Aptamer Library and Selection
Aptamer isolation starts from a degenerate library of ~1×10^15 random oligonucleotides (with fixed primer sequences). This diversity is unparalleled amongst affinity ligand libraries; the closest competitor being phage display libraries (~4.5×10^9 sequences, 222,000x fewer). While proteins and peptides do have potentially more functionality (from 20 different amino acids, compared to 4 nucleic acid bases), additional functionality can be included in aptamers through the incorporation of modified nucleotides.
Nucleic acids are considerably more flexible (more rotational centres per nucleotide, compared to amino acids), meaning that aptamers also have a greater propensity and ability to form complex folds.
Structural elements can be ‘designed in’ to the starting aptamer library if needed; for example if a target is known to bind a stem-loop structure or if the aptamer needs additional functionality (e.g. Spinach Aptamer Beacons or Riboswitches), but most aptamer libraries do not contain these features. These are included as part of our bespoke service offering (where required).
Comparison of typical diversity from Aptamer libraries and other combinatorial libraries. Aptamer libraries are ~222,000 fold more diverse than the closest technology.
Aptamer isolation through in vitro selection
Aptamers are isolated through iterative cycles of in vitro selection. Aptamer-target complexes are formed under conditions appropriate to the customer application, making downstream integration simpler. Target binding aptamers are recovered and amplified to form the starting material for the subsequent generation. Additional steps can be included to accommodate target specificity, complex matrices and other criteria required for the end application.
The starting library is reduced to a ‘handful’ of aptamers, through an iterative process of target specific binding, partitioning (binders from non-binders or unwanted binders), recovery and preferential amplification. This process is best envisaged as an evolutionary process, through which the best aptamers are captured by the target and ‘survive’ the washing processes. These survivors are then amplified and go on to form the next generation. Increasing the ‘selection pressure’ in each round, ensures that only the best aptamers survive.
At Aptamer Group, we understand this evolutionary process and adapt it to suit our customer needs, including application specific parameters into the various steps.
We also recognise that different target types, require different selection processes. We have therefore developed three core processes for generation of aptamers against small molecules, peptides or proteins and cell or tissue based targets .
Most customer projects are based on one of these three core methods, but our experienced team are also on-hand to develop novel process variants as and when required.