FRELEX: Free-Free aptamer selection method
FRELEX is Neoventures’ patented method for aptamer selection. In order to overcome the need to immobilize targets molecules, as required in traditional SELEX, we developed this new method to allow partitioning of bound aptamers from unbound without immobilizing aptamers or target.
The FRELEX aptamer selection method begins in phase I incubating a library of aptamers with a library of 8mers that have been immobilized on a gold chip. If the aptamers are sufficiently open they will form Watson/Crick bonds with the 8mers on the chip. We retain those sequences that bind to the 8mers. We discard all of those sequences that do not bind to the chip. This means all of the sequences retained have the capacity to bind to the 8mers. We elute these off the chip.
In phase II, we incubate the sequences retained from phase I with the target. This complex is then exposed to a fresh 8mer chip. In this phase we retain those sequences that are not bound to the chip with the logic that the reason the sequences do not bind is because they are now bound to a target molecule.
Contrasting channels in a selection strategy
We work with our clients to develop a selection strategy that fits product concept, the diagnostic device or therapeutic need.
The following is a general schematic of a selection project with contrasting channels.
After several rounds of selection the enriched library splits into aliquots and continues positive selection against separate and contrasting targets. We perform next generation sequencing (NGS) on all selection rounds from 7 to 10.
We use mathematical models to assist us in the selection process. One key method is the use of cluster analysis to identify aptamers that covary in their enrichment rates across selection rounds. In general aptamers that are in different clusters have a higher probability of binding to different epitopes on the target protein than aptamers within a cluster.
Each column and row represents a different aptamer across selection rounds. Dark blue represents a correlation of +1, on the diagonal all the spots are dark blue as this represents aptamers correlated to themselves. Dark red represents a correlation of -1. There are two primary classes of aptamers. Upon testing of candidate sequences from each class we found differences in the way that the performed.