The SELEX cycle diagram compares the stages involved in a classical SELEX cycle to those which utilise monoclonal beads or compartmentalised droplets during selection. Rounds are repeated until sequence enrichment occurs at which point aptamer candidates can be sequenced and characterised. Binding sequences are eluted and amplified by the polymerase chain reaction (PCR) which will seed the next round of SELEX. Sequences which bind to the target are then partitioned and any non-binding or low affinity sequences are discarded. A random nucleic acid library is incubated with a target. Generally, SELEX comprises the iterative steps of aptamer library binding, elution of bound species and pool amplification until aptamer candidates with desired criteria are selected. Figure 1 (Orange) shows the basic method for obtaining an aptamer by SELEX. Aptamers are typically selected from a random library through a process called systematic evolution of ligands by exponential enrichment (SELEX). These advantages include greater stability, specificity, ease of chemical modification, lower production cost and less batch to batch variability. Aptamers offer multiple advantages when compared to the analogous protein based affinity reagents, antibodies. Aptamers can be selected against a wide variety of targets including metal ions, small molecules, proteins, and even whole cells. Since aptamers were first described by two independent research teams in 1990, they have been rapidly adapted for various applications in research and biotechnology. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.Īptamers are synthetic oligonucleotides that fold into unique 3-D structures that specifically bind to their targets with high affinity. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS) whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR) and Fluorescence Activated Cell Sorting (FACS) to high-throughput selection techniques. The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols.
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