SELEX Technology Research Articles

Les aptamères ou l'évolution moléculaire dirigée : sélection et applications

Aptamers are short oligonucleotides selected from large combinatorial pools of sequences for their capacity to bind to many different targets ranging from small molecules (amino acids, antibiotics…) to proteins or nucleic acid structures. Aptamers present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers have been shown in many cases to display an inhibitory activity against their targets. Many aptamers are now being developed against biomedical relevant targets, and one aptamer that inhibits the human VEGF165 already received approval for the treatment of age-related macular degeneration. Here we discuss the principles and the practical way of selecting aptamers (SELEX technology) as well as the structural basis for their performance as ligands. A wide scope of applications is described — aptamers have been used as tools for studying nucleic acids/proteins interactions, detecting, purifying or imaging target molecules, regulating gene expression — and includes recent developments of aptamers for therapy and diagnosis.

FluMag-SELEX as an advantageous method for DNA aptamer selection

Aptamers are ssDNA or RNA oligonucleotides with very high affinity for their target. They bind to the target with high selectivity and specificity because of their specific three-dimensional shape. They are developed by the so-called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. We have modified this method in two steps-use of fluorescent labels for DNA quantification and use of magnetic beads for target immobilization. Thus, radioactive labelling is avoided. Immobilization on magnetic beads enables easy handling, use of very small amounts of target for the aptamer selection, rapid and efficient separation of bound and unbound molecules, and stringent washing steps. We have called this modified SELEX technology FluMag-SELEX. With FluMag-SELEX we have provided a methodological background for our objective of being able to select DNA aptamers for targets with very different properties and size. These aptamers will be applied as new biosensor receptors. In this work selection of streptavidin-specific aptamers by FluMag-SELEX is described. The streptavidin-specific aptamers will be used to check the surface occupancy of streptavidin-coated magnetic beads with biotinylated molecules after immobilization procedures.

Aptamers as tools in molecular biology and immunology.

We have listed and described recent promising developments in the field of aptamer research. The properties and the application potential of aptamers propose an exciting future for aptamers either in the clinic or as research tools for various purposes. We have reviewed exciting examples in which the SELEX technology was applied to obtain promising tools that may help to facilitate our understanding of biological processes and to interfere at distinct points in signal transduction cascades. High affinities and specificities of aptamer/target-interactions can now routinely be achieved. Furthermore, a wide spectrum of chemical modifications of nucleotides is known which greatly increase the stability of RNA molecules in biological materials, considerably enhancing their application potential. The aptamer technology shows that the combination of organic synthesis and molecular biology can contribute to interesting and promising new drug leads, which may very soon find their way into daily clinical practice or onto the laboratory benches of many researchers in the life sciences.

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.