Aptamer Library and Selection
Aptamer Library & Selection
- Aptamer selection is an iterative Darwinian process.
- Selection occurs entirely in vitro under controlled laboratory conditions.
- Customer defined conditions (related to end application) are used during ‘complex formation’ steps.
- ‘Challenges’ are introduced throughout selection to find the best binders.
- Remove non-specific aptamers or aptamers binding to undesirable targets.
- Specific release conditions can be included in selection e.g. to allow purification applications.
An aptamer library is 22,000 times bigger than a recombinant phage display library and a billion times bigger than a large combinational chemical library.
Aptamers are isolated from degenerate combinational libraries consisting of approximately 1015 sequences. This huge diversity is derived from a stretch of random nucleotides, typically 20 – 80 nucleotides in length, giving 420to 480possible sequence combinations.
An average combinational chemical library consists of approximately 100,000 classes of molecules, with the better libraries consisting of approximately 1 million molecules. In contrast, recombinant page display libraries (such as those used to make HuCAL libraries) have approximately 45 billion different structures. These libraries are 45,000 times bigger than a large combinational chemical library.
Our selection process uses the principle of selective evolution to develop a highly specific aptamer, with an affinity profile of your choice.
Aptamers with the desired properties are isolated from the ‘non-binding’ sequences through an iterative process of binding, partitioning and preferential amplification known as in vitro selection.
The aptamer library is incubated directly with the target of interest, allowing the formation of aptamer-target complexes to form. Binding complexes are then partitioned away from unbound/ weakly associated species and these species are washed away via elution steps. Target bound oligonucleotides are recovered and amplified by PCR for DNA aptamers, or reverse transcription and subsequent PCR (RT-PCR) for RNA aptamers. The resulting double-stranded DNA is then used to prepare an enriched pool of ssDNA or RNA. This new pool of selected oligonucleotides is used for a binding reaction with the target in the next in vitro selection round.
This process is akin to Darwinian evolution. Those aptamers within the diverse library with the highest affinity ‘survive’ the selection process and go on to the next round. These surviving species produce the next ‘generation’ aptamer population with improved qualities from the last. The ‘selection pressure’ can be increased throughout the process in a variety of ways depending on the desired outcome of the selection. For example, the molecules can be evolved to have exquisite selectivity and specificity by the incorporation of negative selection steps, which minimises the enrichment of non-target bound aptamers and favours the survival of aptamers with the desired properties.
Iterative cycles of this process drive the selection of the aptamer population towards relatively few, optimised sequence and structural motifs. These sequences can then be identified and provided to the customer as an alternative to antibodies.