Aptamers are oligonucleotides (single stranded DNA or RNA) that mimic antibodies in their ability to bind to target molecules. Aptamers are not derived from living organisms, they have nothing to do with life. We start with a library of random sequences and identify those that bind to a specific target using lab based evolutionary process (SELEX). Aptamers are synthesized.
Why should you be interested in aptamers? This depends on what you are doing.
- You work with the identification of antibodies and sale of antibodies:
Industry surveys show that these are your biggest concerns.
- Reproducibility of antibody results
- Validation of antibody specificity
The key issue with reproducibility is that antibody activity is measured by titre. This is because neither the actual binding affinity of the antibody or the amount of the antibody present is not know. The only thing that is known is that one time a certain volume of cell extract successfully bound to a target molecule. Variation definitely occurs from lot to lot of production. It is not known whether this variation is in the amount of antibody produced or the functionality of the antibody that is there. From a commercial point of view this is like selling fish before you cast your net in the water.
Aptamers are chemically synthesized, this means we know exactly how much is produced. This also means that we know their specific binding affinity. This means that every lot can be packaged with confidence that it will perform exactly the same as every other lot.
Antibody validation is becoming an increasingly significant problem. You know that your antibody binds to a specific target protein, but you do not know whether it does not bind to a wide variety of other possible targets. Testing your antibody after it is produced against all other possible targets represents a significant cost. This also presents a large risk, the antibodies that you have focused resources on building a low cost production system are later found to cross-react to something that represents a problem for your clients.
Cross-validation can be performed within the process of aptamer development. We include counter selection against a wide range of other targets while performing positive selection against the desired target. Moreover, at NeoVentures we subject our selected aptamer libraries to positive selection against negative targets including relevant matrices such as plasma or saliva and we characterize these negatively selected libraries by next generation sequencing. We weed out cross-reacting aptamers in the aptamer development process.
- You develop antibody based diagnostic kits:
Your biggest concerns are:
- Reproducibility of antibody test performance
- False negatives due to mutations in target protein
- Low sensitivity with small molecules
- Failure to bind to small molecules in their free form
- Inability to function with sparingly soluble analytes
- Contamination with other antibodies
The intelligent and appropriate use of aptamers can overcome or at least diminish all of these problems.
Reproducibility:
Aptamers are chemically synthesized, this means we know exactly how much is produced. This also means that we know their specific binding affinity. This means that every lot can be packaged with confidence that it will perform exactly the same as every other lot.
False negatives due to target mutations:
Aptamers can be selected and validated against a pool of all known isotopes of any given protein. This means that aptamers can be developed that will bind to all known forms of a given protein. We can also use guided selection techniques to target aptamer development for epitopes that are highly conserved within a target protein.
Low sensitivity with small molecules (< 1,000 da):
Failure to bind to small molecules in their free form:
NeoVentures has developed the proprietary FRELEX process for aptamer identification (US patent 10,415,034) to enable aptamer selection of free target molecules. This allows the aptamers to be selected for binding to all of the charge groups present on the target molecule, leading to higher binding affinities than would be possible otherwise.
Inability to function with sparingly soluble analytes:
At NeoVentures we pioneered the inclusion of organic solvents in the selection process to enable the identification of aptamers for targets such as ochratoxin A that are sparingly soluble in water. We found that aptamers could be selected to bind to such targets in the presence of up to 20% methanol, 10% ethanol and 5% acetonitrile. Aptamers can be developed that will work in solvents that would prevent antibody activity.
Contamination with other antibodies:
It can be very difficult and/or expensive to purify contaminating antibodies from a production run as this requires the use of the immobilized target. At NeoVentures we have experience identifying aptamers for specific antibodies and using these aptamers to support both quality assurance of products, and to enrich the desired antibody.
For more details on any of these applications, and why aptamers could be useful for you please contact us at,
- You distribute antibody based test kits:
Your biggest headache is ensuring that the test kits are shipped with ice or dry ice, and that this ice is still present when your client receives the product.
At NeoVentures we have test data to show that an aptamer immobilized on streptavidin retained 98% of its binding activity after a year of storage at room temperature. Aptamer based diagnostic kits do not require chilling for shipping or customer storage.
