The objective of the Protein Modeling team was to verify aptamer-biomarker interactions to reduce costs in purchasing unnecessary antibodies. As proof of concept, we simulated the docking between aptamer and biomarker via online protein docking server HDOCK and visualized using PyMol. With guidance from the Tokyo University of Agriculture, we were able to show the possibility of our aptamer-biomarker complex.
As proof of concept of our lab project and to show the possibilities of interactions between aptamer and
biomarker, we simulated the docking between aptamer and biomarker via online protein docking server HDOCK
and visualized using Pymol. With guidance from the Tokyo University of Agriculture, we were able to show the
possibility of our aptamer-biomarker complex.
In continuation of last year’s project, ASIJ iGEM 2022 constructed PDB files of aptamers that were tested
for biomarker interaction. The models of following aptamers were created using HDOCK and PyMol: MUCIN 1.3
(2E7V), MUCIN 2.2, STR-T2, STR-T3, STR-T4, and more.
Through open-source websites (UNAFold Web Server and XiaoLabs), PDB files of the folded aptamers were
derived. PyMol was subsequently used to produce visualizations of the aforementioned PDF files. To obtain
the distances of aptamer-biomarker interactions, the necessary biomarker was visualized as well. Using the
ruler function of PyMol, the lengths (nm) between interaction locations of the biomarkers and aptamers were
obtained. (Below is the image of a measured PyMol interaction between biomarker and aptamer)
In biochemistry, an assay is a laboratory method to determine the amount of a target entity such as a compound/molecule. It can provide valuable information to detect, quantify, and study the activity and binding of a biological molecule. The ELISA Assay is used to measure biological molecules in biological samples. In our case, the biological molecule is the biomarker. A biomarker is a substance within an organism that can be traced to the function of an organ or different aspects of health. In our project, we have selected biomarkers that indicate the presence of breast cancer.
In order to calculate the concentration of biomarkers bound to the aptamer, the determination of a constant
Kd value is necessary.
The dissociation constant (Kd) describes the ability of a compound to dissociate or break down to its
constituent components. In our case, it describes the ability of the biomarker to break away from the
aptamer. This value will be utilized in the Hill’s Equation (explained later) to determine the bound
concentration of biomarker to aptamer. There are a number of ways to calculate the constant involving
linearization. Linearization is especially applicable to the ELISA Assay because it does not require direct
ligand labeling or the absolute concentration of the aptamer-biomarker complex. Therefore, a recently
developed linearization procedure was utilized along with the ELISA Assay to calculate the Kd value.
In order to determine the amount of biomarker that is present in the patient’s body, we will have to
calculate the amount of biomarker bound to aptamer. To do this, we will need to calculate the dissociation
constant, Kd which will be used in the Hill’s Equation. The ELISA Assay is a useful essay that can be used
to calculate this constant. Another objective of ELISA is to create a standard curve which is the name for a
fluorescence versus concentration graph. By using ELISA, we can determine the fluorescence of a numerous
biomarker sample using a 96 Well Plate Reader. As a result, we can obtain fluorescence values at varying
concentrations of biomarkers.
However, the 96 Plate Well Reader for ELISA did not produce any conclusive readings. The Kd value necessary
for the Hill Equation was subsequently aderived from compatible literature.
FRET (Förster resonance energy transfer) is the energy transfer between two molecules. Specifically, it refers to the transfer of energy non-radiatively between two light-sensitive molecules from the donor to the acceptor using intermolecular long-range dipole-dipole coupling. FRET is a well-understood phenomenon and is important due to its ability to provide information about protein interactions. However, there are two important requirements for FRET to work. Firstly, the donor and acceptor molecules must be less than 10 nanometers apart. This means that the two fluorophores must be extremely close together for FRET to work. Secondly, the absorption/excitation spectrum of the acceptor must overlap with the fluorescence emission spectrum of the donor. Protein modeling can especially be useful in verifying the first requirement because we can visualize the interaction between the aptamer and biomarker to determine if the distance between them are close enough (<10 nanometers apart) so that FRET can occur properly. FRET was utilized in our project to verify that the biomarker correctly binded with the aptamer, through the presence of fluorescence.
