Spiegelmers: Biostable Aptamers

In the early 1980s the groups of Cech and Altman discovered catalytically active RNA molecules, the ribozymes. This finding showed that RNA has more facets than just being the carrier and transporter of genetic information or serving as a structural element. From now on, nucleic acids could be regarded as molecules which have the potential to actively contribute to the catalytic and regulative processes that maintain cell life. The versatility of nucleic acids, which consist of only four different building blocks, is due to their ability to display distinct and stable 3D structures, thus being capable of forming binding pockets and catalytically active centres. In this respect nucleic acids are comparable to proteins. Nucleic acid molecules have two properties that make them ideal candidates for the intelligent screening of vast combinatorial libraries: Not only are they highly structurally diverse molecules but they are also easily replicated carriers of their own structural information. The structural information of each molecule (the phenotype) is contained within its individual nucleotide sequence (the genotype) so that the amplification of molecules that survive a screening process for a desired phenotype is easily accomplished by standard enzymatic methods. This property of nucleic acids combined with the possibility to generate the required immense structural diversity of up to 10 different sequences by standard solid-phase oligonucleotide chemistry, allowed the establishment of an efficient evolutionary screening technology, the so-called SELEX process (systematic evolution of ligands by exponential enrichment). This technique, which is based on the principles of in vitro selection and in vitro evolution, was published independently by three research teams in 1990. Comparable to Darwinian evolution, nucleic acid libraries of RNA or DNA are subjected to a selection process in vitro. These libraries usually carry a central randomized region that is flanked by fixed sequences to facilitate enzymatic amplification through PCR. When molecules that bind to a given target structure are selected, the library is contacted with the immobilized target. Nonbinding molecules can be partitioned from binders by simple washing in an affinity chromatography-like process. Binding molecules that survive the selection procedure are then eluted and amplified. By executing this procedure iteratively, the complexity of the original library is depleted and target-binding candidates are enriched. As soon as the affinity of the enriched library cannot be further increased, generally after six to twenty selection rounds, the nucleotide sequences of individual binding molecules are determined by cloning and sequencing the enriched nucleic acid library. For target-binding nucleic acids that emerge from the process of in vitro selection, Ellington and Szostak coined the term 'aptamer' from the latin 'aptus', to fit. To date, the sequences and binding characteristics of more than one hundred aptamers against a plethora of different targets that range from small molecules, such as organic dyes, to membrane fragments and cells have been published. The reported dissociation constants are in the picomolar to micromolar range. For an excellent review on aptamers, see the book by James.