Proteins

Isolation of antibodies against protein targets has become widely accepted and the norm in many laboratories across the life sciences. Unfortunately, the antibody-associated lack of specificity, background binding and batch-to-batch variability, has also become the accepted norm.

Nucleic acid aptamers have the potential to solve many of these issues by adapting the isolation.

At Aptamer Group, we addressed this challenge and developed our selection process to include customer requirements and enable the selection of very specific aptamers. Conditions can be tailored to fit customer needs, including specific requirements for buffers, matrices and conditions and these challenges are introduced gradually through the selection process to find the best binders. Aptamer Group’s proprietary high throughput selection process also allows multiple targets to be processed in parallel without compromising on quality. Complex targets, such as those with PTMs, can be selected for and the aptamers produced can detect PTMs and distinguish their occurrence in a protein.

Biophysical Characterisation of the Selection Process

The increase in affinity of the refined aptamer population can be tracked over the course of selection to monitor the progress of aptamer ‘evolution’. Once the final population has reached the required characteristics (specificity etc) we identify individual aptamers.

The increase in affinity of the refined aptamer population can be tracked over the course of selection to monitor the progress of aptamer ‘evolution’. Once the final population has reached the required characteristics (specificity etc) we identify individual aptamers
Individual aptamers are isolated from the population and ranked based on their affinity (or other customer defined criteria). The best performing clones are then sequenced and taken forward for additional optimization or application

Selection of Highly Specific Aptamers

The development of high-quality affinity reagents with strict discrimination between the desired target and closely related proteins represents a major challenge for antibodies, but this can be built in to aptamer selection processes. Especially problematic are targets with small changes such as single amino acid substitutions or post-translational modifications (PTMs). As such, caution is often urged  when using antibodies to detect modified proteins in biological samples.

Careful introduction of these closely related counter-selection targets leads to isolation of a population of aptamers with exquisite discrimination.

Similar processes can be used to isolate a population with broad target recognition. For example, aptamers can be isolated using a family of related proteins. The resulting aptamers should then recognise ‘epitopes’ which are common to all of the targets.

The selected aptamer shows specific binding to the target protein (red) with clear distinction of the counter target (blue) which only differs by two PTMs (citrullination = PTM by deimination of arginine). No binding is seen towards the negative control protein (grey).