The Role of Orthogonality in Genetic Code Expansion

Abstract

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

Keywords: genetic code expansion; genome recoding; nonstandard amino acids; orthogonality; protein engineering; ribosome engineering; translation.

Figure 1

Orthogonality involved in the aminoacylation of tRNAs. First, (blue) an orthogonal aminoacyl-tRNA synthetase (aaRS) can aminoacylate the nonstandard amino acid (nsAA) or a chemically close standard amino acid (sAA); then, (orange) each aaRS∙AA pair can aminoacylate its orthogonal tRNA or another one of the native pool; (green) all of these possible AA-tRNAs leave four potential translation outcomes.

Figure 2

Recoding repurposes the usage of codons. (a) The “universal” genetic code contains 61 sense codons and three stop codons. (b) (green) AGC and (red) UAG are used as sense codon for serine incorporation and stop codon, respectively. (c) These codons can be reassigned to other codons with the same meaning: (blue) leucine, (green) serine, (purple) arginine and (red) UAG stop codons have been reassigned to synonym codons. (d) By removing the native components that use the specific codons and introducing an orthogonal translation system (OTS), a new nsAA can be introduced into proteins using the new open codons: while (green) AGC and (red) UAG formerly encoded for serine or stopping translation, respectively, they are now used to introduce two different nsAAs.

Figure 3

Using orthogonal ribosomes to segregate nsAA incorporation into select proteins. Ideally, when native and orthogonal ribosomes coexist in a cell, they do not interact. The native transcriptome is translated by wildtype ribosomes, whereas orthogonal mRNAs are selectively translated by orthogonal tethered ribosomes (oRiboT). These mRNAs contain reassigned codons that are suppressed by an nsAA-charged tRNA.