Abstract
Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.
Highlights
The method of in vivo nonsense suppression in Xenopus laevis oocytes via chemically aminoacylated tRNA has enabled the site-specific encoding of over 100 different amino acids into ion channels and other proteins[1,2,3]
Multiple tRNA species have been used for delivery in the oocyte system, the most common being a mutated version of the glutamine tRNA from Tetrahymena thermophila, commonly termed THG7318
When THG73 was used as the carrier tRNA for this position, significant hNav1.5 current was generated in the presence or absence of an appended tyrosine amino acid (Fig. 2, top panels)
Summary
The method of in vivo nonsense suppression in Xenopus laevis oocytes via chemically aminoacylated tRNA has enabled the site-specific encoding of over 100 different amino acids into ion channels and other proteins[1,2,3]. The same species of tRNA can be used for encoding the amino acid needed for the experimental inquiry, in contrast to co-injecting an aminoacyl-tRNA synthetase for ncAA aminoacylation[5,6,7] For this reason, chemical acylation of tRNAs is widely used for genetic code expansion in Xenopus oocytes. Depending the functional tolerance at the site of incorporation within the target protein, misincorporation may lead to a mixed population of glutamine and the ncAA at the encoding site (introduced stop codon, usually TAG; amber codon)[19,20] This variability can be controlled for by careful analysis of conditions performed in parallel with non-acylated tRNA (tRNA-CA), varied length of incubation following injection and limited abundance of tRNA, which provides an experimental window in which ncAA rescue precedes any such unintended readthrough event. We assayed the orthogonality of pylT via in vitro chemical aminoacylation and injection into Xenopus oocytes (Fig. 1), using high-resolution ion channel function as a sensitive and quantitative readout of rescue and readthrough
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