Directed evolution of Diels-Alderase ribozymes using in vitro compartmentalisation.

Agresti, Jeremy Jon (2006). Directed evolution of Diels-Alderase ribozymes using in vitro compartmentalisation. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f657

Abstract

Darwinian evolution in nature has produced many remarkable biocatalysts. By emulating the same processes in the laboratory there is great potential to produce catalysts that rival natural enzymes for efficiency, and are tailor made to the needs of science and industry. In addition, it may be possible to gain a deeper understanding of the evolutionary process itself. In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. In this thesis, I demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has until now been unselectable: true intermolecular catalysis. Libraries containing up to 10¹¹ ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By co-encapsulating the gene, RNA, and the substrates/products of the catalysed reaction, ribozymes can be selected for all enzymatic properties: substrate recognition, product formation, rate acceleration and turnover. Starting from a large random library of RNAs, a previous study using SELEX, which is based on selection for single-turnover, intramolecular reactions, yielded Diels-Alderase ribozymes which all contained the same catalytic motif. Selecting exactly the same library using IVC, in addition to ribozymes with the motif found in the SELEX study, it was possible to select Diels-Alderase ribozymes with a completely novel catalytic motif. All the selected ribozymes were capable of catalysing the reaction between dienes and dienophiles in solution in a truly bimolecular fashion and with multiple turnover. Interestingly, the catalytic properties of all the selected ribozymes were quite similar. The ribozymes are strongly product inhibited consistent with the Diels-Alder transition state closely resembling the product. More efficient Diels-AIderases may also need to catalyse a second reaction that transforms the product and prevents product inhibition.