than peptidic origin, is the technology described in the next and final section of this chapter. 2.4 Aptamer Selection 2.4.1 The SELEX methodology Another combinatorial methodology employed in drug design is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and is focused on the selection of high affinity and specificity oligonucleotide ligands, termed aptamers, from vast combinatorial libraries. This process was first described in the same year (1990) by two independent groups in the USA (Tuerk and Gold, 1990; Ellington and Szostak, 1990). The SELEX methodology is based on an evolutionary process characterized by rounds of interaction of an oligonucleotide library with a particular target, partition of bound from unbound species, elution of bound species from target and amplification of those using PCR. Amplified aptamers are allowed to interact again with the target for a number of iterative ‘steps, to result in isolation of higher binding species through competitive binding. This was inspired by the evolution of RNA molecules through the ages into binding specifically to particular target proteins to facilitate biological functions. To reproduce this in the laboratory and within a short time interval, a number of competitive steps may be required to separate the highest binding molecules. Similarly, by counter-selecting and excluding molecules that bind to homologous proteins or isoforms as part of the selection methodology, the process can result in very selective and highly specific molecular entities that offer a high degree of differentiation and can thus target specifically mutant proteins over their native counterparts, thus conferring disease-specific targeting properties to these molecules. In order to start the SELEX process, a single-stranded DNA oligonucleotide pool is chemically synthesized with a random region of nucleotides, varying from 10 to 100 bases, and a fixed primer sequence at either end to allow for amplification by PCR. From this theoretical maximum, which for a 25-base long degenerate region corresponds to 47° or about 10*° sequences, the pool has at least one copy of each possible sequence and structure to be selected for optimal affinity towards the target. Following the synthesis of the oligonucleotide pool, a selection method based on an evolutionary approach of repeated steps of interaction, elution and amplification is applied. This methodology includes the exposure of the target molecule to the library in order to allow for interaction of all binding aptamers with this target. The target is exposed to the aptamer library in a number of ways, usually attached to a solid support such as agarose or sepharose in affinity chromatography matrices, magnetic beads or polymers, but often also in solution, separated by filtration and molecular weight differences. The sequences that do not bind to the target are washed away. The bound oligonucleotides are then eluted via a chosen elution method, which can be temperature, alterations of pH, salt concentration, use of chaotropic agents or another method of choice. Eluted aptamers are pooled for amplification via a PCR (DNA) or RT—PCR (RNA) amplification protocol. The selection, eution and amplification protocols comprise a SELEX round. In order to select for the best binder, a number of rounds ranging from 6 to as many as 15, have been reported to allow, through competitive binding against a limited target, the displacement of all non-specific or weak binders by the best binding aptamer. The method was conceived as an evolutionary process that allows for the selection of better, higher affinity binders. To exclude aptamers that may bind to similar proteins and ensure specificity and selectivity, counter-selection rounds are also included at this stage, allowing aptamers that bound to the target, but not to related molecules, to be amplified for the next selection round. After the aptamer selection process, the PCR products of the last round are double stranded and cloned into a vector and, subsequently, sequenced to allow for identification of the best binding sequence, which can then be chemically synthesized. The SELEX methodology is summarized in Figure 2.4. There are features of the technique that need to be tailored to suit the requirements of each selection. First consideration is the choice of the nature (RNA, DNA or unnatural bases) and length of the nucleic acid library. The second stage is the definition of the method used for the selection process. The molecules can be selected in a variety of in vitro environments/methods that can be chosen and modified to fit the need. Such selection methods include immobilization of the target in affinity chromatography matrices, magnetic beads or other support systems, or the selection in solution and separation of bound complexes from unbound species with the use of nitrocellulose filters of appropriate pore size. Finally, the number of SELEX rounds may need to be optimized, depending on the stringency of the selection required and the amount of target available for competitive binding. Following the identification and synthesis of the best binding aptamer, further studies are carried out to determine the binding affinity (Kg) and specificity of the selected molecule against the target and, depending on the application, if desired, the molecule can then be ‘fashioned’ (e.g. 5' or 3' modification for chelator bioconjugation or nuclease resistance) for the required end use. Further additional chemical modifications, such as PEGylation (Floege et al., 1999), or liposomes, can be easily made at desired positions of the aptamer to improve its pharmaceutical, therapeutic and/or diagnostic application. Attachments of such molecules serve to decrease the renal clearance time of aptamers, as the pharmacokinetic properties of these molecules are limited by their small size (5-25kDa) and hydrophilic nature. Signalling molecules such as fluorophores can also be incorporated into the aptamer for imaging/signalling purposes, while the attachment of drugs can facilitate the therapeutic effect of the aptamer, if required (Floege et al., 1999). Although chemical modifications can be completed relatively easily, it is important that such alterations to the backbone structure of the aptamer do not affect its binding to the target, particularly given that binding is largely governed by shape-shape interactions between the two molecules. 