Discovery, implications and initial use of semi-synthetic organisms with an expanded genetic alphabet/code

Abstract

Much recent interest has focused on developing proteins for human use, such as in medicine. However, natural proteins are made up of only a limited number of canonical amino acids with limited functionalities, and this makes the discovery of variants with some functions difficult. The ability to recombinantly express proteins containing non-canonical amino acids (ncAAs) with properties selected to impart the protein with desired properties is expected to dramatically improve the discovery of proteins with different functions. Perhaps the most straightforward approach to such an expansion of the genetic code is through expansion of the genetic alphabet, so that new codon/anticodon pairs can be created to assign to ncAAs. In this review, I briefly summarize more than 20 years of effort leading ultimately to the discovery of synthetic nucleotides that pair to form an unnatural base pair, which when incorporated into DNA, is stably maintained, transcribed and used to translate proteins in Escherichia coli. In addition to discussing wide ranging conceptual implications, I also describe ongoing efforts at the pharmaceutical company Sanofi to employ the resulting 'semi-synthetic organisms' or SSOs, for the production of next-generation protein therapeutics. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Keywords: expanded genetic alphabet; expanded genetic code; protein therapeutics; semi-synthetic organisms; unnatural base pair.

Figure 1.

The natural base pairs, dG–dC and dA–dT and three UBPs, dNaM–d5SICS, dNaM–dTPT3 and dCNMO–dTPT3, developed to expand the genetic alphabet. TAT1 is only used in the ribonucleoside triphosphate form shown (see near end of §4 for details).

Figure 2.

Structure of UBP (a) in free duplex DNA [67] and (b) during formation in the active site of a polymerase [54]. (Online version in colour.)

Figure 3.

(a) Error elimination system to increase UBP (X–Y) retention, where Cas9 cleavage is directed by single-guide RNAs that target it to sequences that have lost the UBP (lower left plasmid (red); N = natural nucleotide), leading to degradation of the corresponding plasmid [78]. (b) Proposed mechanism of in vivo replication of DNA containing a UBP. In well replicated sequences, replication is mediated by the main replicative polymerase, Pol III (tan sphere), with errors eliminated by methyl-directed mismatch repair (MMR). In more challenging sequences, the stalled Pol III replisome is rescued by Pol II (blue sphere), which results in retention of the UBP, or by RecA (light pink hexagons), after which the UBP is lost during recombinational repair of the stalled fork. Pathways through which retention is increased (Y–X UBP product) or decreased (N-X product) in the optimized SSO are shown in bold green and orange arrows, respectively. Adapted with permission from [80]. (Online version in colour.)

Figure 4.

Components of an SSO with an expanded genetic alphabet and code. The unnatural nucleotide triphosphates of the UBP are indicated as (d)XTP and (d)YTP; the encoded non-canonical amino acid (ncAA) and nucleotide transporter (PtNTT2) are indicated; RNAP, RNA polymerase. (Online version in colour.)

Figure 5.

Natural IL-2 (left) preferentially binds the trimeric IL-2Rαβγ receptor relative to the dimeric IL-2Rβγ receptor (indicated by thick and thin arrows, respectively). The site-specifically incorporated PEG of THOR-707 (right) interferes with IL-2Rα binding and thus better stimulates cell populations bearing the dimeric IL-2Rβγ. (Online version in colour.)