Directed evolution of ribozymes with novel catalytic functions

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Alternative systems for the selection of ribox/combox variants able to catalyze the reduction of benzyl alcohol have been set up. The first one aims to identify in an initial step variants of the ribozyme able to bind with higher affinity to the target substrate (benzyl alcohol). The system is based on capture-SELEX, and variants identified with the system will be screened for catalytic activity in a second step using a fluorescence-based assay. Additionally, we have also identified alternative substrates likely to have a higher reactivity. These include benzyl alcohol and benzaldehyde derivatives with one or more nitro or carboxyl substituents in the aromatic ring. Iterative rounds of capture-SELEX with the different substrates are currently being performed.The second strategy is based on the previously mentioned selection system which exploits the requirement of oxidized NAD+ for anaerobic growth (Selles Vidal, Murray and Heap, 2021). The library of combox variants, as well as wild-type combox, have been cloned into the appropriate plasmids for the selection system. Additionally, a classical protein benzyl alcohol dehydrogenase has been also cloned into the same vector. We have demonstrated that cells transformed with the protein benzyl alcohol dehydrogenase can grow anaerobically in medium supplemented with benzyl alcohol, proving that this pair of substrate and enzymatic activity are suitable for the selection system. We are currently applying the system to the RNA-based benzyl alcohol dehydrogenases.