From a large pool of mutated aminoacyl-tRNA synthetases the enzyme that loads tRNAs with their corresponding amino acids Wang selected the one that would attach a desired artificial amino acid to a tRNA that recognizes one of the stop codons. Every time the stop codon appeared in the genetic code, the new tRNA would insert the artificial amino acid.
But doing the same trick in mammalian cells becomes way more complicated. Simply transferring the bacterial genes into mammalian cells doesnt work since they flat out refuse to produce bacterial tRNAs. While it is easy to screen large numbers of mutated aminoacyl-tRNA synthetases in bacteria and yeast, it cant be done in mammalian cells in the same way. But Wang and his team got around both obstacles.
We found that we could coerce mammalian cells to express bacterial tRNAs by using the H1 promoter, says first author Wenyuan Wang, Ph.D., a postdoctoral researcher in Wangs laboratory. Relying on yeast to do the dirty job of finding a synthetase that recognizes tRNA and attaches the right unnatural amino acid helped them to overcome the second challenge. Using yeast for the selection process and then transferring the enzyme for use in mammalian cells may sound like a nave idea, but members from the same kingdom behave very similarly in terms of tRNA synthetases and it worked, he adds.
After a green fluorescence protein-based functional assay in various mammalian cells and neurons literally gave them the green light, Wang teamed up with Slesinger, who studies ion channels in the brain, to illustrate that this technology can solve othe
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