Engineering Genetic Codes

Somewhat recently I blogged about a paper¹ under the headline "The Most Interesting Paper I've Ever Read." I settled on that title because of the stunning ingenuity of the authors' approach and because rewiring the genetic code is an exciting and worthy goal. When I approached that earlier blog post I failed to appreciate how much work had already been done in incorporating non-standard amino acids into proteins, which I'll give a brief treatment.

What Isaacs et al. (2011)—"the most interesting paper I've ever read"—offered was a way to eliminate one codon from the genetic code. The genetic code is an ordered list, a cipher, where three nucleotides of DNA/RNA information can be translated into a single amino acid. Since there are four nucleotides and three positions in the code, the genetic code can accommodate 4³, or 64 messages. Living organisms only use 21 messages: 20 amino acids plus one message that means "stop." Despite that, cells use all 64 positions in the code. We call the genetic code degenerate because multiple code words mean the same thing (TAA, TAG, and TGA all mean "stop," for example).

The main goal we want to reach by engineering the genetic code is to incorporate non-standard amino acids into proteins. Cells only use 20 amino acids, all left-handed, whereas chemists can synthesize a much broader suite in the lab. In order to incorporate non-standard amino acids into proteins, you have to incorporate it into the organism's genetic code by assigning it a codon. If we work within the existing (degenerate) genetic code, the only choice is to pick a codon to assign doubly since all 64 combinations are already assigned. Even with this limitation it's possible to incorporate non-standard amino acids into the genetic code². Though not ideal, this approach still works surprisingly well. We have a good understanding of how tRNAs—the molecules that actually translate the genetic code—work. Labs working in this area have been able to build tRNAs that suppress the amber stop codon (TAG), the least frequently used codon, by instead incorporating a non-standard amino acid. Thus, when the suppressor tRNA reads TAG it suppresses the stop message by instead incorporating a non-standard, 21st amino acid.

Using methods like this, biologists have successfully incorporated a wide variety of exotic amino acids into host genetic codes². Using our knowledge of biochemistry, we can use these new amino acids to study protein structure and function in the test tube and more excitingly, living cells. For example, some synthetic amino acids are fluorescent by nature. Using fluorescence to study proteins in cells has become immensely popular thanks to green fluorescent protein (GFP). The gene coding for GFP can be fused to another protein gene, so that the (relatively small) GFP molecule hangs off the side of the other protein. The GFP, and therefore your protein of study, is wherever you see green spots. The down side is that, though small for a protein, the GFP might interfere with the function of the other protein. Replacing a single amino acid of the protein with a synthetic, fluorescent amino acid is a very attractive alternative since proteins fold with rather high fidelity, so a single (educated) amino acid substitution is unlikely to make any difference. The amino acid at right, above, has already served this purpose².

A more practical application of this technology has been to incorporate amino acids with novel chemical tricks². For instance some synthetic amino acids are very good at chelating metal ions (at right, bottom image). Many natural proteins are already able to accomplish this, but by creating a pocket of several amino acids—often not near one another within the protein sequence—with a proper charge to bind the ion. Since we have a very poor ability to predict protein folding, we can instead call on amino acids that individually have this property. As our knowledge improves, we can use this technology to refine the chemical reactions that take place within living cells that synthetic biology may well provide. Fairly recently an exciting first step has even been made in engineering the genetic code of an animal, every developmental biologist's best friend Caenorhabditis elegans³. Who knows what's next?

Image Credits: Created using PubChem Sketcher; structures listed in Xie & Schultz (2006)

References:

1. Isaacs, F. J. et al. Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement. Science 333, 348–353 (2011).

2. Xie, J. & Schultz, P. G. A Chemical Toolkit for Proteins-An Expanded Genetic Code. Nature Reviews Molecular Cell Biology 7, 775–782 (2006).

3. Greiss, S. & Chin, J. W. Expanding the Genetic Code of an Animal. Journal of the American Chemical Society. Published online 8 August 2011.