At Aptamer Group, our isolation and characterisation processes are tailored to meet our customers’ needs. We ‘Start with the end in mind’; meaning that we consult with every customer to identify the specific requirements for the aptamers to perform in the end application. We then adapt our aptamer isolation and characterisation processes to include these application parameters; ensuring that our aptamers are fit for purpose.
Our high-throughput liquid handling platforms allow us to undertake aptamer selections against multiple targets in parallel. These platforms also allow us to apply Design of Experiment principals to each of our aptamer selection projects. This approach allows us to ‘explore’ several selection stringencies in parallel and study the effect of each. Using the most optimal parameter from each round increases the likelihood of successfully isolating a population of aptamers with the required functional properties.
Every project is split into three distinct Phases, with defined deliverables that are tailored to fit each customers’ individual application requirements. This approach ensures that our aptamers are fit for purpose and meet the customers needs; not only for an affinity ligand but for a tool that actually does its job.
Phase 1 – Feasibility Study
In all aptamer selection projects involving protein targets, an initial piece of work is carried out answer three key questions;
1.Is an aptamer stable enough in the customer application matrix?
Aptamers (like any nucleic acid) are susceptible to degradation by nucleases. We therefore assess the stability of our aptamer library in a matrix appropriate to the customers end application e.g. plasma. If the aptamer library is not sufficiently stable, then backbone modifications can be incorporated into the aptamer to increase resistance to nuclease activity.
A Stability Study is used to monitor aptamer degradation in customers sample matrices. The unmodified ssDNA library (left) shows degradation over the time course shown. The modified aptamer library (right) does not show this degradation (within the study duration).
2. What buffer components promote the strongest interaction between our aptamer library and the customer target?
We carry out a binding assay using a panel of buffers with different salts, additives etc. The buffer which promotes the strongest interaction between the aptamer library and customer target, is taken forward. Customer requirements are also taken into consideration e.g. metal ions or cofactors required for protein stability; absolute buffer requirements for the end application etc.
A Buffer Screen is used to identify a binding buffer which promotes a stronger interaction between our aptamer library and the immobilised target (green bars), compared to background binding to the blank support (red bars).
3. Does the target show concentration dependant interaction with the aptamer library?
If it is possible to promote an interaction between our aptamer library and the customer target, then that interaction should be concentration dependent. We carry out a binding assay to demonstrate that this is the case.
A binding assay is used to assess concentration dependant binding between our aptamer library and the immobilised target.
Other studies are carried out for small molecule targets, including;
- A solubility study is carried out to check that the target is soluble (and remains in solution) in an appropriate selection buffer.
- Selection compatibility study is carried out to determine if the small molecule target has any inhibitory effects on the processes needed for aptamer selection.
The results of this Feasibility Study are used to guide the early staged of Phase 2 – Polyclonal Aptamer selection. Our customers also find these results are reassuring as we have demonstrated that it is possible to promote an interaction between an aptamer library and their target. This data may be used to support a Business case for internal funding, grant applications etc.
We also use this information to triage projects and terminate projects which are unlikely to reach a successful outcome. This minimises customer risk as it prevents commitment to a project that is unlikely to succeed.
Phase 2 – ‘Polyclonal’ Aptamer Selection
When we have completed our Phase 1 Feasibility Study and demonstrated that aptamer selection is likely to be possible; we use the identified conditions in our semi-automated high throughput aptamer selection approach.
A standard assay plate has 96 wells, which allows us to carry out aptamer selections against multiple targets in parallel. This also allows us to apply the Design of Experiment principals to study the effect of different selection pressures in each selection round. This approach enables us to identify and apply the optimum selection pressure without risk of loosing aptamer material. This strategy greatly increases the likelihood of successfully isolating aptamers and reduces the number of rounds (and hence time and resources) required to reach a population of aptamers with the required binding parameters.
When our aptamer selection data shows signs of enrichment (along with any other customer specified criteria); we carry out a preliminary binding assay to demonstrate that our enriched aptamer population has the required target specificity (or cross reactivity) and is functional in the customer defined matrix e.g. plasma. Other binding parameters (specified by the customer), may also be assessed at this point e.g. tailored binding and release conditions required in our AptaBind™ Aptamer Mediated Affinity Chromatography (AMAC) process. This data is used to show that our aptamer population meets the customers functional requirements. Once these criteria are met, we proceed to Phase 3 – ‘Monoclonal’ Aptamer Isolation and Minimal Fragment Identification (MAFI).
Our automated, Design of Experiments (DoE) approach to aptamer selection allows us to study the effect of multiple selection parameters in parallel. We choose the optimum conditions to take forward to subsequent selection rounds. This allows us to ‘find the fruit’ faster.
A functional population binding assay is carried out at the end of Phase 2 (Polyclonal Aptamer Selection) in every project. We use this assay to demonstrate that the isolated aptamer population meets the customer specificity requirements.
Here the BioLayer Interferometry (BLI) assay demonstrates that the aptamer population shows clear and preferential binding to the target peptide (red trace), compared to a counter-selection target (blue trace), and that the binding response was not significant in the unselected aptamer population (green trace).
Phase 3 – ‘Monoclonal’ Aptamer Isolation and Minimal Aptamer Fragmnent Identification (MAFI)
Once we have obtained data to demonstrate that the isolated aptamer population meets the customer requirements; we proceed to identify individual aptamers through a functional assay approach.
At Aptamer Group we believe that the best approach to demonstrate our aptamers performance, is to test them for binding to the customer target. We therefore screen our individual aptamers by function, rather than relying on circumstantial evidence obtained by approaches such as Next Generation Sequencing. By taking this approach, we can be sure that the isolated clones are indeed aptamers, rather than any other form of sequencing artefact. Only aptamers which are shown to possess the required binding characteristics are taken forward.
When we have isolated a functional individual aptamer(s), we then perform a screen to identify the minimal aptamer fragment (which we call an Optimer™) which retains the required target binding parameters. These Optimers™ are delivered to our customers for use in their assays. At Aptamer Group, we also work with our customers to help integrate aptamers into their respective workflows. We can carry out additional process development such as integration of the aptamers into purification columns for Aptamer Mediated Affinity Chromatography (AMAC), various assay developments such as ELISA-like assays etc.
Individual aptamers are screened and ranked based on their performance in a functional binding assay, using customer defined binding parameters. In this case, binding to the target is assessed; specificity, cross-reactivity or buffer induced release can all be monitored as success criteria. The best performing aptamers (green traces) are taken forwards.
Aptamer fragments are screened using the same assay format as their ‘parent aptamer’, and ranked based on their performance in the same functional binding assay. Each fragment is compared with the parent aptamer (orange trace). Fragments which retain their function (Optimers™, green traces) are further characterised. Non-functional sequences (red traces) are discarded.
Phase 4 – Supply and Beyond
Examples of the additional process developments include integration and assessment of the Optimer™ in a purification column for Aptamer Mediated Affinity Chromatography (AMAC); development of a quantitative assay, such as ELISA-like assays; assessment of the Aptamers / Optimers™ in cell-based assays such as flow cytometry or FACS, fluorescence microscopy or tissue imaging.