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It took Schultz and his team approximately 5 years and many dead ends to solve the problem of generating an orthogonal tRNA/synthetase pair. They then tackled the job of altering the synthetase/amino acid specificity using a number of different in vitro evolution strategies. Recently they hit on a general method which after a few rounds of molecular evolution allowed Wang to generate a synthetase that loaded an orthogonal tRNA with 99.99 percent fidelity. "It worked surprisingly well," says Schultz.

"You just need to add the novel amino acid to the culture and grow the cells," says Wang.

Using this method, they incorporated O-methyl-L-tyrosine into proteins with fidelity greater than 99 percent, which is close to the translation fidelity of natural amino acids. "We really wanted high fidelity, and we thought if we could get it with O-methyl-tyrosine or tyrosine or phenylalanine, then one could probably get high fidelity with [almost] any amino acid," says Schultz.

Some wondered whether the feat could be repeated. One of the reviewers of the paper told Schultz that the result was amazing but a dead end. "He said O-methyl worked because it is very similar to tyrosine and we would never be able to do it again ," says Santoro.

"The next day," says Schultz, "Lei came in and he had just added [a] napthyl alanine to the code." Since that day, Schultz's group has added a host of novel amino acids to the E. coli genetic code.

Novel X and Four-Base Codons

There are novel amino acids that contain fluorescent moieties that are smaller than green fluorescent protein. These can be used in place of GFP to label proteins and observe them in vivo. Others novel amino acids useful as molecular probes have side chains that can be phosphorylated or that contain spin labels.

There are novel hydrophobic amino acids, which should be useful for probing structure, and novel nucleophiles, heavy metal-binding amino acids, and photoisomerizable side chains, all of which should confer new activity to the proteins. There are novel glycosylated amino acids that could be used to make therapeutic proteins, and there are novel amino acids with keto groups that can be used to selectively label proteins with practically any molecular group of interest.

There are also groups that contain photoaffinity labels that could be used for covalently cross-linking proteins to one another in a photoinduction proteomics experiment.

"The idea," says Postdoctoral Fellow Jason Chin, "is that you will put photo-crosslinking groups into a specific site in a protein. You could then see what the protein interacts with in living cells. And you will be able to look at weak interactions that are difficult to detect by current methods."

Putting in many different novel amino acids is the next step, and Schultz is working on generalizing the method used for the O-methyl-tyrosine so that he can routinely do this.

Schultz believes the key to easily inserting a new amino acid is changing the specificity of the aminoacyl-tRNA synthetase. Schultz describes the process as "gutting" the active site—putting in a hole that can be filled with a combination of a new amino acid and a new protein side chain. However, not all pegs fit in the same hole, and some synthetases will not be able to take certain novel amino acids. But by making several of these tRNA/synthetase pairs, it should be possible to put in almost any amino acid.

One pair that is underway is the leucine synthetase pair, which Schultz and his graduate student Christopher Anderson are working on now. This pair is interesting because it may be used to expand the technology beyond the amber codon, which, though successful and robust, is limited. "That only lets you use one or potentially two [different] amino acids [per protein]," says Schultz.

"We're developing the leucine synthetase system to recognize a four-base codon," says Anderson.

The difficulty, though, is that most anticodon loops are key recognition elements of the synthetase, and this recognition becomes intolerably perturbed due to the structure of the four-base tRNAs. "You have to be able to change the anticodon loop of the tRNA and still be able to have the synthetase recognize the tRNA," says Anderson. In the leucine synthetase system this is not a problem because the synthetase recognition occurs at another site.

The strategy involves using molecular evolution experiments to select for tRNAs with anticodon loops that recognize four or five bases. The advantage of using the longer codon is diversity—there are 256 four-base codons possible, for instance, and many of these can be re-assigned to a new unnatural amino acid. They are now building new tRNA/synthetase pairs that decode four bases at a time.

Life with Many Amino Acids

Another interesting question the group is working out is how to transfer the technology to eukaryotic cells. "Right now all this work has been done in E. coli," says Santoro. "It would be much more interesting to be able to express proteins containing unnatural amino acids in mammalian cells."

" The ability to do unnatural cell biology by introducing unnatural amino acids such as flurophores and photocrosslinkers into proteins in eukaryotic cells will provide a powerful arsenal of tools to dissect and understand how these cells and even whole organisms work," says Chin, who is working on expanding the eukaryotic code. "We will be able to probe protein interactions involved in human disease with unprecendented precision in living cells."

Another follow up project applies the technology in a random way, adding novel amino acids to the genetic code of cells, allowing the cells to use them. The team will see how these changes affect the organism's ability to, say, evolve in response to stress.

To answer this, Schultz and his colleagues plan to do a random unnatural amino acid mutagenesis of the entire E. coli genome and subject the new cells to some sort of selective pressures—like heat or antibiotics—and see what happens. Could a 21-amino-acid bug be more robust than a 20-amino-acid bug?

"If you put that cell under some stress, will the cell be able to evolve faster to deal with that stress?" asks Schultz.

A further application of this line of research that Schultz is contemplating is to randomly mutate proteins with several unnatural amino acids in many places simultaneously. And yet another possibility would be to add the metabolic pathway for the synthesis of the novel amino acid to the cells so that it would not have to be added to the growth medium—something that Wang, Anderson, and Zhang are addressing at the moment.

Polyester Proteins

Schultz and his team are also asking whether we need amino acids in the first place. Can you use some other polymer building block, like hydroxy acids, as a protein constituent and will the resulting protein fold?

Whereas novel amino acids differ in side chain chemistry but have the same amide backbone as natural amino acids, hydroxy acids would have a completely different backbone structure. Instead of a polyamide, they would form polyesters.

One of the first things they will try is simply to get a single hydroxy acid incorporated site specifically, using the same technology they worked out for the O-methyl-tyrosine system. Then with this proof of principle out of the way, they will proceed to trying to use two or more tRNA/synthetase pairs that incorporate hydroxy acids that are based on hydrophobic and hydrophilic amino acids in the hope of making a folded polyester protein.


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"We're developing the leucine synthetase system to recognize a four-base codon," says graduate student Christopher Anderson. Photo by Jason S. Bardi.








"It's hard to find synthetases that recognize novel amino acids because nature has designed them to recognize only the natural ones," says Research Associate Steve Santoro. Photo by Jason S. Bardi.







"We will be able to probe protein interactions involved in human disease with unprecendented precision in living cells," says Research Associate Jason Chin. Photo by Jason S. Bardi.













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