ReviewSELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands
Introduction
Since its first description in 1990, the SELEX technology is widely applied as an in vitro selection method to evolve nucleic acid ligands, called aptamers, with new functionalities. The term aptamer is derived from the Latin word “aptus”—which means fitting (Ellington and Szostak, 1990) and the Greek word “meros” meaning particle. Aptamers are short single-stranded nucleic acid oligomers (ssDNA or RNA) with a specific and complex three-dimensional shape characterized by stems, loops, bulges, hairpins, pseudoknots, triplexes, or quadruplexes. Based on their three-dimensional structures, aptamers can well-fittingly bind to a wide variety of targets from single molecules to complex target mixtures or whole organisms (Fig. 1). Binding of the aptamer to the target results from structure compatibility, stacking of aromatic rings, electrostatic and van der Waals interactions, and hydrogen bondings, or from a combination of these effects (Hermann and Patel, 2000). The first aptamers developed consisted of unmodified RNA (Ellington and Szostak, 1990, Tuerk and Gold, 1990). Later on, single-stranded DNA aptamers for different targets were described (Ellington and Szostak, 1992) as well as aptamers containing chemically modified nucleotides (Green et al., 1995). Chemical modifications can introduce new features into the aptamers, improve their binding capabilities or enhance their stability (Gold et al., 1995). Numerous variants of the original SELEX process were described to select aptamers with high affinities and specificities for their targets. Many of the selected aptamers show affinities comparable to those observed for monoclonal antibodies. In addition, aptamers can distinguish between chiral molecules and are able to recognize a distinct epitope of a target molecule (Michaud et al., 2003, Jenison et al., 1994). Thus, the differentiation between closely related targets (e.g. theophylline and caffeine) is possible. A further revolutionary aspect of the SELEX technology is the selection of ligands beyond natural systems by use of chemically produced oligonucleotide libraries, without the constraints imposed by having to be selected or produced in a living organism. The big variety of 1015 different oligonucleotides within the SELEX library and the amplification steps of target-binding oligonucleotides during the selection process facilitate far higher possibilities to select ligands with highest affinity than natural selection. Moreover, the in vitro selection process for aptamers can be carried out under conditions akin to those used in the assay for which the aptamer is being developed—the aptamer will maintain its structure and function in the final assay and will not dissociate, which can be a problem with antibodies (Mukhopadhyay, 2005). The SELEX conditions can be further modified to direct the selection to aptamers with desired features. This stands in contrast to the classical production of antibodies, where it is not possible to influence such parameters and therefore leaving it limited to physiological conditions (Jayasena, 1999).
This review will give an overview of the SELEX technology. The different steps of a SELEX process are discussed in detail with regard to the selection of target-binding aptamers. Advantages and limitations, as well as the versatile application potential of aptamers are described. The in vitro selection of nucleic acids with catalytic activity, like ribozymes and DNAzymes are not included in this review.
Section snippets
General principle
Combinatorial chemistry is an important technology for industry as well as biotechnological and pharmaceutical research to discover new materials or molecules with desirable properties, new drugs, and catalysts. It is characterized by the synthesis and simultaneous screening of large libraries of related, but structurally distinct compounds to identify and isolate functional molecules. Nucleic acids are very attractive compounds for combinatorial chemistry, because they are able to fold into
Post-SELEX modifications
Post-SELEX modifications are executed either in order to increase the stability of the selected aptamers or to optimize binding parameters to the target or relevant molecules. For further applications, modifications with functional groups for detection or immobilization are possible.
In order to enhance biostability of the aptamers, it is possible to introduce – after a successful selection – similar chemical modifications as was the case for initial oligonucleotide libraries above-mentioned in
Automated SELEX
In order to develop aptamer sensor arrays that can be used for analyzing molecular mixtures, a fast and parallel development of multiple aptamers is desirable. Therefore, considerable efforts aimed at the development of automated platforms for aptamer selection. Automation of the selection procedure means the integration and automation of different molecular biology methods (for binding, partioning, elution, amplification, conditioning) and is thus a very complex automation challenge.
A robotic
Advantages and limitations of aptamers and their selection technology
The basic SELEX technology is a powerful and universal tool to evolve aptamers able to bind very tightly and specifically to a wide variety of targets. Hundreds of publications concern the selection of aptamers and their applications, which reflect the great interest for this research field and its enormous potential for pharmacy and medicine, as well as environmental analytics. The main advantages of aptamers and their selection technology are summarized in the following listing; some of them
Fields of applications of aptamers
In spite of the very encouraging promises, research on aptamers is still at the beginning. One disadvantage is the unavailability of a standardized protocol for aptamer development which is applicable without specific modifications for different targets. But aptamer research is catching up for faster results. Medical and pharmaceutical basic research as well as clinical diagnostic and therapy (e.g. inhibition of enzyme activities, blocking of receptor binding sites) offer a big field of
Future perspectives
Aptamers for therapeutic applications promise a good stroke of business among the aptamer applications. In this area we find the furthermost advanced commercialization with Macugen®, a therapeutic against wet age related macula degeneration, produced by Pfizer Inc. This first demonstration of a successful aptamer based drug development will prod other companies like Archemix, SomaLogic, Regado Biosciences, Noxxon, NascaCell, AptaRes and others to follow with their own aptamer based drugs.
Acknowledgement
We thank Nadia Nikolaus for helpful discussions and critical reading of the manuscript.
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