BioTechniques News
Maddy Chapman
In the quest to improve our ability to produce more complex non-standard proteins, a research team has broken from conventional wisdom to reveal some fascinating insights into our evolutionary history.
Last week, a team of researchers at the Wyss Institute (MA, USA), led by senior author George Church, took a dramatic step forward in the production of synthetic proteins from an ‘expanded genetic alphabet’. The alphabet features 14 non-standard amino acids that can be applied in cell-free systems. By engineering tRNAs and ribosomes, the team were able to run two genetic codes in a single automated cell-free platform that they named AGENTEX (Automated Genetic tRNA Expansion), which could lead to the production of novel therapeutic proteins. In the process, the team also exposed vital flaws in the prevailing wisdom surrounding the highly conserved CCA ending of tRNAs and its impact on the molecule’s functionality.
While there is nothing new about incorporating non-standard amino acids (nsAAs) into proteins, challenges arise when you try to incorporate multiple nsAAs into a single polypeptide or change the corresponding amino acids for multiple sense codons in a single genome. To overcome these issues, Church’s team tinkered with several aspects of the traditional translation system.
The first key component was the design of a compressed genetic code. In nature, 64 different codons code for 20 amino acids, a start and three stop codons (with some overlap between the coding sense codons and the start and stop codons). The team engineered a library of tRNAs to encode the 20 amino acids, a start and a stop codon from just 20 codons, using 14 other codons to incorporate for 14 different nsAAs into the translation system.
Traditional cell-free translation platforms either use lysate extracted from cells or a PURExpress system that uses individually purified components for translation. PURExpress offers you greater control over the contents of the system but lacks the natural components for tRNA modifications that can contribute to their function. Lysate, however, is vulnerable to crosstalk between the cells’ natural translational system and your fancy new engineered one.
To overcome these issues in one fell swoop, the team looked to the interaction between tRNAs and the ribosome. In nature, tRNAs all have a highly conserved end sequence of CCA and ribosomes will not bond to tRNAs with a different ending (otRNAs). As the CCA ending is so highly conserved, it has been assumed that tRNAs that differ in this region are completely dysfunctional and cannot be aminoacylated by aminoacyl tRNA synthetases (aaRSs), a process referred to as ‘tRNA charging’. This assumption has been backed up in the literature, mostly by investigations in simplified cell-free PURExpress-like systems.
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Church and his team developed two methods – tSCAN (tRNA sequencing of charging by automated NGS) and tSCAN-M (tRNA-species charging analysis by mass) – to quantify the aminoacylation of synthetic tRNAs in cell-free translation systems and identify which amino acids were attached to which tRNA, respectively. Using these methods, they found that when produced in cell lysate, where they can be post translationally modified, most otRNAs could be aminoacylated by aaRSs.
However, the hard block of the impossible interaction between otRNAs and the ribosome remained, prompting questions about the potential insight that this discovery could provide into evolutionary history. The authors stated that understanding “why the CCA sequence is universally conserved may also provide insights into the evolution of the translation system and the origins of life,” later suggesting that strong selective pressures would normally prevent the emergence of alternate genetic codes as several changes would have to occur to ribosomes and tRNAs simultaneously.
With this discovery under their wing, the team were able to robotically produce their 34-codon genetic library with otRNAs and introduce them into a cell lysate taken from cells engineered to produce a ribosome that would accept them. The resulting cell-free system therefore contained any natural tRNAs and ribosomes in the cell and altered otRNAs and ribosomes, each set incapable of interacting with the other, essentially eliminating crosstalk between the two.
Ultimately, the team was able to develop AGENTEX, an automated platform that enables you to enter your genetic code design for it to then construct the appropriate otRNAs, engineered ribosomes and cell lysate. Following this building phase, it can then mix the components, run the translation and synthesis of your novel proteins and analyze your genetic codes’ performance, determining the degree of aminoacylation with tSCAN and confirming the identity of the amino acids attached to each otRNA with tSCAN-M.
As recent developments, including the publication last month of 16 AI-designed bacteriophages, have sparked revitalized discussions about biosafety, the team was keen to highlight that the cell-free nature of this system builds biocontainment into the workflow, conveying an innate degree of biosecurity. This boosts the translatability of the platform, showcasing its potential impact on the production of novel therapeutic peptides and biopolymers, plus, with two translational systems working alongside each other in a single cell, it could even lead to the construction of “organisms with radically divergent properties from extant life”, as the authors conclude in their discussion.
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