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 Mg2+ concentrations (>50 mM) for the full function. For instance, lowering the Mg2+ 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 Mg2+ 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 Mg2+ concentration. Thus, this new flexizyme opens a new avenue to construct an mdFx–transcription–translation coupled system.