Background

All living organisms synthesize proteins according to blueprints stored in deoxyribonucleic acid (DNA). In this process, it is known that only 20 types of amino acids are used. The scientific community is increasingly focusing on genetic code expansion (GCE) technology, which aims to incorporate non-canonical amino acids (ncAAs), not found in nature, into proteins to develop new drugs and materials. Conventional GCE methods have involved artificially editing the genome of living Escherichia coli. However, direct genome editing is time-consuming and can reduce cell viability. Cell-free protein synthesis (CFPS) technology, which produces proteins in an environment without cell membranes, has emerged as an alternative. Yet, it still requires manual creation of custom transfer RNA (tRNA), making it relatively slow.

Key Discovery

A joint research team led by Felix Radford from Harvard Medical School and George Church from the Wyss Institute developed an automated platform called Automated Genetic tRNA Expansion (AGENTEX), which significantly shortens the process of genetic code redesign. This platform operates using a liquid aspiration pipetting robot and autonomously performs all steps from gene template purification to protein translation. The research team focused on challenging the existing biological consensus that ribosomes recognize only the three-nucleotide CCA sequence at the end of tRNA. Using an analytical tool called tSCAN, they demonstrated that non-standard tRNAs with alternative terminal sequences, such as CGA, can be used without issues in protein synthesis. The team is working to construct a protein factory structure that combines modified ribosomal large subunits (LSUs) with non-standard tRNAs to go beyond the range of natural amino acids. In actual experiments, the AGENTEX platform successfully synthesized proteins by reassigning 34 codons using 34 different aminoacyl-tRNA synthetases. This represents a pathway to control the genetic translation system in vitro without modifying living genomes.

Significance and Outlook

This research lays the foundation for rewriting genome blueprints in test tubes, independent of cell survival. The cycle from digital genome design to physical protein synthesis is expected to be dramatically shortened from days to hours. In particular, research into pharmacologically active peptide drugs and highly stable new materials is anticipated to gain significant momentum. The research team's automated experimental methods and software have been released as open-source, which is expected to accelerate follow-up studies in academia. However, a limitation is that the final protein yield in cell-free systems is lower than in living cells. Additionally, further technical improvements are needed to enhance translation accuracy to ensure seamless decoding of more than 34 different codons.