Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons

Abstract

We have created a bacterial semisynthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a noncanonical amino acid synthesizes proteins containing the noncanonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon–anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and, in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.

Posttranscriptional Site-Directed Spin Labeling of Large RNAs with an Unnatural Base Pair System Under Non-Denaturing Conditions

“…The resulting dsDNA templates containing dNaMs in the template strand are used to explore the site-specific modification of RNAs with UBP (Figure 2D). Although the TPT3-NaM UBP has been successfully utilized in creating an Escherichia coli and an eukaryotic semisynthetic organisms [44][45] , the ribonucleotides of TPT3 and TPT3 CO have not been tested in in vitro transcription. We therefore first characterize the ability of the T7 RNA polymerase to incorporate rTPT3 or rTPT3 CO into the RNase P RNA (Fig- ure 2D).…”

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

“…Although the in vivo replication and transcription of TPT3-NaM UBP have been validated recently in an Escherichia coli and an eukaryotic yeast semi-synthetic organisms by Romesberg group (45,46), the ribonucleotides of TPT3 and TPT3 A have not been tested in in vitro transcription with T7 RNA polymerase. The dsDNA templates containing dNaMs in the template strand for 3'SL and DENV-mini are used to explore site-specific incorporation of UBP into RNAs.…”

Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions

“…been successfully utilized in creating an Escherichia coli and an eukaryotic semisynthetic organisms, 48,49 the ribonucleotides of TPT3 CO have not been tested in in vitro transcription. We therefore rst characterize the ability of the T7 RNA polymerase to incorporate rTPT3 or rTPT3 CO into the RNase P RNA (Fig.…”