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SELEX has been the basic method for identifying aptamers for the last thirty years. It is time for an improvement.<\/p>\n
The key constraint implicit in the SELEX method is that it is not reproducible.<\/p>\n
Every time a new starting library is selected from a synthesized library we start with new sequences. This means that even if we are selecting aptamers for binding to the same target we end up with different sequences.<\/p>\n
The problem with this approach is that it requires that counter selection for aptamer specificity must be performed within the same selection. This creates a particular difficulty when we consider counter selection against serum where the concentration of serum albumin averages 600 uM. Counter selection works very well for aptamers that exhibit strong binding, but much less well for aptamers that bind weakly to the counter target. It would be preferred if we could perform selection against counter targets like serum albumin with a reproducible aptamer library.<\/p>\n
We have achieved this goal with our development of the Neomer selection library and method. The Neomer library is based on the combination of two modules in one single stranded DNA molecule. Each module contains eight random nucleotides, thus the total number of sequences in a Neomer library is 4.29 billion. This is a number of sequences that represents a subset of the number of sequences we start selection with. Thus, we can start selection with multiple copies of every sequence. The remaining problem was how to characterize the frequency of each of these 4.29 billion sequences with next generation sequencing where we can capture on average only 5 million sequences per library?<\/p>\n
We achieve this by incorporating a restriction site between the Neomer library modules. We cleave the library, and process each of the module single strands separately in next generation sequencing.<\/p>\n
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Each module has eight random nucleotides resulting in 65,536 possible sequences per module. With NGS coverage of 5 million reads this means that on average we are observing 76 copies of each sequence. The product of the frequency of a sequence in module A by the frequency of any sequence in module B gives us the frequency of that sequence in the original intact library. Thus by processing the two modules separately we can characterize the frequency of all 4.29 billion sequences. In fact, we could add additional modules and characterize the frequency of even more sequences.<\/p>\n
The Neomer selection method is a closed sequence system.<\/p>\n
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This closure means that we can characterize the sequence frequency of all sequences in all selections. We can determine from the same starting library which sequences exhibit affinity for a desired target and do not exhibit affinity for serum albumin. We can also do all this in a single round of selection.<\/p>\n
Welcome to the next generation of aptamers! We are prepared to provide this method as a service for the selection of aptamers for your targets. We are also prepared to license this method to others. Please contact us for more information.<\/p>","protected":false},"excerpt":{"rendered":"