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Protein-binding aptamers: the what and the how

When developing an aptamer to a protein target it’s important to keep in mind how you want to use it as much as what you want to bind.

When developing aptamers to protein targets the ‘how’ is just as important as the ‘what’.

The ‘what’ is what you need your aptamer to bind to. Whether you are working with multimeric proteins, tiny peptides or post-translational modifications, you want high target selectivity, no cross-reactivity, matrix functionality etc. Of course, we take all this into account in developing your discovery approach.

What we also consider is how the aptamer will be used and we have experience with many different approaches.

We can develop high affinity, high selectivity aptamers, but we also develop aptamers that can bind in specific ways to your protein target. Depending on your intended application, we will vary the aptamer discovery approach to account for:

  1. Protein detection
  2. Protein inhibition
  3. Protein activation
  4. Protein binding & release
  5. Protein binding & cell internalisation
  6. Blocking protein-protein interactions
  7. Aptamer-aptamer pairs
  8. Aptamer-antibody pairs

Protein detection

If you need to detect or quantify a protein in a sample you may be exploring its presence/absence, localization or the level of a specific protein present. You may want to target a specific payload to the microenvironment where the protein is expressed, such as in the case of precision chemotherapy.

In these situations, you are typically looking for a high affinity binder, with a rapid on-rate and slow off-rate, that is functional in the assay environment, whether in your assay buffer, diagnostic sample matrix such as blood, or in vivo. This will allow you to quickly detect the protein and ensure that your aptamer remains bound to the target for the period of the assay.

Target selectivity is also a must. You need to know that the aptamer you use binds only to the target(s) you are interested in and is not skewing experimental results through cross-reactivity.

There are applications where multiple aptamers to different parts of the target protein can be used together in the manner of an oligoclonal mix to increase signal and sensitivity for proteins of low expression or to increase protein characterization. Another solution to increase assay signal can be through a

Protein inhibition

Inhibiting a protein target, such as an enzyme, can offer insights to the protein’s function or can be used as a therapeutic strategy to overcome disease-associated activity within the body. To develop inhibitory aptamers targeting the active site of the enzyme during the discovery process is crucial and we have experience in modifying our discovery process to make sure aptamers are directed to this site.

High affinity aptamers, with slow off-rates, and high selectivity are essential to ensure selective inhibition of the target enzyme. For this function the small size and flexible nature of aptamers offers a benefit over protein binders as they can fit into crevices, such as enzyme active sites, more easily, to inhibit a broader range of protein targets.

Following targeted development, a panel of the resulting aptamers are screened for their inhibitory potential via in vitro biochemical and cell-based assays to ensure they meet the standard required.

Protein activation

Agonistic aptamers can allow probing of protein function in biological systems or may act as a direct therapeutic. Whether acting as stimulatory or inhibitory agonists, aptamers with both high and intermediate affinity for the target protein should be investigated.

Recent studies demonstrate that homo-dimeric aptamers with intermediate affinity around 50nM may show optimal agonistic function, through receptor clustering. In this situation aptamers with rapid on- and off-rate would be beneficial. This is because, for receptor clustering, each aptamer in the dimer needs to bind to two receptors at the same time for a short period of time and then repeat this process continually.

Alternatively, high affinity aptamers that bind highly selectively to known agonistic sites on a target, such as an allosteric protein, may offer good agonistic potential.

Depending on the target protein of interest and its mechanism of action, aptamers with the profile of interest can be developed and further screened through biochemical assays to determine activity.

Protein binding & release

In the development of affinity chromatography ligands or wearable biosensors, you need more than high selectivity from your aptamer.

For proper functionality as an affinity chromatography ligand, the selected aptamer must bind the target protein with a slow off-rate in the column loading and wash conditions, to ensure the target remains bound to the aptamer-conjugated resin. The aptamer also must then release the protein target with a rapid off-rate upon changing the conditions to allow purification.

We have experience in working with buffer changes as small as half a pH unit between binding and release. These small changes can enable increased functional yields of fragile proteins.

For wearable biosensors, aptamers can work as recognition ligands by binding the target protein from the blood. To ensure regeneration, the aptamer must be able to release the protein target following binding. In such cases, the aptamer must show high on-rate and medium/high off-rate to allow the sensor to regenerate quickly.

Protein binding & cell internalisation

Receptor-mediated endocytosis involves binding to a target receptor on a cell membrane and the induction of endocytosis into the cell. This strategy has been adopted for the targeted delivery of diverse payloads to specific cell types. Aptamers can bind the cell membrane receptor of interest and support internalization of conjugated payloads from small molecule drugs to gene therapies and nanoparticles.

In developing aptamers that support cell internalization, selectivity to the protein target is the critical factor over affinity. We can incorporate multiple counter-targets into the discovery process to ensure the selectivity of the aptamer. This would be followed by further development using cells expressing the protein receptor to ensure the aptamers induce internalization.

Blocking protein-protein interactions

Whether as research tools or as therapeutics, aptamers as protein-protein interaction inhibitors are a newly emerging strategy. While it was long thought that protein-protein interactions were ‘undruggable’, there is increasing evidence that with the correct approach this is a viable strategy for a range of interactions.

Aptamers are membrane permeable via several mechanisms, from pinocytosis to receptor-mediated endocytosis, allowing easy access to the interior of the cell, where unlike many protein-based molecules, aptamers remain functional within the reducing conditions of the cytosol. These features make aptamers a good candidate for disrupting protein-protein interactions.

When developing inhibitors of protein-protein interactions, researchers typically aim for KD <1 µM. We have developed aptamers with affinities ranging from pM to high nM according to the desired application, so affinity for protein-protein interaction inhibition is unlikely to be an issue in aptamer development.

High target selectivity is critical for the aptamer to ensure it inhbits the desired protein-protein interaction but does not exhibit off-target interactions. This will ensure the accuracy of experimental results and reduce off-target effects of potential therapies.

If guidance is available regarding the specific interaction site, through crystal structures etc,  then orthosteric aptamers would be the development route of choice to ensure the required selectivity and function. However, if this isn’t the case aptamer discovery can be performed with both interacting proteins in the process and the inhibition of the aptamer confirmed through post-discovery screening.

Aptamer-aptamer pairs or aptamer-antibody pairs

In ELISA, lateral flow tests or proximity ligation assays, binder pairs act as the workhorses of the assay. However, binders that work well individually may often not work well as a pair. A major challenge of finding good binder pairs is finding a good detection binder that reacts with the antigen with high affinity and reacts to an epitope that does not interfere with the antigen binding of the initial capture binder. Other considerations to keep in mind include buffer compatibility, stability and the orientation of the binder on the solid phase.

We have experience in developing both aptamer-aptamer pairs for a wholly aptamer-based system, and aptamer-antibody pairs for when you have one functional antibody but need an additional binder.

The capture aptamer requires high affinity with a slow off-rate, to ensure the target protein remains bound throughout the assay and any potential buffer changes. Target selectivity is also primarily denoted by the capture aptamer so this is a critical feature, meaning multiple counter-targets and assay matrix should be incorporated into the discovery process to drive selectivity.

For the detection aptamer, a high affinity binding with a rapid on-rate and slow off-rate is needed. While the detection aptamer should also show sufficient target selectivity, if the capture aptamer is selective enough, this job will already be taken care of in your assay, through the initial aptamer-target binding.





While all of the listed ways of working with protein-binding aptamers do require at their most basic form binding of the aptamer to the protein, ensuring the right functionality for the final aptamer requires knowledge and careful planning of the discovery strategy. We have experience with all of these different approaches and more and would be happy to talk to you about how we can support your needs for better protein binding reagents.

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