Aptamers are selected from large libraries of random sequences, thus aptamers themselves are not designed but the libraries that they are selected from are. The standard method of aptamer design has been for SELEX selection. This involves a synthetic library with a random region flanked by two primer recognition sites.
The Random Region
The random region is usually 40 nucleotides in length. There are however substantial constraints with this library design.
The random sequence needs to be at least 40 nucleotides in length in order to adequately explore structure space. If the random region is shorter than this, then there is too much potential for the random region to fold back onto the primer recognition regions. With a longer random region, there can be fold back onto the fixed sequences while still allowing sufficient variation in secondary structure with non-fold back regions.
The problem with this is that with 40 random nucleotides there are 1.2E24 possible sequences (4^40). This is equivalent to kilograms of DNA and is too much to use in a selection reaction. A general approach is to start with 1E15 sequences. This sounds like a lot, but it represents a very small subsampling of the possible sequences.
Limitations of SELEX
1E15 sounds like a lot of sequences but this is really so small a subsample of the 1.24 possible sequences that it is improbable that any two aliquots of 1E15 sequences will even have any of the same sequences in them. This means that SELEX is not reproducible. Any selection for a given target will result in different aptamer sequences being selected.
Each of the 1E15 sequences will be present as a single copy. This means that in selection it is probable that up 99.999% of the sequences are lost in the first selection round, including potentially the very best sequences.
Starting with one copy of each sequence means that reiterative rounds of selection are required to increase the copy number of the best sequences. This process of reiterative selection and PCR amplification is prone to bias by the amplifiability of the sequences in the library. Sequences with higher secondary structure may not be amplified as well as sequences with less secondary structure.
Antibody development has a system built in called immune tolerance to eliminate antibodies that bind to molecules that are produced by the host (self-avoidance). This means that antibodies will not bind to other antibodies or to abundant common targets like serum albumin. With SELEX it is possible to perform counter selection and remove aptamers that bind to counter targets. The difficulty with this approach is that counter selection works very effectively for aptamers that bind strongly to the counter target and does not work effectively for aptamers that bind weakly to the counter target. This is a problem with counter-targets such as serum albumin that are present at an average concentration of 600 uM in blood. If a target protein is present at a concentration of 600 pM, the difference in abundance is a billion-fold. If the binding affinity of an aptamer for the target protein is 1000X less for serum albumin, the presence of the serum albumin will still saturate it and interfere significantly with binding. There is a need to introduce an immune tolerance system into aptamer selection.
The Solution – NEOMERS
To overcome these constraints, NeoVentures has developed and validated a new approach to aptamer selection that we call Neomers. Neomers are not merely improved aptamers based on truncation of SELEX aptamers, like proprietary options from other companies. Neomers represent a breakthrough in aptamer development with reduced cross-reactivity to common targets in biological fluids.
The proprietary Neomer library design consists of 16 random nucleotides interspersed with fixed nucleotides.
This is a breakthrough in aptamer selection science as we can determine if any given sequence in our library binds even weakly to abundant targets in relevant matrices such as:
- Serum albumin
We are building more knowledge with every Neomer project. We are building a database of Neomer fingerprints and proteins and metabolites that cause them. Neomers will enable more successful diagnostic applications of aptamers.
Learn more about the advantages of Neomers by getting in touch with our team. Click here to contact NeoVentures.
Dr. Gregory Penner academic training was a blend of very practical plant breeding theory combined with molecular biology. He has used this blend of biology and mathematics to first develop and lead a cereal biotechnology research team with the government of Canada and subsequently as a global research leader with Monsanto Inc. He has been a thought leader in aptamer development globally for the last twenty years as CEO and President of NeoVentures. He has led this company to financial stability without outside investment with an integrated approach to aptamer discovery and commercialization. In 2015, he co- founded a second company, NeoNeuro in Paris France, focused on an innovative approach to identify Aptamarkers for complex diseases.