In a previous blog I stressed the need to further improve aptamer performance after SELEX identification in order to develop aptamers that are effective enough to be commercialized. I promised that I would describe how we do this at NeoVentures in more detail in a future blog.
First, it is important to realize that a full length aptamer is in flux among possible shapes at room temperature. In the picture below there are four shapes formed by the same aptamer sequence.
All of these shapes are not only formed by the same aptamer sequence, they are constantly in flux with each other at room temperature. The following image is an energy landscape. Each node represents a shape, and the length of each vertical line represents the energy barrier between this shape and the other shapes.
We use this energy landscape and the structures of the aptamers to make hypotheses about specific sub-element structures. We truncate the aptamers down to these sub-elements, and stabilize the desired form with nucleotide substitutions. In this way we can design specific sequences that will only be present in the form that binds to the target molecule.
The affinity of binding between aptamers and their targets is governed by on rate of the formation of the complex, and the off rate of the complex coming apart. The on rate is largely driven by the probability that the aptamer will collide with the target molecule. In an aptamer based lateral flow application the target is forced to interact with the immobilized aptamer. However, if the aptamers is in a conformation that is not conducive to binding when the collision occurs then binding will not occur. If we can fix the aptamer into the binding shape, then we will increase the on rate of complex formation, and increase binding affinity. In addition, this process also decreases the off rate, as the aptamer is less likely to be flipping into states that do not bind the target. Again, this will increase the binding affinity.
Intelligent, software driven, optimization of aptamers is a crucial NeoVentures step in the development of aptamers that can be effectively commercialized.