Mariners knew that there were dangers in uncharted waters and those that wandered there may not come back. It is safe to work with aptamers in controlled buffers and many papers have been published detailing how well they work close to the shore where maps exist. There are however also many stories about how they are lost in voyages that wander off the shore into the unknown of real matrices like plasma and saliva. In this blog I will provide our report back from our journeys off the known map and what needs to be done in order to travel there safely with aptamers.
The introduction of a real matrix, plasma or saliva or perspiration or intracellular fluid or urine or cerebrospinal fluid is not causing the noise to increase because this is not an issue with antibody based tests. The application of the aptamer in a matrix is failing due to a loss of signal because the aptamer is binding to non-target material in the matrix.
Antibodies are intrinsically evolved not to do this. They would be pretty useless in the immune system if they were distracted by other molecules in blood. Aptamers are synthetic and do not know better. There is a reason we refer to our initial synthesized aptamer libraries as naïve… The key culprit in plasma is human serum albumin (It would appear that Microsoft has an issue with the abbreviation HSA and has strict rules against in all of their software). It is not just that HSA is sticky, it is designed to be sticky with binding regions against a diverse array of target molecules. It acts as the micro sponges of the circulatory system collecting all of the floating debris.
At NeoVentures we have definitely encountered this problem and we have evolved a series of approaches to deal with it.
- Counter selection and positive selection.
It is important to include the presence of the matrix as a counter selection agent (preferably depleted of the target molecule) and as a presence in the positive selection. The inclusion in the presence of the positive selection is often even more important. This ensures that aptamers that are enriched by the selection process are enriched because they have the capacity to bind to the positive target in the relevant matrix.
- Next generation sequence analysis of simultaneous matrix specific selection channels.
Once we have obtained an enriched aptamer library we will make aliquots of the library and continue selection as we have against the positive target with one aliquot. We will use another aliquot to continue positive selection against the matrix without the target. All of these selected libraries are characterized by next generation sequencing. Often the highest copy number sequence is enriched in both the matrix alone and the target selection channels. This is not a candidate aptamer that we want to use.
- Aptamer optimization
The optimization of aptamers following selection is an essential part of all NeoVentures aptamer development projects. This involves truncation and nucleotide substitution to create a minimal functional stable structure. The minimal concept is important here, because it is often the portions of the aptamer that we remove that contribute to non-specific binding.
- Empirical testing
We will try if possible to incorporate binding assays or aptamer performance assays into our aptamer development projects that include assays in the presence of the relevant matrix. With surface plasmon resonance imaging this is difficult because the complexity of the matrix overwhelms the binding signal, but there we will always do a test for binding against HSA alone. For small molecules, the matrix is too complex for isothermal titration calorimetry analysis, but we can do binding assays in other ways with immobilized target to evaluate performance in the presence of the relevant matrix.
It is possibly to sail calmly through unknown and uncharted waters with aptamers. “…for there is nothing mysterious to a seaman unless it be the sea itself…”
― Joseph Conrad