We will be developing competitive lateral flow aptasensors for a small molecule target. However, we are concerned about the strength of the DNA aptamer- target interaction versus the interaction of DNA aptamer and its complementary sequence in the test zone. Are there ways to initially assess these factors?
You can evaluate these two types of interaction with theoretical modes, such as Molecular Dynamics simulations. First, build a model that contains the complex of DNA aptamer and its DNA binding site. Then, do MD simulations and calculate the interaction energy (or binding free energy for better accuracy) between the aptamer and its DNA binding site. Repeat this procedure for aptamer and the complementary sequence you can compare these two interactions by comparing their interaction energies or binding free energies.
Here is an example for your reference.
Article Tethered-Hopping Model for Protein-DNA Binding and Unbinding...
Classical thermodynamics experiment: Differential scanning calorimetry (DSC) run on each of the different complexes would be a direct, unbiased and model-independent measurement. Measuring 'melting' allows you a direct way to calculate enthalpy.
You might also be able to set up a measurement system in an optical instrument (UV absorbance) or a titration calorimeter. Start with a fixed concentration of one complex, and titrate in the third molecule as a competitor. This approach will give you a direct measurement under your desired operating conditions for your sensor, but you likely will have to spend time developing and optimizing that assay before getting the answer you seek.
Also, with competitive assays you probably need to understand dissociation kinetics of the initial complex. You may be disappointed to find that once either complex is formed that it will not easily switch to the other complex when the third molecule is introduced, especially if you expect your sensor to operate at room temperature. Achieving a system where all of the binding equilibria are nicely balanced is not easy.
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