In vitro evolution of new ribozymes with polynucleotide kinase activity

We have isolated a large number of polynucleotide kinase ribozymes from a pool of RNA molecules consisting of an ATP-binding domain flanked by regions of random sequence. Different classes of kinases catalyse the transfer of the γ-thiophosphate of ATP-γS to the 5′-hydroxyl or to internal 2′-hydroxyls. An engineered version of one class is able to catalyse the transfer of thiophosphate from ATP-γS to the 5′-hydroxyl of an exogenous oligoribonucleotide substrate with multiple turnover, thus acting as a true enzyme.

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