In vitro evolution of flexizymes that function under the conditions in translation system

Flexizyme is an artificial ribozyme capable of acylating any kinds of tRNAs with virtually no limitation of acid donor substrates. Although the flexizyme system is a highly flexible tRNA acylation tool, it requires relatively high Mg(2+) concentrations (>50 mM) for the full function. For instance, lowering the Mg(2+) concentration to 10 mM, where the in vitro translation system generally functions, resulted in significant decrease in activity. To overcome this limitation, we implanted a new random domain into a part of flexizyme to aim at selecting a new functional domain that would exhibit acylation activity at lower Mg(2+) concentrations. Indeed, in vitro selection of active species from such an RNA pool afforded a new flexizyme, called mdFx, showing a 3-fold higher yield at 10 mM Mg(2+) concentration. Thus, this new flexizyme opens a new avenue to construct an mdFx-transcription-translation coupled system.