Furthermore, downstream identification and characterization of aptamer‐candidates is greatly facilitated by 2nd generation sequencing and high‐throughput characterization 10. These modifications include optimized motif distribution, degenerated libraries of a known binding motif or secondary structure and the use of artificial nucleotides with an extended nucleotide alphabet 11, 12. Therefore, an increasing effort has been put on modifying starting libraries. Another key factor for SELEX success is the starting library, as the structure of an aptamer and its possible interactions with the respective target directly depends on the aptamer's sequence. Today, highly efficient microfluidic, bead‐based, or electrophoretic partitioning techniques are most utilized in aptamer selection, while traditional SELEX relied on nitrocellulose filter binding 9. A core step in SELEX is the separation of bound and unbound aptamer‐candidates after incubation with the target. Here, we only give a short overview of major improvements, while in‐depth reviews on these topics can be found elsewhere 1, 9, 10. Since the beginning of aptamer development many technologies have been applied to overcome inherent barriers and limitations of SELEX. Despite employing only standard methods the success rate of conventional SELEX is only about 30% 9. Once identified, affinity and specificity can be assessed for each aptamer candidate individually. After sufficient enrichment the aptamer‐candidates are commonly identified by cloning and sequencing. Typically, it takes 8–15 rounds of selection to enrich high affinity aptamers from a randomized library 8. Following a strand separation, the enriched library of aptamer candidates is subjected to the next round of selection and amplification. Subsequently, these recovered aptamer‐candidates are amplified using PCR. After incubation of library and target, unbound oligonucleotides are washed away, while bound oligonucleotides are later recovered by elution. All oligonucleotides are composed of a central randomized region that is flanked by defined priming regions. SELEX typically is initiated by incubation of a target molecule with an oligonucleotide library consisting of 10 14–10 15 different oligonucleotides. Consequently, aptamers do display high specificities for their target, due to an excellent structural match with the target.Īptamers are generated via an iterative selection process called systematic evolution of ligands by exponential enrichment (SELEX). The aptamer‐target binding is based on electrostatic interactions, van der Waals interactions, and hydrogen bonding that are nonspecific by nature, but the synergistic combination of these interactions may result in a high affinity binding. Aptamers are usually described as high affinity ligands with a distinct specificity for their corresponding target, which makes them excellent ligands for affinity purification 2, 3, 4 and offers great potential in sensing applications 5, 6, 7. In their folded state aptamers are able to bind to a variety of targets such as ions, small molecules, and peptides as well as to proteins or cells 1. buffer composition) these aptamers fold into specific 3D structures. Depending on their sequence and applied conditions (e.g. One type of artificial ligands is represented by single‐stranded oligonucleotides, termed aptamers.
However, in the past 20 years the generation of artificial ligands has made substantial progress. Historically, this field is dominated by the use of antibodies that are produced by either immunization of animals and subsequent harvesting of antibodies or by large‐scale cell culture. Modern biology and medicine rely heavily on the use of specific ligands for molecular recognition of biomarkers or other molecules.
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