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By using a reproducible aptamer library, we can predict the structures of all sequences within the naïve library. This shifts the analysis of aptamer enrichment during selection from the sequence level to the structural level. We have mapped the structural space needed to interpret selection results, allowing us to leverage next-generation sequencing data to identify the structural elements being selected. This insight guides truncation with scientific precision, and we are actively advancing these capabilities to further mimic antibody production through an in silico approach. As a result, NeoVentures is driving the creation of even better aptamers.

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“Less is more” is definitely true for aptamer truncations.

In the picture above, the aptamer has long dangling ends on both the 5’ and 3’ sides. Why is this a problem? Single stranded DNA or RNA has a biphasic nature. The phosphate backbone has a positive charge and is strongly hydrophilic. The nucleotides on the other hand have mixed charges and are hydrophobic. This means that unhybridized single stranded oligonucleotide regions in the presence of water are driven to adhere to surfaces. This satisfies the hydrophobicity and hydrophilicity of both sides. Unfortunately, this also drives aspecific binding to other targets. The removal of such regions results in an increase in the specificity of binding.

Truncation of these regions also tends to lead to an increase in affinity as well. Why would that be?

The prediction of the structure shown above was performed with RNAFold. The colour coding corresponds to the probability of each nucleotide being in the position predicted, with red meaning highly probable and green to blue meaning improbable. This improbability is due to the potential for these regions to hybridize to other regions of the aptamer other than the ones in the predicted structure. In this case there are low probability prediction on the position of certain nucleotides in the dangling ends. This means that a certain amount of the time these nucleotides are hybridized to others resulting in a different overall structure. It is a safe assumption that only one of the aptamer structures binds to your favourite target. By removing the dangling ends the number of possible structures is reduced, thus the structure that binds to your target is present a higher proportion of the time. This is why aptamer truncation leads to higher affinity and specificity.

In subsequent blogs we will discuss how we use our Neomer library process to scientifically guide truncation. In keeping with Mies Van der Rohe, I will end this blog with one of his other sayings: “No design is possible until the materials with which you design are completely understood”.

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The global market for ELISA based assays was estimated at $2.52B in 2023 but what has happed with the sister assays, the aptamer based ELISA or ELONA? Why are they no FDA or CE mark approved tests for Enzyme-linked Oligonucleotide Assay?

We spend a lot of time talking, working and developing cutting edge sophisticated diagnostic assays based on microfluidics, electrochemistry and fluorescent switches and lateral flow assays. It may make sense to pick some of the low hanging fruit, like the ELONA, prior to reaching out on limbs for the ones that are more difficult to capture.

In a lateral flow assay, the target is forced to interact with the immobilized aptamer, but then it is subject to constant washing as more solution passes over. The ELONA assay is attractive compared to lateral flow because it is possible to allow much more time for target/aptamer binding, and because it is possible to amplify the colour signal. In an ELONA, there is no loading of a capture zone, but it is possible to allow 30 minutes to an hour for complexes to form.

The key to any diagnostic approach is to maximize the signal to noise ratio. We have achieved this with ELONA applications by functionalizing gold nanoparticles with both aptamers that bind to the target and oligos that are labeled with a biotin for the capture of streptavidin/horseradish peroxidase (Strep/HRP) conjugates.

First, we conjugate a capture aptamer to streptavidin with a biotin on the aptamer and a long enough spacer such that the aptamer is able to rise above the surface of the protein. This is passively immobilized to the surface of a microtiter well. The target in a sample is added, along with the dual functionalized gold nanoparticles described earlier.

Unbound functionalized GNPs and target are removed with a wash step. Streptavidin/HRP is added, unbound Strep/HRP is removed with an additional wash step. Then, colour is developed with the addition of TMB.

An example of results obtained at NVB with this approach is provided below.

With this approach we were able to detect the presence of 1 fmole of target (confidential protein) from the absence of the protein with a t-test significance of 0.014 across three replications.

There are two keys to our success with this application:

• The enablement of a higher than 1:1 relationship between Strep/HRP and binding events.
• The use of NEOMERS, aptamers developed by NeoVentures with our non-SELEX selection process.

For success it is necessary to use aptamers with high binding affinity and specificity as the ELONA process requires several wash steps.

For more information on our Neomer aptamer development process or our experience with aptamer applications in diagnostics contactez-nous

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At NeoVentures, we do not consider ourselves as a (CRO). We approach what we do more as co-entrepreneurs with you. Clients come to us with problems that might be solved with aptamers. We discuss the project in depth under confidentiality and together we develop an aptamer selection and application strategy. Sometimes we tell clients that what they have envisioned is not possible yet with aptamers. There are many reasons for this, sometimes the limit of detection required is simply not possible given the nature of the target.

We have learned that some selection processes and targets do not work well. For example:

• Selection of aptamers for specific cells, especially prokaryotic cells, based on cell selection alone.
  • We think that this is because there is an abundance of poor targets on the surface.
  • We prefer to design selections with a double positive round for a known protein followed by selection on living cells.
• Lipids, fatty acids, molecules less than 300 Daltons are not good aptamer targets.
  • We have tried selections against these targets and are not satisfied with the results.
• Peptides
  • We have generally thought that selection of aptamers for peptides should work well, but we have been consistently disappointed with the results. We think the issue is the selection on the peptide. We are now modelling the structural flux of peptides with software like PEP-FOLD4, but proteins will always be better targets.

In terms of aptamer applications, we have confidence in systems like qPCR and fluorescent switches for diagnostics. In our experience, electrochemistry platforms with existing commercial chips are not ready for commercial applications. We are working together with solution providers to make this work better and improve the chip to chip variability.

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Now, if you were an antibody, what would your wishes be?

• To be chemically synthesized instead of produced in cells.
• To be capable of being PCR amplified.
• To be small enough to undergo allosteric shifts when binding to a target.

Three pretty simple wishes, that I bet have occurred to almost everyone that works with antibodies. Of course, you know where I am going with this, for these reasons antibodies really wish they were aptamers.

Chemical synthesis means reliable production from lot to lot. This also means that no animals are involved or sacrificed during the process. Also, the ability to be synthesized removes the need for production schedules. This allows just in time inventory.

PCR amplifiability allows for highly sensitive detection. Once a target binds to a detection aptamer, If the amount of detection aptamer could be quantified in a qPCR assay, this would be the method of choice for all diagnostic assays for everything. This is the basis for next generation proteomics, translating information from protein abundance to DNA code.  (see NVB Europe for more on this).

Smaller size enables change or displacement of structures upon binding to targets. This is the basis of fluorescent switch and electrochemistry applications. Fluorescent switch platforms are the assay of choice for high throughput analysis in bioproduction. There is no need to immobilize targets or ligands, results are rapid and sensitive. Electrochemistry is the future for point of care applications (POC). This will be enabled with the cost-effective production of consistent gold surfaces, bringing this promising technology to commercial reality.

If you asked an aptamer what it wished for, what would it answer?

It will only want to be a Neomer, so it could enjoy all the perks of being an aptamer plus the specificity of an antibody.

Neomers:a new aptamer selection method developed by NeoVentures

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