Investigating the hidden meaning of synonymous codon choices
With the ability to synthesize genomes de novo using REXER and GENESIS, we are interested in the design of a synthetic genome with certain synonymous codons removed by recoding to alternative synonymous substitutions. Such recoded codons could be subsequently reassigned to encode unnatural amino acids. In addition to the TAG amber stop codon, we have identified 10 sense codons in 5 subsets (two codons each subset, which need to be removed simultaneously to enable subsequent reassignment) that could serve both as a complete set of codons targeted for genome-wide removal and as a model set to study synonymous codon choices using REXER mediated recoding (Figure 2).
The recoding study will not only identify the best synonymous substitution for each of the target codons but more importantly shine a light on the meanings of synonymous codon choices. Nature chooses one triplet codon from up to six potential synonyms to encode each amino acid at each position in the genome; this choice can define transcriptional or translational regulatory elements, translation speed, mRNA folding, gene expression, co-translational folding, protein production levels, and is likely to have further undiscovered roles. Synonymous codons may have distinct roles at different sites in the genome, and there may be epistatic interactions amongst codons within and between genes.
We would like to perform REXER using the 11 target codons to systematically investigate synonymous codon functions on an operon scale, and gradually expand to alternative and longer genomic regions. It would be very challenging to implement this idea using conventional methods, not only due to an inability to efficiently introduce a very high density of synonymous codons across many essential genes but also because of the need to precisely pin-point allowed and disallowed synonymous codon changes with high throughput and accuracy. REXER serves as a promising tool to fundamentally address all these challenges and to systematically and exhaustively investigate hidden meanings of synonymous codons across the genome. It would mark the first study on high density and systematic synonymous codon substitutions being attempted on a genome scale. This study will reveal numerous positions where synonymous codon choices are not random but a ‘deliberate' evolution choice, and help to unravel the possible hidden code within the codon choices along the open reading frames of many genes.
Figure 2. 11 codons targeted for removal by recoding. Identifying target codons for removal (grey), and all possible synonymous substitutions to which they can be recoded. Numbers indicate the total number of different recoding options for a given subset of target codons with fixed projections between targets and synonyms throughout the genome.