit is meant to be as convenient as possible, removing the long periods of time needed for incubation and washing greatly streamlines the procedure and makes it realistic for this to be administered at home or in a clinical setting. Furthermore, we decided to use aptamers instead of traditional antibodies due to their superior stability and ease of synthesis, thus allowing this proof-of-concept to be more feasibly implemented in the future. In addition, aptamers exhibit complicated structural changes upon binding with the substrate (in this case, the biomarker in question), which we were further able to exploit to create a light-switching fluorescent probe that fluoresce when bound to the biomarker of interest. One can then determine the original concentration of the biomarker by reference a standard curve, similar to how one would quantify the data of an ELISA or Bradford assay. Furthermore, this form of liquid biopsy has been shown through experiments to offer satisfactory measurements and data.

For more information, please see our Human Practices page.

Selection of Aptamers from Literature

We reviewed the papers of Ferreira et al and Agnihotri et al to find aptamers and their binding affinities for Mucin 1 and CA 15.3 respectively. Both papers indicated that said aptamers were highly selective for the biomarkers of interest, and the sequences were relatively short and easy to synthesize de novo. However, there currently seems to be no published research regarding synthetic antibodies for CA 27.29. Therefore, we selected Mucin 1 and CA 15.3, as well as their corresponding aptamers, for this proof of concept.

Design of Aptamer Probe

For the design of the probe, we consulted with Dr. Kazunori Ikebukuro of Tokyo University of Agriculture and Technology and made extensive use of the aptamer modeling software UNAFold to design our probes.

Our probes were based on the FRET principle and fluorescence quenching using donor-acceptor pairs and heavily inspired by similar research by Yang et al and Wu et al, who designed FRET-based aptasensors for the detection of PDGF and Helicobacter pylori respectively. In both instances, the fluorophore-labeled aptamer resulted in detection that was highly specific and did not require a long incubation period, unlike ELISA or other immunoassays of similar specificity and sensitivity. And in both groups, fluorescence upon binding with the substrate was achieved by a conformational change of the aptamer, which changed the distance between the fluorophore and the quencher attached to the ends of the probe and resulted in a weakening of the FRET effect and increased fluorescence. The final fluorescence was then quantified using a well-plate reader under excitation and compared to a standard curve derived from standard solutions of the biomarker/cell of interest.

For our project, we based the design of the probe upon this concept, and designed two different probes for the detection of Mucin 1 and CA 15.3. Using UNAFold, we were able to predict the conformational change of the aptamers upon binding with our biomarkers, and were able to select one aptamer for each protein that changed in such a way as to significantly weaken the FRET effect between the donor-acceptor pair attached to the ends upon binding, but not when suspended unbound.

We envision this project to be used in a clinical setting, where multiple samples may be tested at once (and thus lowering the cost).

For a more detailed discussion, please refer to the Modeling page.

Principles of FRET (Forster Resonance Energy Transfer)

For our project this year, we decided to employ the use of a light-switching aptamer probe, which will change shape and fluoresce under electromagnetic excitation when bound to the biomarker of interest. To that end, we have decided to make use of the Förster Resonance Energy Transfer principle, which allows for non-radiative energy transfers between two molecules, commonly known collectively as a donor-acceptor pair. Normally, FRET is often used in elucidating the structure of cellular components due to the effect’s extreme sensitivity to distance; beyond an angstrom distance of 100 Å, the interactions between the donor and acceptor greatly decrease. Therefore, monitoring FRET interactions is a useful way to gauge the distance between said donor and acceptor.

Donor-Acceptor Pairs

FRET is often used in the context of elucidating protein structure. This is done through the use of what are known as donor-acceptor pairs, which are molecules which exhibit FRET when within close proximity to each other. A subset of this general phenomenon involves a fluorophore and a quencher (instead of two fluorophores), whereby FRET is shown by the decrease in fluorescence by the fluorophore when in close proximity to the quencher, instead of the emission of light from the fluorophore that is not being excited. In our case, we are using a quencher and a fluorophore, which means that FRET interactions will decrease fluorescence of the fluorophore, while decreased FRET interactions will increase the fluorescence of the fluorophore. Below, we will outline the specific design for each probe.

ssDNA Aptamer Probe using BHQ and Cy5

For our ssDNA aptasensor for the detection of Mucin 1, we settled on the donor-acceptor pair BHQ1-Cy5. Normally, Cy5 (being a dye), is excited and fluoresces under near-infrared light. However, this donor-acceptor pair dictates that within the distance in which FRET is effective, the BHQ1 quencher will “eat up” the fluorescence of the Cy5 dye, due to their overlapping absorbance spectrums. Therefore, when attached to the ends of our aptamer, the FRET interaction will take place and the fluorescence of Cy5 will be quenched, due to the fact that the aptamer forms a hairpin shape and the distance between Cy5 and BHQ1 is less than 100 Å as a result of Watson-Crick base pairing. However, when the aptamer is bound to the biomarker, the aptamer is predicted to change shape and separate the ends. This increases the distance between the Cy5 dye and BHQ1 beyond 100 Å, which results in a drastic weakening of the FRET effect and the fluorescence of Cy5 under near-infrared light as a result. Thus, the change in fluorescence/conformation of the aptamer indicates whether it is bound to Mucin 1 or not. The higher the concentration of biomarker, the more aptamer will fluorescence, thus leading to an increase in measured fluorescence.

dsDNA Aptamer Probe using FAM and Guanine Nucleotides

For our dsDNA aptasensor for the detection of CA 15.3, we settled on the donor-acceptor pair FAM-guanine. We chose this because guanine is easier to synthesize and attach to our aptamer than other synthetic quenchers, and FAM is also widely used. Furthermore, the FAM-guanine pair is excited by UV light, which is different from our single-stranded probe. Therefore, this design allows us to test a different wavelength and see if it is more effective.

Our dsDNA aptasensor relies on a strand displacement assay, similar to the ones used by XMU China in their 2018 project. After designing a partial complementary strand to our aptamer (please see the Modeling page for more details), we attached a FAM group to the 5’ end of our complementary strand, which would be naturally quenched by the guanine present on the 3’ end of the aptamer. As the complementary strand pairs with the aptamer in a Watson-Crick base pairing scheme, the distance between the FAM group and the guanine is less than 100 Å, which is enough for the FRET effect to be significant. However, when the partial complementary strand is displaced by the biomarker, the distance between FAM and guanine drastically increases, which decreases FRET efficiency and results in fluorescence of the FAM group under UV light.

As a further test, we also attempted to design a double stranded probe with Cyanine 3 dye and BHQ2 quencher in place of FAM and guanine respectively.

Limitation and Testing

Since we did not have access to human samples, we used the PBS buffer as a replacement for serum and saliva in our final proof of concept, as it closely models physiological conditions. However, this does not take into account the background fluorescence caused by extraneous proteins and lipids normally present in serum and saliva.

We were able to resuspend both aptamers successfully and derive standard curves using standardized solutions of our biomarkers. The results are shown below. For more information, please refer to our Modeling page.

Proposed Method to Detect Multiple Biomarkers using Different Aptamer Probes in a Single Solution through Proof-of-Concept

As the ssDNA and dsDNA DNA probes detect different biomarkers and our excited by different wavelengths of light, we envision a design that involves having both biomarkers in the same buffer solution, which can now detect the concentrations of both biomarkers when a sample is added, simply by exciting the resulting mixture with UV light or near-infrared light for the detection of CA 15.3 and Mucin 1, respectively.

References

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  2. AlphaLISA Immunoassays. https://www.perkinelmer.com/category/immunoassays-alpha. Accessed 7 Oct. 2022.
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