High yield, low magnesium flexizyme reactions in a water-ice eutectic phase

Flexizymes enable the stoichiometric acylation of tRNAs with a variety of compounds, enabling the in vitro translation of peptides with both non-natural backbones and side chains. However, flexizyme reactions have several drawbacks, including single-turnover kinetics, high Mg(II) carryover inhibiting in vitro translation, and rapid product hydrolysis. Here we present flexizyme reactions utilizing an ice-eutectic phase, with high yields, 30X lower Mg(II), and long-term product stability. The eutectic flexizyme reactions increase the ease of use and flexibility of flexizyme aminoacylation, and increase the in vitro protein production.