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Using directed evolution, chemists have created a novel ribosome that can insert multiple unnatural amino acids into a single protein in cells.

Until now, the lack of available codons—he three-nucleotide "words" of the genetic code—has constrained the number of unnatural amino acids that could be added to a single protein. With nearly all codons spoken for, scientists have been largely limited to trying to repurpose just one stop codon when inserting unnatural amino acids in proteins, says Jason W. Chin, whose team at the Medical Research Council Laboratory of Molecular Biology, in Cambridge, England, developed the new ribosome.

Now, Chin's group has used directed evolution to generate a ribosome that can read four-nucleotide or "quadruplet" codons in addition to the normal triplet codons (Nature, DOI: 10.1038/nature08817). In the new ribosome, the portion of the ribosomal RNA that recognizes the transfer RNA, which shuttles amino acids to the protein-synthesizing machinery, has been altered so that it can bind quadruplet codons. Although these alterations would be lethal in the natural ribosome, they are allowed in Chin's orthogonal ribosome, which does not have to make all the proteins needed to keep the cell alive.

The ribosome's ability to read both triplet and quadruplet codons allows insertion of not only the canonical amino acids but also multiple unnatural amino acids into a single protein. The researchers used the new ribosome to insert two unnatural amino acids into the protein calmodulin.

Those amino acids contain an azide and an alkyne which then undergo a Cu(I)-catalyzed Huisgen's [2+3] cycloaddition reaction when brought together by the protein's structure. The success of this cross-linking reaction shows that the ribosome can be used to program new properties into proteins, the researchers note.

The ability to read quadruplet codons opens the possibility of new words in the genetic code that can be assigned to new amino acids. Chin and colleagues are currently exploring how many of the 256 potential quadruplet codons can be assigned to new amino acids.

But the ribosome is only part of the story. "Once you have a ribosome that furnishes a whole series of blank codons, the next question is where on Earth do you find synthetases and tRNAs to allow you to put in so many amino acids," Chin says. With this goal in mind, Chin and coworkers have begun to build aminoacyl-tRNA synthetase/tRNA pairs from scratch (J. Am. Chem. Soc., DOI: 10.1021/ja9068722).

The combination of both papers "shows that we are scientifically at a new era of biopolymer engineering," says Ryan A. Mehl, an expert on unnatural amino acids at Franklin & Marshall College, in Lancaster, Pa. "Chin's group has shown that one can take an unbelievably complex biological system—translation—and reengineer it."

Tom W. Muir of Rockefeller University notes that others have used quadruplet codons but that Chin's study is the first to use a ribosome specifically engineered and optimized for that purpose. "The technology seems to work very well, offering a marked improvement in the yields of bis-modified proteins," Muir says. "This could open the floodgates to all manner of exciting applications."