Expansion of the genetic code

With few exceptions, the canonical genetic code is preserved in all three kingdoms of life and encodes 20–22 common amino acids. A general method for incorporating unnatural amino acids (Uaas) into proteins in live E. coli cells was developed by the PI during his graduate study mentored by Dr. Peter G. Schultz, effectively expanded the genetic code for the first time. This method involves the generation of an orthogonal tRNA/synthetase pair, which does not crosstalk with endogenous tRNA/synthetase pairs but is functionally compatible with the protein translational machinery (Figure 1.1). The orthogonal synthetase is engineered to charge the desired Uaa onto the orthogonal tRNA, and the tRNA decodes a unique codon (such as the amber stop codon UAG) to incorporate the Uaa into proteins through translation.



Figure 1.1: A general method for genetic incorporation of unnatural amino acids in live cells

To study various biological processes and disease mechanisms, mammalian cells, multicellular organisms and animal models are desirable. Two impediments had stymied intensive efforts to incorporate Uaas in mammalian cells for years: the difficulty in expressing prokaryotic tRNAs and the infeasibility of evolving synthetases in mammalian cells. We solved the first problem by devising an external type-3 polymerase III promoter (e.g., H1 and U6, Figure 1.2) and the second problem with a transfer strategy. These new approaches enabled Uaas to be genetically incorporated into proteins in various mammalian cells, including primary neurons (Nat. Neurosci, 10, 1063-1072, 2007). These methods have subsequently proven generally applicable to a variety of eukaryotic cells and multicellular organisms, which are now widely used. The incorporation efficiency was further increased by optimizing the affinity between the orthogonal tRNA and synthetase (Mol. Biosyst. 5, 931-934, 2009) and by enhancing cellular uptake of Uaas with chemical modifications (ChemBioChem, 11, 2268-2272, 2010).


Figure 1.2: A general method for functional expression of prokaryotic tRNAs in mammalian cells: a type 3 polymerase III promoter (e.g., the H1 promoter) drives the prokaryotic tRNA without the last trinucleotide CCA followed by a 3’ flanking sequence (tRNA4 in the figure).

The low incorporation efficiency of unnatural amino acids in yeast had hindered effective applications. We found that orthogonal E. coli tRNAs expressed in yeast using the conventional method are not competent in translation. To solve this problem we developed a new functional expression method by using internal leader polymerase III promoters. In addition, we demonstrated that disabling nonsense-mediated mRNA decay markedly increases the incorporation efficiencies of unnatural amino acids in yeast. These new strategies increased the yield of unnatural amino acid-containing proteins from tens of micrograms to tens of milligrams per liter in yeast (J. Am. Chem. Soc. 130, 6066-6067, 2008), and enabled effective application of unnatural amino acids in yeast for research (e.g., J. Am. Chem. Soc. 131, 14240-14242, 2009).

internal leader polymerase III promoter

Figure 1.3: The internal leader polymerase III promoter drives the functional expression of prokaryotic tRNAs in yeast. A yeast strain deficient in Nonsense-mediated mRNA Decay (NMD) was generated by knocking out UPF1, which increased the incorporation efficiency of unnatural amino acids.

Stem cell lines stably incorporating unnatural amino acids provide not only novel means for studying stem cell biology but also an attractive source of unnatural amino acid-encoding mature cells that are otherwise difficult and expensive to procure. We developed a lentiviral-based method and achieved stable unnatural amino acid incorporation in neural stem cells, throughout their differentiation, and in the differentiated neurons. We did not observe notable interference with differentiation by incorporating unnatural amino acids, suggesting the suitability of using unnatural amino acids to study differentiation. This method should be generally applicable to other stem cells (Stem Cells 29, 1231-1240, 2011).


Figure 1.4: Neurons differentiated from neural stem cell HCN-A94

Although single cells are valuable models, research into development and various intercellular signaling processes necessitates multicellular organisms. We developed new strategies to address every challenging aspect of unnatural amino acid incorporation in C. elegans, a multicellular model organism extensively used for studying basic biology and human diseases. We have generated a series of stable transgenic worms capable of genetically encoding various unnatural amino acids (ACS Chem. Biol. 7, 1292-1302, 2012).


Figure 1.5: Stable transgenic worms with unnatural amino acid incorporated into mCherry in muscle cells

Mammal models are invaluable for studying basic biology and human diseases. Using in utero electroporation and various new strategies, we genetically incorporated unnatural amino acids into embryonic mouse, representing the first success in expanding the genetic code of mammals (Neuron 80, 358-370, 2013). We activated ion channels in mice neocortical neurons with light.

electroporation and genetic incorporation

Figure 1.6: In utero electroporation and genetic incorporation of unnatural amino acid Cmn in embryonic mouse