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RMA library

affinity chromatography with immobilized target molecule elution of binding ligands

RMA library enzymatic amplification affinity chromatography with immobilized target molecule discard unbound RNA

elution of binding ligands

Scheme B.23.1.

lection of active species from nucleic acid libraries by separation from unreactive molecules and enzymatic amplification of the enriched library. Because of these two key steps the method is called SELEX (systematic evolution of ligands by exponential enrichment). Two aims can thus be attempted. The first is the selection of molecules with specific binding properties, called aptamers [1]. In this approach the target molecule of interest is immobilized on a solid matrix (Scheme B.23.1). The combinatorial nucleic acid library is then applied to the affinity matrix in a suitable buffer. Unbound nucleic acid molecules are washed away and the binding molecules are retained. These are later competitively eluted with a buffer containing the free target molecule. The eluted molecules undergo enzymatic amplification as a result of addition of a template-dependent polymerase. Thus, several thousand copies of each selected nucleic acid molecule are generated. This results in a library in which molecules with high affinity for the target are enriched. This process must be repeated several times until molecules with the desired properties dominate the enriched library. The members of this library are then identified by sequencing.

The second aim is selection of molecules with new catalytic function, called ribozymes. Two different approaches are used to find catalytic nucleic acids. One is to synthesize a transition-state analog (TSA) of the corresponding reaction [2]. The TSA is then used as target molecule in the affinity selection scheme described above. The selected aptamers are screened to find molecules that catalyze the respective reaction that proceeds via this transition state. This concept has been successfully used for catalytic anti-

B.23 SELEX: Systematic Evolution of Ligands by Exponential Enrichment I 435

B.23 SELEX: Systematic Evolution of Ligands by Exponential Enrichment I 435

Scheme B.23.2.

bodies; unsuccessful attempts have usually been reported for nucleic acids. The most successful method for identification of nucleic acid catalysts is direct selection in which members of a combinatorial DNA or RNA library are isolated if reaction with a substrate X is accelerated (Scheme B.23.2). X carries an anchor group so that RNA molecules that react with the substrate acquire the anchor group and can subsequently be isolated by affinity chromatography on a suitably derivatized matrix [3]. Unreacted RNA does not bind to the matrix and can be removed by washing. Again this cycle is repeated until active molecules dominate the library. One limitation of this method is that the isolated molecules are self-modifiers (i.e. not true catalysts) and perform the reaction once only. In this book chapter a method is described that enables this limitation to be overcome, enabling generation of nucleic acid catalysts for true bimolecular organic reactions.

References

1 Famulok, M.; Szostak, J. W. Schultz; P. G. Science 1994, 264, Angew. Chem. Int. Ed. Engl. 1992, 1924-1927.

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