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Directed Evolution of Ribo-X and Ribo-Q

In our past research, we have utilized directed evolution approaches to evolve novel ribosomal decoding capacities to redefine the amber stop codon and create quadruplet codons to encode multiple unnatural amino acids in vivo. Based on orthogonal translation machinery that is insulated from the expression of the proteome by the endogenous ribosomes and mRNAs (Figure 4a), we evolved the decoding property of the orthogonal ribosome to firstly efficiently decode the UAG amber stop codon as a new sense codon

(Ribo-X, Figure 4b), and then to decode quadruplet codons (Ribo-Q, Figure 4c). We then evolved a set of four quadruplet tRNAs that can efficiently decode their cognate quadruplet codon (TAGA, AGGA, AGTA, or CTAG) on orthogonal mRNAs by Ribo-Q for incorporation of multiple distinct unnatural amino acids (Figure 4d). These experiments established that it is possible to evolve the ribosome and the tRNAs to drastically enhance the efficiency of unnatural amino acid incorporation in response to a synthetic orthogonal quadruplet code (Figure 4). 

Kai 3 Figure 4.  Development of a synthetic orthogonal quadruplet code for efficient incorporation of unnatural amino acids. 

a.    The O-ribosome and O-mRNA run orthogonally to their wildtype endogenous counterparts.
b.    Evolving the decoding property of O-ribosome to decode UAG as a sense codon (riboX) for unnatural amino acid.
c.    Creation of riboQ with the ability to decode quadruplet codons.
d.    Systematic directed evolution of quadruplet decoding tRNAs to encode multiple distinct unnatural amino acids in response to a riboQ based synthetic orthogonal quadruplet code with maximally 44=256 new quadruplet codons for unnatural amino acid incorporation.