Our project depends heavily upon the conformational changes of aptamers upon binding to the biomarker to trigger a weakening of the FRET effect and cause fluorescence. Therefore, it is crucial for us to accurately predict the shape of aptamers both when bound to and not bound to our biomarker. In order to fulfill this goal, we made extensive use of UNAFold and HDOCK to predict the shape of our aptamers in closed conformation, and were able to choose two that exhibited the desired qualities in the buffers used in their original papers.
In continuation of ASIJ iGEM’s 2021 Project, 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. 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.
For the single-stranded aptasensor for the detection of Mucin 1, we modeled the aptamer in UNAFold. The results showed that the aptamer formed a hairpin shape
when not bound to the biomarker, which indicated that FRET would be taking place in a closed (unbound)
conformation, and thus satisfying our first requirement. However, upon consultation with Dr. Ikebukuro, we
were told that existing software is not able to successfully model aptamers in an open (bound) conformation
accurately, but that the structure would change drastically. Thus, we conducted the experiments to see if
the two aptamers in question are feasible for the purpose of quantification and potential uses in diagnosis.
However, the modeling still offered us valuable insight into choosing an aptamer with the right conformation
when unbound. Shown below is the shape of our single-stranded DNA probe Mucin aptamer S2.2, with a melting
temperature of 36.5 degrees Celsius when suspended in the buffer used in the original paper.
Furthermore, for both aptamers, we used UNAFold to calculate the melting temperature of the aptamers, or the
temperature at which Watson-Crick base-pairing interactions are broken. According to Dr. Ikebukuro,
biomarkers with a strong affinity for their respective aptamers can break the Watson-Crick base-pairing
interactions if the melting temperature is 50 degrees Celsius or less, assuming the Kd value of the
aptamer-biomarker interaction is in the nM range. Therefore, using UNAFold, we have shown the two aptamers
we chose for the development of DNA probes both had melting temperatures in the range required. Shown below
is the complementary base pairing of the partial complementary strand with a moiety of the double-stranded
aptamer probe Clone 2, with a melting temperature of 36.9 degrees Celsius when suspended in the buffer used
in the original paper with an aptamer concentration of 100 nM.
In particular, the double-stranded aptamer seen on the Proof-of-concepts page required us to design a
partial complementary strand to the aptamer, which would be displaced by the biomarker in a strand
displacement assay similar to the one detailed by Nutiu and Li in their paper Structure Switching Signaling
Aptamers. Using UNAFold, we designed a strand complementary to a looped portion of the aptamer (when folded
up in its single-stranded conformation) and calculated its melting temperature, which was in line with the
requirements specified above.
Thus, modeling has played a very big role in the successful development of our project. Through our tests,
we have successfully validated the predictions of our aptamer-biomarker interaction.
