Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells

Knowledge of protein function is of pivotal importance to life science research. It can guide conventional drug development programmes and lead to novel strategies to address currently non-targetable systems [1–3]. In order to understand the precise role and interacting network of a protein, it is essential to analyse it within its native environment. For a mammalian protein, its function often also depends on its host cell (e.g. cell type and cell cycle stage), specific subcellular location and post-translational modifications. In addition, a protein of interest often exists in the presence of other closely related homologues (e.g. proteins within the same family), making it difficult to decipher the precise function of a specific protein in cells. Targeting the protein by small-molecule inhibition is often not possible in these cases, as protein homologues will also be affected. To tackle this problem, over the last two decades there has been a drive to develop and refine the technique of genetic code expansion which allows researchers to exploit the cellular translation machinery for site-specific incorporation of unnatural (non-canonical) amino acids into target proteins [4–14]. Consequently, this enables the use of building blocks beyond the 20 canonical amino acids and incorporation of unnatural amino acids with unprecedented functionality into target proteins in live cells. The repurposing of the translational machinery by this approach has paved the way for revealing the functions of proteins under physiological conditions [15–19]. For example, the technique can be used to site-specifically introduce an unnatural amino acid into the homologue of interest, whereby unique functionality (on the unnatural amino acid) can be used for selective activation, inhibition, or reversible regulation of the target homologue [7].

At the molecular level, the mechanism of protein translation is highly conserved in all organisms, where the cellular machinery ‘translates’ every nucleotide triplet as a codon consecutively on the mRNA into the corresponding amino acid. In nature, the endogenous aminoacyl-tRNA synthetase (aaRS)/tRNA pairs within the cell decode 61 of the total 64 codons to 20 canonical amino acids. The remaining three codons (UAG, UGA and UAA) are used for translation termination, and hence they are also known as ‘stop’ codons. In order to achieve site-specific incorporation of an unnatural amino acid, an orthogonal aaRS/tRNA pair is needed, which must decode a codon that does not correspond to any canonical amino acid, a so-called blank codon (Figure 1). Stop codons are most commonly used as a blank codon in genetic code expansion, and decoding of a stop codon is known as ‘suppression’ because it suppresses the translation termination. The amber stop codon (UAG) is often used as the blank codon due to its minimal occurrence in most organisms.

Within the concept of genetic code expansion, ‘orthogonality’ refers to the non-reactivity of the orthogonal aaRS/tRNA pair with the endogenous pair and canonical amino acids in the host cell. The orthogonal synthetase must only acylate the orthogonal tRNA with the designated unnatural amino acid; neither canonical amino acids nor endogenous tRNAs are substrates of the orthogonal synthetase; similarly, neither the unnatural amino acid nor orthogonal tRNA is a substrate of the endogenous synthetases (Figure 2).

Besides the amber codon, other stop codons [20–26] and different four-nucleotide codons [27,28] have been used as a blank codon. The use of four-nucleotide codons expands the theoretical codon numbers from 4³ (64) to 4⁴ (256) so that multiple different unnatural amino acids can be incorporated at the same time. However, decoding a four-nucleotide codon by the ribosome is less efficient than decoding the normal three-nucleotide codons. Although this issue has been addressed in Escherichia coli through ribosome engineering [29–31], the lower efficiency in decoding four-nucleotide codons remains an issue in mammalian systems [27,28].

To date, many unnatural amino acids (1–110, Table 1) can be site-specifically incorporated into proteins produced by mammalian cells using genetic code expansion [5,32]. While the amino acids are structurally diverse, the majority of them can be incorporated through only a few orthogonal synthetases and their mutants. The Pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl pairs from archaea species Methanosarcina barkeri (Mb) and Methanosarcina mazei (Mm) have proven to be extraordinarily useful pairs [4]. The tRNA^Pyl naturally recognises the UAG codon and thus engineering of this tRNA is not needed. In addition, this pair is orthogonal in both E. coli and mammalian cells; hence, it facilitates the engineering of PylRS in E. coli and subsequently using the engineered PylRS mutant for incorporation of the designated unnatural amino acid in mammalian systems. As shown in Table 1, a wide range of amino acids has been incorporated into proteins in mammalian cells through only a few point mutations on the PylRS gene.