Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation

Here we describe a novel technology that allows users to charge nonnatural amino acids onto any tRNA. This technology is based on a resin-immobilized ribozyme system, called Flexiresin. It enables users to readily and rapidly synthesize misacylated tRNAs with a wide variety of phenylalanine analogs. Since Flexiresin is reusable and little effort is necessary for regeneration, it is economical and convenient. Moreover, it can adapt to virtually any tRNA chosen by the user, and can therefore be applied to not only a single site mutation but also multiple sites with designated nonnatural amino acids when both the amber and programmed frame-shift mutations are utilized. The original ribozyme utilized for Flexiresin was artificially generated in vitro, and thus the technology in principle could be broadened from Phe analogues to essentially any amino acid.