We have performed an extensive literature review of aptamers for Mucin 1 and CA 15.3 (our two biomarkers of
interest) and have added the sequences to the registry, along with pertinent information from the UNAFold
DNA modeling software various tests we conducted in our lab. For more information, please see our
Engineering page.
Building on a paper by Nutiu and Li discussing a simple method for the design of fluorescent aptamer probes,
we consulted Dr. Kazunori Ikebukuro of Tokyo University of Agriculture and Technology to develop a set of
guidelines for aptamer probe design.
For the design of double-stranded probe DNA, one usually targets the loop part of the hairpin structure of
the aptamers. Then, one designs the full match to the loop part, the deletion mutant lacking one base at the
both 5' and 3' end, and the elongated mutant having additional one base complementary to the aptamers
sequence at the both 5' and 3' end.
Then, one can calculate the melting temperature of those hybrids (aptamer-cDNA complex) and compare those
with the aptamers' folding melting temperature using UNAFold.
One can usually choose the hybrids showing melting temperatures 3 to 5 degrees celsius higher than that of
the aptamer itself. However, that depends on the Kd of between the aptamer and the target protein. If the Kd
is smaller that 10 -8 M (in the nM range), the hybrid with the melting temperature 10 degrees celsius higher
than that of the aptamer folding temperature itself can be chosen. The important part is the equilibrium
between the hybrids and the aptamer-target complex. If the melting temperatures still do not work, it is
preferable to try 2 bases or more deletion of both 5' and 3' end and those elongations. It also depends on
the length of the loop parts.
Furthermore, the binding of biomarker with aptamer of Kd of nM levels can usually break the hybridization
with Tm of 50 degree celsius. The binding with Kd of micro M can usually break the hybridization with Tm of
less than 40 degree celsius.
We followed established protocols for ELONA, an ELISA assay modified to accommodate aptamers, in order to
determine the dissociation constant. We used a protocol modified from Sang et al to determine the Kd value
using the Hill equation, a Scatchard plot, and non-linear regression. However, the results were inconclusive
so we decided to find our Kd value through the 3D modeling software, UNAFold.
The results demonstrated that the aptamer probes produced satisfactory results and aligned with our
mathematical predictions, thus validating our design guidelines. Please refer to our results page for
further details.
Lastly, we have devised a simple workflow for the detection and quantification of our biomarkers of interest
using the Hill equation. For more information, please see our Modeling page.
We reviewed common protocols for the resuspension of aptamers and optimized those protocols for the
resuspension and preparation of aptamers for Mucin 1 and CA 15.3 under physiological conditions.
Furthermore, we also created a standard curve for the fluorescence of our probes, which may be used by
future teams as reference when using our technology to determine the concentration of biomarkers in an
unknown solution.
For more information, please refer to our lab notebook and Modeling page.
We were able to conduct a series of experiments on our aptamers of interest to understand their behavior and
properties. We conducted tests on the shelf life and specificity of our aptamer probe solutions and reported
the results on our parts pages. There was a statistical difference between the fluorescence values of the
varying concentrations of biomarker solutions, confirming that the aptamer probes still successfully bind to
biomarkers and fluoresce after 2 weeks in the refrigerator. Since the results indicated that the properly
handled aptamers kept in dark in tinfoil showcased reasonable shelf life, it further highlights the
practicality of our design in a clinical setting. Furthermore, we have also demonstrated the superior
durability of aptamers over antibodies, thus also making a strong case for the feasibility of this
technology.In short, we believe that the information gleaned from our experiments is extremely useful for
future teams hoping to use our aptamers in a project.
For more information, please see our Parts page and Lab Notebook.
We developed a short program that can take in inputs of the fluorescent value to quantify our biomarker
concentration. We tested the program with our experimental results, and it functions properly. This would be
useful for future teams working with FRET and a mechanism involving the interaction between two binding
molecules, as one could merely change the initial values of Kd, L, and P0. This can be used in a variety of
applications, such as the detection of another biomarker through its complementary aptamer and finding the
real concentration of the biomarker-aptamer complex in solution.
As for implementing our proposed detection mechanism in the real world, we propose using a 96 well plate. The wells will be prefilled with buffer solutions with the specific aptamer probes we have identified and discussed above suspended in them. After blood samples are collected from patients, they can be added to this plate so its fluorescence values can be measured with a 96 well plate reader. We deemed using this method appropriate as our goal is to administer these tests in a clinic; clinics and hospitals should have access to 96 well plate readers. Furthermore, using this method will allow us to do these screenings on a mass scale, rather than individually, increasing the efficiency of screenings.
