Textbook Rule That ‘tRNA Requires a CCA’ Broken, Opening New Path for Genetic Code Expansion

The textbook principle that a CCA sequence is required at the 3' end of tRNA—a barrier to genetic code expansion—has been overturned. A study found that tRNAs without CCA termini can also transport amino acids normally.

According to the international academic journal Nature on the 26th local time, a research team led by George M. Church, a professor at Harvard Medical School in the United States, said it had developed the “AGENTEX” system, which can transport amino acids without the CCA sequence at the 3' end of tRNA.

Living organisms use 64 codons to specify 20 amino acids. Scientists have sought to add nonstandard amino acids (nsAAs) to design cells that produce new materials, drugs and catalysts.

Existing technologies have introduced more than 400 types of nonstandard amino acids, but their use has been limited to a small number of codon sites. The reason researchers have been unable to systematically redesign the entire genetic code was the prevailing belief that amino acids could be attached only when a CCA sequence was present at the 3' end of tRNA.

Using robotic technology and next-generation sequencing, the research team examined the full set of Escherichia coli tRNAs. The results showed that many tRNAs with modified termini, including CUA and CAA, could be loaded with amino acids by all Escherichia coli aminoacyl-tRNA synthetases (aaRSs) without additional modification.

Based on this finding, the team built an orthogonal dual-translation system by combining tRNAs with modified anticodons and ribosomes engineered to recognize only non-CCA termini. Using the system, the researchers tested a minimal code that compressed the genetic code into 34 codons and 34 aaRSs.

However, AGENTEX is currently limited to a cell-free lysate system. The research team explained that problems involving off-target editing, cytotoxicity and aminoacylation efficiency—the key challenges in genome recoding in living cells—have not yet been resolved.

The research team analyzed, “If the CCA restriction can be easily bypassed, it raises the question of why natural selection has maintained the universality of this terminus,” adding, “This conservation may be a mechanism that ensures the accuracy and robustness of translation.”