Abstract
High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics. Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies. Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments. They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies. Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules. This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target. This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool. Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents. However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers. As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed. These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps. Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of novel aptamers.
Highlights
High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics
This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and reselected against the desired target
Systematic Evolution of Ligands by EXponential enrichment (SELEX) involves generating a library of nucleic acid molecules (e.g., DNA or RNA) with random sequences ($1015 different sequences), screening the library for nucleic acids that bind to the target of interest, partitioning the bound molecules from the unbound molecules, and amplifying the bound molecules into a new pool enriched for good binders (Figure 1)
Summary
The basic principles for the in vitro evolution and selection of aptamers were described over twenty years ago.. Given its simplicity and familiarity among many laboratories, affinity chromatography has become the dominant method for small molecule selections.46–50 This method generally requires large quantities of target to achieve sufficiently high loading onto the entire column and can suffer from non-specific binding of the nucleic acids to the resins, requiring extensive washing or negative selections.. This method generally requires large quantities of target to achieve sufficiently high loading onto the entire column and can suffer from non-specific binding of the nucleic acids to the resins, requiring extensive washing or negative selections.46 This method requires the incorporation of an affinity tag to target molecules for immobilization; and this is commonplace for proteins, it can be difficult to achieve for small molecules and restricts the modes for aptamer binding. The separation step is slow and can be far from equilibrium, providing opportunities for bound complexes to dissociate during the lengthy process
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