Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

Abstract

We systematically evaluate potential platforms for site-specifically incorporating two distinct noncanonical amino acids into proteins expressed in mammalian cells with optimal fidelity and efficiency – a technology that will have many enabling applications.

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

“…Additionally, the EcTyrRS/tRNA pair have been used together with the pyrrolysyl pair to site-specifically incorporate two distinct Uaas into proteins expressed in mammalian cells. (Xiao et al, 2013; Zheng et al, 2017a) The scope of this technology would be further expanded with the ability to incorporate a broader set of useful Uaas using this pair.…”

“…(Dumas et al, 2015; Italia et al, 2017a; Italia et al, 2017b) It also limits the scope of the nascent technology for concurrently incorporating multiple different Uaas into proteins in eukaryotes, each of which must be charged by a distinct orthogonal pair. (Italia et al, 2017b; Xiao et al, 2013; Zheng et al, 2017a; Zheng et al, 2018)…”

Playing with the Molecules of Life

“…87, 132 These new tRNA/aaRS pairs have allowed multiple, different amino acids to be incorporated in both E. coli 123, 126, 133 and eukaryotic systems. 134, 135 These experiments suggest that the number of orthogonal pairs that can be discovered from natural sequence diversity does not place a limit on the number of distinct building blocks that may be simultaneously genetically encoded in cells. An active area of research now focuses on reassigning degenerate codons in the genetic code to encode additional ncAAs.…”

Expansion of the genetic code via expansion of the genetic alphabet

“…Though these methods enable incorporation of up to two, different ncAAs in both prokaryotic [14] and eukaryotic [15] cells, the heterologous recoded tRNAs must compete with endogenous release factors (RFs), or in the case of quadruplet codons, normal decoding [16], which limits the efficiency and fidelity of ncAA incorporation. To eliminate competition with RF1, which recognizes the amber stop codon and terminates translation, efforts have been directed toward removal of many or all instances of the amber stop codon in the host genome [17,18] or modification of RF2 [19] to allow for the deletion of RF1.…”