2.4.2 Non-SELEX methods for the selection of aptamers SELEX has proved to be a robust and powerful methodology for the isolation of many aptamers directed towards a variety of targets. However, a number of attempts have been made to improve, or bypass this technique, both to overcome some of the drawbacks associated with traditional SELEX procedures and to bypass the patent stronghold on SELEX by Archemix and Somalogic in the USA. Thus, ‘non-SELEX’-based methods for the selection of aptamers have recently been put into practice. The utilization of capillary electrophoresis has demonstrated to be a highly efficient approach into the partitioning of aptamers with desired properties from arandomized pool (Drabovich et al., 2005; Berezovski et al., 2005). Using this technique, aptamers to h-Ras, a protein involved in the development and progression of cancer, were isolated with predetermined kinetic parameters (Berezovski et al., 2005). The isolation of aptamers with predefined kinetic and thermodynamic properties of their interaction with the target has so far been obstructed by the standard SELEX technology. Furthermore, this method of aptamer selection only employed the partitioning steps of SELEX without the need for amplification between them. Hence, one of the most significant advantages of this non-SELEX method is its application to libraries which ere difficult or cannot be amplified, thus overcoming the problems associated with using modified oligonucleotide libraries, as mentioned above. As well as the relative simplicity and easy-to-use nature of this procedure, aptamers are selected within only a few hours, which contrasts the several days or weeks needed for standard SELEX systems. Exploitation of computational methods has also led the way into the development of non-SELEX methods for aptamer selection. More importantly, computational methods have been powerful in selecting aptamers with inhibitory activities or sequences that undergo ligand dependent conformational changes, a property useful for the design of molecular and aptamer beacons. One of the major drawbacks associated with SELEX is the selection of aptamers that may not have any inhibitory activity towards its target, since the selection of aptamersis based on affinity. Consequently, researchers have used this drawback to drive the engineering of alternative selection methods based on inhibitory activity of aptamers. Algorithm methods have shown to be sufficiently effective in selecting aptamers with such properties. This computational method has been used to predict the secondary structure of nucleic acids under different conditions e.g. in the presence and absence of a ligand (Hall et al., 2007). In general, algorithmic methods use aptamers with known structures and/or features (such as aptamers that undergo ligand-induced conformation changes) to rapidly select oligonucleotides from virtual pools, which may present similar properties or adopt similar structures. Hence, sequences are selected that match a defined profile. One of the most valuable structures applied to computational selection is that of the G-quartet. Such structures are suggested to have important implications in the biology of cancer and thus aptamers that adopt such configurations are of great interest. An aptamer selected for thrombin has been thoroughly investigated and is known to adopt such a configuration. Consequently, this aptamer has been used as a model to investigate the potential of new selection methods, based on inhibitory activity. By randomizing the sequence of the duplex region of the anti-thrombin aptamer, Ikebukuro and co-workers (2006) selected inhibitory aptamers using genetic algorithm on a library of limited sequences. In another report, evolution mimicking algorithms were used to select aptamers with potent inhibitory activity from a pool that was designed to form G-quartet structures and contain a limited number of sequences (Noma and Ikebukuro, 2006). Computational selection methods of aptamers require detailed information on the prerequisites of the basis of selection, for example the structure of the aptamer that new aptamers are to be modelled on or the structural changes that the profile aptamer undergoes upon ligand binding. Given that this is not always feasible and that gaining this information can sometimes be a lengthy process, computational selection of aptamers may be delayed in materializing as a potential option for ligand identification. Meanwhile, SELEX itself is still widely used for aptamer selection and is constantly advancing to overcome many of its initial challenges.
Key Takeaways
- SELEX methodology involves iterative rounds of interaction, elution, and amplification to select high-affinity aptamers.
- Counter-selection steps ensure specificity by excluding molecules that bind to similar proteins or isoforms.
- Computational methods can be used as alternatives to traditional SELEX for selecting aptamers with specific properties.
Practical Tips
- Utilize SELEX methodology for precise and high-affinity aptamer selection in drug design.
- Implement counter-selection steps to enhance the specificity of selected aptamers.
- Explore computational methods for alternative aptamer selection when traditional SELEX is not feasible.
Warnings & Risks
- Be cautious with chemical modifications that may affect binding affinity.
- Ensure detailed information on target structure and ligand-induced conformational changes for effective computational selection.
Modern Application
The SELEX methodology described in this chapter remains a cornerstone of modern drug design, particularly in the development of targeted therapies. While the basic principles have been refined over time, the core process of iterative selection and optimization continues to be vital. Modern advancements include improved computational tools for predicting aptamer properties and more efficient screening methods like capillary electrophoresis. Understanding these techniques is crucial for developing effective treatments against cancer and other diseases.
Frequently Asked Questions
Q: What is the SELEX methodology used for in drug design?
The SELEX methodology is a combinatorial approach used to select high-affinity aptamers from vast libraries of oligonucleotides, which can be tailored to bind specifically to target proteins or molecules.
Q: How does the SELEX process ensure specificity in selected aptamers?
The SELEX process ensures specificity by including counter-selection steps that exclude aptamers binding to similar proteins or isoforms, thus focusing on high-affinity and specific binders.
Q: What are some alternative methods to traditional SELEX for aptamer selection?
Alternative methods include the use of capillary electrophoresis and computational algorithms that can predict aptamers with desired properties without the need for amplification steps.