How to Generate Artificial RNA and DNA Catalysts

The vast majority of in vitro selection experiments, however, did not focus on catalysts but on RNA molecules that spedlically bind a given target molecule, so-called aptamers. The most crucial step in any variant of this technique is selection, meaning an effective distinction between desired and undesired species. It is in the selection step that the most decisive advances in ribozyme discovery were made, as will become clear in the discussion below. The standard selection event in aptamer SELEX is a binding event, and consequently the desired molecules can be distinguished from the rest by physical separation, namely by affinity chromatography. 

Better acceleration rates and catalysis of more complex reactions were obtained by direct selection. In this strategy, selection is not based on a binding event rather it is based directly on the desired catalytic event For example, if the to-be-catalysed reaction is phosphodiester hydrolysis, the RNA library is immobilized on solid support. The selection event is thus directly tailored to hydrolysis active molecules cleave their covalent linkage to the solid support and thus dissociate from it The catalytic event thus enables spatial separation of active and inactive molecules. Direct selection has therefore allowed to isolate catalysts for numerous RNA-modilying reactions, from hydrolysis and ligation to RNA alkylation, acylation, phosphorylation and many others (see below). 

The use of reactants other than RNA in direct selection allows targeting of more complex reactions. An essential prerequisite is the attachment of a (non-RNA) reactant X to the RNA molecules, i.e., conversion of the RNA library into a RNA-reactant library. Assuming a desired RNA catalyst of the chemical reaction X Z, which may include simple addition as in X + Y Z, the selection has to be designed in a way to isolate 

Depending on the reactivity of reactant Y towards the functional groups contained in RNA, its conjugation to species of the RNA library will occur with more or less site-specificity to the conjugated X-reactant. More reactive Ys may add to RNA at positions 

The strategies to generate catalytic DNA molecules are almost identical and start with the same type of libraries. While one would intuitively expect that these selections are easier to perform since transalption and reverse transcription are eliminated from the selection cycles, in practice they are not, since the double-stranded DNA libraries resulting from PCR-type amplifications are catalytically incompetent, and their conversion into high-quality single-stranded DNA libraries is not trivial. 

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