An alternative approach to Protein Engineering

An alternative approach to Protein Engineering

The central dogma of molecular biology is a well-known phenomenon to biotechnologists around the world. In the process of molecular translation, sequence-defined polymerisation of amino acids into protein happens using an mRNA (messenger) template. This implausible catalytic activity of biosynthesis of protein motivates scientists to develop engineered proteins. However, in nature, only limited monomers of amino acids are utilized and this constricts the diversity of proteins. If there is some way to expand these set of monomers, then we could yield new versions of designer therapeutics, genetically encoded materials, artificial enzymes as well as new organisms with new functions, forms and biocontainment strategies.

Conservatively, the genetic code which establishes a relationship between the DNA bases in a gene and the protein sequence it codes is degenerate (i.e. 64 codons code for 20 conventional amino-acids and 3 stop codons). Fisher et al., in their work have developed the first living organism which has 67 triplet-based codons and can code 3 unnatural base-pairs into multiple non-canonical amino acids into one protein. How can such a strategy be possibly achieved? The first method can be by substantially reprogramming the natural transfer RNA (tRNA) charging systems to accentuate these efforts. Alternatively, a quadruplet codon can be used. However, the machinery of translation has been delicately fine-tuned to perform optimally with a triplet codon. A compelling strategy can be by developing extended genetic alphabets.

In this particular study, an unnatural base pair was developed that relies on hydrophobic and packing interactions arising from unnatural nucleotides dNaM (an artificial nucleoside containing a 3-methoxy-2-naphthyl group instead of a base) and dTPT3. This base-pair led to the foundation of increased storage of information in the DNA of a semisynthetic organism which can be faithfully retrieved as mRNA. However, the translation efficiency of such an unnatural base-pair is yet to be assessed. It is interesting to note that there needs to be at least one G–C pair in the codon. This is to compensate for the weaker interactions among the unnatural base-pairs.  This elegant work by the authors represents an exciting frontier for using an expanded genetic alphabet in a living organism, but it also raises many questions. For example, how far can the technology be pushed to enable high-yielding expression of proteins?

Additionally, what is the maximal number of codons that can function in a living organism? Could more than 100 codons be decoded in a single organism? What about 200? Recently, a genetic system based on ‘Hachimoji’ DNA and RNA was developed, comprising two new base pairs, named S and B as well as P and Z, based on hydrogen bonding rules. The 4 additional bases made available by this advance theoretically creates 512 potential codons (8³), moving such questions out of being purely hypothetical and one step closer to being possible.

Though many interesting questions remain, the potential of this technology for repurposing molecular translation is clear. The unnatural base pair technology described liberates the genetic code from evolutionary constraints imposed by the decoding needs of the host organism. This offers exciting opportunities to vividly intensify the resolution at which we can manipulate the protein biosynthesis machinery to design new rules for constructive biology. We can look beyond what does exist to what can exist. Very soon, we may see a world where organisms are engineered to decipher entirely alternative genetic codes.

- Sanket Mukherjee
4^th Year
Department of Biotechnology,

References:

Fischer, E.C., Hashimoto, K., Zhang, Y., Feldman, A.W., Dien, V.T., Karadeema, R.J., Adhikary, R., Ledbetter, M.P., Krishnamurthy, R. and Romesberg, F.E., 2020. New codons for efficient production of unnatural proteins in a semisynthetic organism. Nature Chemical Biology, pp.1-7.

Image source- Nature Chemical Biology journal