Expanding the genetic code of mammalian cells

Abstract

In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology.

Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes

“…(Chin, 2017; Dumas et al, 2015; Italia et al, 2017b; Mukai et al, 2017; Young and Schultz, 2018) To co-translationally incorporate an Uaa, it is encoded by a repurposed nonsense codon, which is suppressed by an orthogonal (i.e., does not cross-react with its host counterparts) Uaa-selective aminoacyl-tRNA synthetase (aaRS)/tRNA pair. (Chin, 2017; Dumas et al, 2015; Italia et al, 2017b; Mukai et al, 2017; Young and Schultz, 2018) Eukaryote or archaea derived aaRS/tRNA pairs are orthogonal in bacteria and used for Uaa incorporation, while those derived from bacteria are typically orthogonal in eukaryotes (Figure 1A). (Chin, 2017; Dumas et al, 2015; Italia et al, 2017b; Mukai et al, 2017; Young and Schultz, 2018) To create Uaa-specific variants of aaRS/tRNA pairs through directed evolution, two cell-based selection systems have been developed, using Escherichia coli(Santoro et al, 2002; Wang et al, 2001) or Saccharomyces cerevisiae (yeast)(Chin et al, 2003a; Chin et al, 2003b) as selection hosts, for engineering pairs that are orthogonal in bacteria or eukaryotes, respectively.…”

A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation

“…15 Using this platform, we expressed superfolder green fluorescent protein incorporating 5HTP at a permissive surface exposed site (sfGFP-151-5HTP; Figure S11). Incubation of sfGFP-151-5HTP with 40 μM 4CDz resulted in its rapid and complete covalent modification (Figure 3A, Figure S12) within 30 min, as observed by mass spectrometry analysis, while a nearly identical wild-type sfGFP (Tyr at 151 position) protein remained unmodified upon the same treatment (Figure 3B).…”

Synthesis at the interface of virology and genetic code expansion

“…[51] One central limitation comes from the suboptimal efficiency of the engineered aaRS/tRNA pairs at the core of this technology. Additionally, the overwhelming majority of applications of this technology in mammalian cells rely on transient transfection for the delivery of the genetic components, which has significant limitations.…”

“…Additionally, the overwhelming majority of applications of this technology in mammalian cells rely on transient transfection for the delivery of the genetic components, which has significant limitations. [51,52] The factors that limit the Uaa incorporation efficiency, and the principles for the design of an optimal mammalian expression system also remain incompletely understood. [51,52] Virus-based tools have played a significant role in overcoming these challenges in recent years.…”

“…[51,52] The factors that limit the Uaa incorporation efficiency, and the principles for the design of an optimal mammalian expression system also remain incompletely understood. [51,52] Virus-based tools have played a significant role in overcoming these challenges in recent years.…”