Using multiple discovery methods in aptamer development provides better binders. Learn why multiple discovery methods are important, which is right for you and how they can be combined for best effect.

Aptamers can be generated for a wide range of target types. Starting at small molecules, we can scale through peptides and oligonucleotides to proteins and protein complexes right through to viruses, bacteria, and eukaryotic cells. But the best way to ensure functional ligand whether aptamer or antibody is to make sure the discovery process is aligned with your specific target type.

Why do we need multiple discovery methods?

If you are looking for an affinity ligand to your target of interest, whatever it may be, then considering how it is developed can be key.

Most times that you look for affinity ligands they have been generated using one single method that is used for all target types. Whether this method to make the affinity ligand is the immune system of a host animal for antibodies, a phage display platform for protein scaffolds or a standard SELEX process for aptamers, sometimes, you might need more.

Discovery processes that are tuned for each target type can offer improved results in discovery and greater performance from the resulting affinity ligand. Combining multiple discovery methods can also improve outcomes for enhanced performance. Nobody wants to waste time trying to optimise things that are never going to work, and we would all rather less optimisation and more results, so it is worth thinking about.

Our Optimer platform contains three separate discovery processes for small molecules, proteins and cells, which can be used in parallel for rapid results or combined for increased validation and performance.

Multiple discovery methods tuned for your target

While most affinity ligand developers use just one discovery method, typically oriented to protein target discovery, using multiple discovery methods improves the performance of the final aptamer. We’ll have a quick look at each discovery method below to see the benefits of each and why you may need more than a simple one-size-fits-all approach.

Protein targets

Proteins are the most commonly studied targets in life sciences. Looking at antibodies, as they represent the major ligand used across the life sciences, they are typically generated to a protein target by injecting the target into an animal with the hope of raising an immune response. The antibodies generated through this immune response are then purified and applied in research. Antibodies have a success rate of approximately 50% with this strategy. Using aptamers, we can open up more of the target space, allowing ligands to be developed to new target types, including:

• toxic targets like venoms that might kill the host animal before the immune response
• little or non-immunogenic targets that don’t provoke an immune response
• highly conserved proteins where the degree of resulting cross-reactivity in the antibodies might mean you can’t directly study your protein of interest
• proteins that perform vital functions in the body where antibody generation might kill the host animal or antibodies can’t be generated in the first place

Enabling researches to explore these protein targets with affinity ligands for detection, quantification, localisation and function allows scientists to explore new biomarkers and applications.

Small molecule targets

Small molecule targets are trickier to develop affinity ligands for.

Due to their small size, they are not taken up, processed, and presented on MHC class II complexes by antigen-presenting cells, and so they can’t be presented to T cells to induce a sufficient immune response for antibody generation. To overcome this, small molecules are often conjugated to a carrier protein for antibody generation. Small molecules plus carrier proteins mean bigger molecules, allowing their recognition and processing by the immune system. But, constraining small molecule targets in this way limits the available chemically reactive groups for interaction (as they are already conjugated to the carrier protein) and often results in limited target affinity.

Our aptamer discovery process for small molecule targets is performed in vitro with the target in solution, meaning we overcome these issues and deliver better-performing ligands. The aptamer wraps around the small molecule target for increased engagement, selectivity and affinity, so your research is easier and provides the results you need.

Cell targets

Obviously, when performing discovery to cell targets, the ligand does not bind to the whole cell (that would be some mighty ligand!). Rather it binds to specific proteins, lipids or carbohydrates on the cell surface.

Antibodies to prokaryotes can be created through standard immunisation of host animals and downstream analysis to determine the specific antigens that they bind to. However, human cells may not raise a response, and are often of more interest to researchers. Aptamer development is performed in vitro, so there are no immunogenicity issues, allowing all cell types to be targeted.

Cells can serve as a useful target source for discovery. For example, if purified protein targets are hard to source, cells over-expressing the specific target might be used instead. Or cell discovery processes can be used in combination with protein discovery, to assess aptamer binding to the target as a recombinant protein and also in its cell-expressed format, making sure that the ligand can recognise cell receptors in their folded conformation and that they don’t bind to portions of the target that might be inaccessible within cell membranes.

Finally, cell targeted discovery can be used to discriminate between different cell populations eg healthy vs disease to identify aptamers that target a specific cell population, with the potential to identify the target biomarker in later stages using techniques like mass spectrometry.. This is useful if a biomarker is not known or you want to explore new biomarkers for a specific cell type. The cell discovery process can identify biomarkers on cell populations and deliver a functional affinity ligand to allow downstream research all within the same process.

How we combine multiple discovery methods for the best ligands

At Aptamer Group, we offer three separate discovery processes, each uniquely tuned to the target type. These multiple discovery methods can be run in parallel to get answers quickly or combined to support the best aptamer development programme.

1.     Small molecules – With small molecules in solution, allowing availability of all reactive epitopes and interaction with the target as it would be in your assay for maximum performance.

2.     Proteins – From peptides to protein complexes and even viral capsids, we can work with your target in our automated, high-throughput discovery process.

3.     Cells – Viruses, bacteria and eukaryotic cells can all be used as part of our cell selection process, allowing native display of targets for increased assurance of epitope interaction or the ability to discriminate between different cell populations with subsequent identification of the selected biomarker.

We use these discovery processes in parallel and sequentially to offer the best aptamer selections and ensure, for example, that protein-targeted aptamers recognise the required target when expressed on cells for applications such as flow cytometry or ICC.

For example if you want aptamers to a peptide we can use small molecule and protein discovery methods and examine the performance of the resulting aptamers to offer the greatest range of binders quickly. Equally, for aptamers to a cell receptor, we may start with protein discovery methods to ensure selectivity and then progress to cell discovery methods that will ensure the aptamers bind the receptor as it is expressed on the cell surface. We can even combine this with samples like fixed tissues to make sure you get the best performing reagents for applications like immunohistochemistry and spatial biology.

If you need affinity ligands with the best performance matched to your target and application, get in touch with our technical experts today to find out how we can help.

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