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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.
Figure 1. Misacylation of orthogonal tRNA for genetic code expansion. (a) Target protein cRNA containing a site-directed stop codon (nonsense codon) is subsequently suppressed by an orthogonal tRNA - THG73 or pyrrolysine tRNA (pylT). The grey highlighted nucleotides “CA” represent ligated pdCpA (see methods). (b) Co-injection of tRNA and cRNA into the Xenopus oocyte enables electrophysiological two-electrode voltage clamp characterization of ion channels and receptors containing ncAA.
Figure 2. Rescue of an introduced stop codon into the human cardiac voltage gated sodium channel. Voltage-induced currents upon co-injection of hNav 1.5 S571TAG with either acylated (left) or unacylated tRNA (right) variants THG73 (top) or pylT (bottom). Xenopus oocytes expressing sodium channel variants were subjected to membrane depolarizations (steps from −80 mV to −20 mV). Traces show development of rapidly inactivating sodium currents as downward deflections. The level of zero current for each cell is indicated by a black dashed line.
Figure 3. pylT can be efficiently ligated to dinucleotide-amino acid substrates in vitro. TBE UREA tRNA gels show successful ligation of substrates to pylT. Approximately 2 µg of tRNA was run per well. Each lane represents an independent ligation. Note the consistent gel shift representative of ligation of the dinucleotide-amino acid substrate to the truncated pylT. Abbreviations: Ala (alanine), Tyr (tyrosine), Aah (alpha-hydroxy alanine). Examples of additional substrate types are available in the Supplementary Figures.
pylT enables the faithful encoding of lysine into position K1237 of rNav 1.4. (a) Example traces of rNav 1.4-K1237TAG cRNA coinjected with either lysine-acylated (top) or unacylated full length (bottom) PylT. Oocytes were held at −100 mV and pulsed from −80 mV to +40 mV with 30 ms depolarizing steps. Voltage gated sodium channel activity is evidenced by increasingly large downward deflections in the traces in response to depolarization. The level of zero current for each cell is indicated by a dashed line. (b) Normalized current-voltage relationship plots comparing the WT rNav 1.4 channel to that of the rNav 1.4 K1237TAG rescued with lysine.
Figure 3. pylT can be efficiently ligated to dinucleotide-amino acid substrates in vitro. TBE UREA tRNA gels show successful ligation of substrates to pylT. Approximately 2 µg of tRNA was run per well. Each lane represents an independent ligation. Note the consistent gel shift representative of ligation of the dinucleotide-amino acid substrate to the truncated pylT. Abbreviations: Ala (alanine), Tyr (tyrosine), Aah (alpha-hydroxy alanine). Examples of additional substrate types are available in the Supplementary Figures.
pylT enables the faithful encoding of lysine into position K1237 of rNav 1.4. (a) Example traces of rNav 1.4-K1237TAG cRNA coinjected with either lysine-acylated (top) or unacylated full length (bottom) PylT. Oocytes were held at −100 mV and pulsed from −80 mV to +40 mV with 30 ms depolarizing steps. Voltage gated sodium channel activity is evidenced by increasingly large downward deflections in the traces in response to depolarization. The level of zero current for each cell is indicated by a dashed line. (b) Normalized current-voltage relationship plots comparing the WT rNav 1.4 channel to that of the rNav 1.4 K1237TAG rescued with lysine.
Figure 4. pylT enables the faithful encoding of lysine into position K1237 of rNav 1.4. (a) Example traces of rNav 1.4-K1237TAG cRNA coinjected with either lysine-acylated (top) or unacylated full length (bottom) PylT. Oocytes were held at −100 mV and pulsed from −80 mV to +40 mV with 30 ms depolarizing steps. Voltage gated sodium channel activity is evidenced by increasingly large downward deflections in the traces in response to depolarization. The level of zero current for each cell is indicated by a dashed line. (b) Normalized current-voltage relationship plots comparing the WT rNav 1.4 channel to that of the rNav 1.4 K1237TAG rescued with lysine.
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